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METHODS OF

BIOCHEMICAL ANALYSIS

Edited by DAVID GLICK Professor oC Physiological Chemistry l ' ui\·ersily of Minnesota, Minneapolis

VOLUME

VII

INTERSCIENCE PUBL I SHERS, I NC . , NEW YORK INTER CIENCE PUBLISHERS LTD., LONDON

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Copyright

© 1959 by Interscience Publishers, Inc.

Library of Congress Catalog Card Number 54-7232

Interscience Publishers, Inc., 250 Fifth Avenue, New York I, N. Y. For Great Britain and Northern Ireland: InterBcience Publishers Ltd., 88/ 90 Chancery Lane, London, W. C. 2

PRINTED IN THE UNITED STATES OF AMERICA BY MACK PRINTING CO., EASTON, FA.

METHODS OF BIOCHEMICAL ANALYSIS Volume VII

Methods of BIOCHEMICAL ANALYSIS Volume 7

CONnNTS, I..... un...lectropho....lc Analysis. 8y Pie"e Grabor

fho A_lysis of laslc Nltrogo..ous Co ... pounels of To,dcological Importanco. 8y A. S. Curry

Spectrophotomotry of fro.slueollt liologlcal Matorial.; Opal Gla.. Mothod. 8y ICa%uo Shibota

Tho De"rml_tfoft of Illolitol, Ithaftolamlno, and Serino In lipldo.. 8y John M. Mcl(Jbbin

Tho Assay of L1poprotoln Llpa.. I. VI"o and in Vitro.

8y Edward D. ICorn

De.. ralnatfoft of Croatlalfto and dinl ... Compound..

ltelaf~

G.aal.

By John F. Van Pi/rum

n.. Detor.Jnatioa of Ethyl Alcohol In Blood and

n._s.

Iy Franlc Lundquist De......n.tf08 of Hoparin.

ay Lou;, 8. Jaqua and Hel.n J. B.II

Advisory Board: S. BERGSTR(jM, Unitlersily of Lund, Sweden A. M. BR E , Argonne :\"alional Laboratory, Lemonl, Illinois G. O. BU RR, Erperiment Station, Hawaiian Sugar Planters' Association, 'lonolulu

R. CO SDE ,The Cana.dian Red Cross Memorial llospital, Tap[ow, /Ilaidenhead, Berluhire, England A. B. HAST[ G, Harvard Medical &hool, Bo.~toTl H. nOLTER, Carlsberg Laboratory, Copenhagen, Denmark R. D. HOTCHKl S, The Rockefeller Institute for Medical Hesearch,:.rVew York J. K. N. JO E , Queen's University, l{ingston, Ontario, Canada C. G. KI G, The ]\-ulrition Foundation, ew York H. A. LARDY, University of Wisconsin, Madison 11. C. LICJJSTEI ,University of Minllesota, Minneap()lis G. F. MARRIAN, Unitersity of Edinburgh, Scotland B. L. 0 ER, Food Research Laboratories, New York J. ROCHE, Col/ege de France, Paris W. C. RO E, University oj Illinois, Urbana A. TISELIUS, University of Uppsala, Sweden D. D. VAN SLYKE, Brookhaven National Laboratory, Upton, Lollg IsicUtd, New York

METHODS OF BIOCHEMICAL ANALYSIS

PREFACE TO THE

VOLUME VII

ERIES

Annual review volume dealing with many different fields of science have proved their value repeatedly and are now widely used and well established. These reviews have been concerned primarily with the results of the developing fields, rather than with the techniques and method employed, and they have served to keep the ever-expanding scene within the view of the investigator, the npplier, the teacher, and the student. It is particularly important that review services of this nature should now be extended to cover metbods and techniques, because it ifl becoming increa. ingly difficult to keep abreast of the manifold experimental innovations and improvements which constitute the limiting factor in many cases for the growth of the experimental sciences. Concept· and yj ion of creative scienti ts far outrun that which can actually be attained in present practice. Therefore an emphasis on methodology and instrumentation i. a fundamental need for l11n,lerial achienment to keep in sight of the advance of useful ideas. The cu rrent volume is the first. of a serie which i designed to try to meet thi need in the field of biochemical analysif'. The topics to be included are chemical, phYf'ical microbiological and, if necessary, animal assay, as w 11 as basie techniques and instrument..'),tion for the determination of enzyme, yitamin " hormones, lipids, carbohydrates, proteins and their products, minerals, antimetabolite, etc. Certain chapters will deal with well-established method or techique which hl1\-e undergone sufficient imprm'ement to merit recapitulation, reapprai ~ al, and new recommendation, . Other chapters will be concerned with e senti ally new Dpproaches which bear promi. e of great u efulne s. Relatively few subjects can be included in any single volume, but as they accumulate these volumes should comprise a self-modernizing encyclop dia of methods of biochemical analy. is. By judicious selection of topics it is planned that most subject of current importllnce will receive treatment in the e volumes, v

Vl

PREFACE

The general plan followed in the organization of the individual chapters is a discussion of the background and previous work, a critical evaluation of the variou approache, and a pre entation of the procedural details of the method or methods recommended by the a.uthor. The presentation of the experimental details is to be given in a manner that will furnish the laboratory worker with the complete information required to carry out the analyses. Within this comprehensive scheme the reader may note that the treatments vary widely with respect to ta te, style, and point of view. It is the editor's policy to encourage indiyidual expre sion in these pre entations becau e it i stifling to originality and justifiably annoying to many authors to submerge them-elves in a standard mold. cientific writing need not be a ' dull and uniform as it too often is. In certain technical details a con. istent pattern i followed for the sake of convenience, as in the form u_ed for reference citn.tions and indexing. The success of the treatment of any topic will depend pl'imarily 011 the experience, critical ability, and capaciLy to communicate of the author. Those invited to prepare the re pective chapter' are scientists who either have originated the methods they di CUR. or have had intimate personal experience with them. It is the wi 'h of the Advisory Board and the editor to make thi series of volumes as u eful as possible and to this end uggestions will always be welcome, DAVID GLICK

Minneapolis, Minnesota

METlJOD

0 1" BIOCHEMI CAL ANALYSI

OLUME VII

CONTRIBUTORS

IIELEN J.

BELL, Department of Physiology and Pharmacology, University of Saskatchewan, askatoon, Canada A. . CURRY, IIome O.ffice Forensic cience La))oratory, IIarrogale, Yorkshire, Engl,and PIERHE GHABAR, Chef du Sen'ice de Chimie III icrobienne, I nstitut Pasu-'Ur, Paris, France LoU! B. JAQ"(;E', Department of Phy[:'iology and Pharmacology, UnilJ('1'Sit!J of 'askatchewan, asl.-aloon, Canada EUWARD D. Kon:-\, Laborator!J of Celhtlar Phys-iology and ]letabolism, .Vational Hcart institute, National Institutes oj Health, U. S. Department of Health, Edttcalion and Welfare, Bethesda, Maryland FRANK LUNDQUI. T, Cnil'crsity Institute of Forensic Jledicine, Copenhagen, Denmark JOlIN 1\1. l\1CKIBDlN, Department of Biochemistry, State Cnil ersity of New York ('cllpge of Jf cdicine, SyraClt e, New York l{Azuo llIJlATA, The Tol-ntgawa Institute for Biological Re carch and The Tokyo Institute of Technology, Tok!Jo, Japan JOHN VAN PIT, lIM, De7Jartment of Physiological Chemistry, Un iller ity of ,M~innesota, Jl! inl1ea.polis, .~[innesola

METHODS OF BIOCHEMICAL ANALYSIS

VOL ME VII

CO TE TS

Immunoelectrophoretic Analy is.

By Pierre Graba,r

1

The Analy is of Basic Nitrogenou_ ompound of Toxicological Importance. By A. S. Curry . . . . . . . . . .

39

pectrophotometry of Translucent Biological Materials-Opal Glass Tran mission Method. By Kawo Shibata . .

77

The Determination of Inositol, Ethanolamine, and Serine in Lipide. By John M. McKibbin . . . . . .

III

The As ay of Lipoprotein Lipase in T'ivo and in Vitro. By Edward D. [(om . . . . . . . . . . . . . . .

145

Determination of Creatinine and Related Guanidinium Compound. By John F. Van Pilsum . . . . . . .

193

The Determination of Ethyl Alcohol in Blood and Ti ue . By Frank Lundquist . . . . . . . . . . . . . . . .

217

Determination of Heparin. Bell.

By Louis B. Jaqu es and Helen J. 253

Author Index .

311

Subject J ndex

331

Cumulative Index

345

METHODS OF BIOCHEMICAL ANALYSI

VOLUME VII

IMMUNOELECTROPHORETIC ANALYSIS PIERRE

RABAR,

Inslilul Pasleur, Paris

I. Preparation of the Gels. . . . . . .. .............................. . 1. Nature of Gels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Purifiention of Agar . . . . . . . . . . . . ....................... A. Purification b~' Heating and Washing. . . . .. ......... B. Purification by FJ·t't'zing aud Alcoho!i(, Precipitation . . . . . .. . , 3. BufTerl.'d Agar GI.'I ~ ... " .......... 4. Preparation of Pla tes .. . . . . . . . . .. . . . . . . . . . . . . . .. .......... II . Electrophoresis in a Gl.'lifil.'d Mediulll . . . . . . . .. .,. ............ 1. EIl.'clric Current . . . .. . . . . . . . . . . . . . .. . . ........ 2. Buffer Solulion .. . . . . . . . . .. .... . ..... 3. Ele('troendosrnosi8. . . . . . .. . . . ................... . ..... 4 . Apparatus.... . . . . .. . . . . . . . . . . . . . . . . .. .. A. Cunent OUI'('('. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . B. Electrode's ... ..... ... . . . ........... . .. .... .... '. EI ctrode Trou~h F ........................... . .......... III . Hperific Preeipitalioll . . . . . . .. ....... 1. lmmunc :-;CI'!l.. . . . . . . . . . . • • . • . . . . . . . . . . . . . . • . • . .. . . . . . . 2. Use of Immune era . ... . ....... ................... ..... 3. Formation of perifiC' PJ'f'ripit Jlt e. . . . . . . . . . . . . . . . .. .......... IV. R c('ording of R esults. .. ...... . . . . . . . . .. ........... 1. Direct Photographic R ecord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Washing and Drying of Gel. . . . . ....................... 3. Colorations ... . ........... , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. taining for Proteins . . . . .. . .. ' ... . . . . . . . . . . . . . . . . . . . B. tail1ing for Lipoproteins. . . . . . . . .. ....... . .............. . taining of Glycopl'oteins or Pol~' sa('rharj d('s . . . . . . .. ....... V. Standard Techruque and Micro Variall t s. . . . . . . . . . . . . . . . . . . . . . . . . 1. tandnrd Techniqlle .. ... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Micro Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Interpretation and Discussion of R esults. . . . . . . . . . . . . . . . . . . . . . . . . 1. Determination of Jumber of Constituents in 0. Mixture .......... 2. Definition and Identification of Constituents of a Mixture . . . . . . . A. Appearance and Position of tho Arc. . . . . . . . . . . . . . . . . . . . . . . . B. Identification by Immunological Reactions . . . . . . . . . . . . . . . . . . C. haracterization by pecial Reaction .. .......... ......... VII. Conclusions . . ....... ,...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .. . . . . . .. . •. . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

3 6 6 7 7

8 12 12 13 14 ]5

]5 ]6 16 17 17 19 21 22 22 23 24 24 24. 25 25 26 27 2 28 30 31 33 35 36 37

2

PIERRE GRABAR

Important progress has been obtained in studies on proteins in general, and particularly on mixtures of proteins, through the use of various methods of electrophoresis. However, the possibility of resolving a mixture is limited by the sensitivity of the method used for the detection of the individual proteins of the mixture. It is also known that proteins possessing either similar or identical mobilities may exi t. These considerations have provoked the idea of introducing a very sensitive method, that of specific precipitation by antibodies for the detection of various proteins after their elecphoretic

................. f. ....... ,~

,.

\

..

~.

...

; ......__....."../."'" t

• • • • • • • •

t

I

Fjg. 1. Schematic representatjon of the precipitin arc formation.

separation. The principle of the method (17), which ha been called "immunoelectrophoretic analysis" (lEA), follows: The mixture to be studied is subjected to electrophoresis in a gel. When the dispersion of the constituents by the electric field is judged to be sufficient, electrophoresis is stopped and a precipitating immune erum is allowed to diffuse perpendicularly to the axis of migration. The antibodies present in this serum and the antigenic con tituents of the analyzed liquid diffu e into the gel (see the arrows, Fig. 1), and when they meet in proper relative proportions (corre ponding to the optimal zone for their mutual precipitation) insoluble antigen-antibody complexes appear and arcs of specific precipitation become vi ible. Each antigen or hapten present in the analyzed solution reacts with its homologous antibody specifically and independently. Thus even substances possessing identical mobilities can be differentiated. The

IMMUNOELECTROPHORETIC ANALYSIS

3

great sensitivity of the specific precipitation reaction allows the detection of minute amounts of antigen, thus making it possible to perform analyses of very small amounts of mixtures or to detect the presence of traces of impurities. lEA permits one to e tabli h the minimal number of antigenic (or haptenic) constituents of a liquid and to identify them by their specific reactions with the homologou antibodies. This double definition can also be supplemented by other means of characterization, such as color reactions, enzymic actions, etc. ince most proteins are good antigen , this method is particularly suited to the study of proteins and of complexes formed by proteins with carbohydrates, lipides, and metals. Moreover, orne of the polysaccharides, although not always capable of eliciting the formation of antibodie , are haptens, capable of reacting with their pecific antibodie and thereby detectable by the precipitin reaction. Therefore lEA can al 0 be appli d to the analysis of liquids containing polysaccharides or their complexes with proteins. During the elaboration of thi method, particular care has been taken to simplify it to the utmost and to avoid complicated apparatus in order to allow it application to diver e area of re earch. everal of the factor which are involved can be varied at will, 0 that many modifications and variants of the original method can be envisaged. The different tage of the method will first be described and discu ed. Then, a short de cription of some details of the standard technique as generally u ed in this laboratory, and of the micro technique of Scheidegger (33), will be given as examples. Finally the potentialities of lEA, the interpr tation of the re ults obtained, and the advantages, disadvantage, and limitations of its use will be di cussed.

I. PREPARATION OF THE GEL 1. Nature of Gels

For the purpose of lEA a gelified medium has a double advantage. Its very high liquid content allows an electrophoretic tran port iroilar to that in a liquid medium, and its structure slows down the free diffusion of macromolecular substances during and after the end of the electrophoretic separation. For practical uses, the choice of a convenient gel is limited by the following considerations: (a) the sub-

4

PIERRE GRABAR

stance which forms the gel should be a potent gelifier, i.e., it should be able to form a real gel even at very low concentratioD; (b) the gel hould be formed in an aqueous medium, which is the normal biochemical one; (c) the gel-forming substance hould be neutral, i.e., it should have as few ionized groups as possible in order not to be influenced by the electrical field; and (d) the gel should be tran parent in order to allow the observation and photographic recording of specific precipitation arcs. o far, the best results have been obt.o'tined with agar gels, and in the following descriptioDs of the technique only agar gels will be mentioned. Among other substances tested which are capable of forming true gels in water, one, pectin, gave satisfactory results (15). It even pre ents some advantages over the agar, such as formation of a gel at lower concentrations, the po sibility of easy elution by mean of enzymic breakdown of pectin, and formation of the gel by enzymic activity. However, the enzyme involved seems to be unstable and the proper quantity must be established each time by a series of preliminary experiments. Moreover, the necessity of introducing calcium ions entails some disadvantages. Therefore, pectin gels have not yet been widely employed. Many other sub tances, often said to be capable of forming gels, in reality form only very vi cous solutions. Devoid of elasticity, such solutions will flow and are therefore of limited utility in this case. Some interesting re ults have been obtained with starch. Compared with agar, starch has the disadvantage of being opaque, and if it is used for immunoelectrophoresis, supplementary manipulations are required. Slater (37), after electrophoresis in tarch, produces an agar gel in which the precipitin reactions take place at the side of the starch block. Other authors carry out the immunochemical reactions on eluates from starch electrophore is. It seems simpler to avoid supplementary manipulations and to perform both the electrophoretic separation and the precipitin reaction in the same medium, a is done in the present method. Another important factor that must be mentioned i the concentration of the substance used as support. The concentrations of starch necessary for formation of a suitable supporting medium are much higher than those of agar. The possibility of nonspecific interactions with the medium is thereby increased. It eems well established that the mobilities of some proteins in starch gels are significantly different

IMMUNOELECTROPHORETIC ANALYSIS

5

from tho. e which these substances show in liquid media. This difference is probably due to an adsorption of the substance by the tarch. In some instances this property may have value in separation of certain constituents with the same mobilities in liquid media. Mixed gels containing a very low concentration of agar (e.g., < 0.5%) and starch (e.g., 5%) seem to be useful; the elution from them is easy and it is possible to obtain nearly transparent films upon drying. To the author' knowledge only preliminary experiment have been performed with such supports (16,21). Instead of gel, Kohn has proposed the use of cellulose acetate membmnes for lEA and has obtained result which seem to be valid (24,25); but the relatively high content of dry sub tance in these membrane. and the knowledge available of their structure induce some restrictions on their regular u e. In fact, it is pos ible to imagine that parallel to the electrophoretic transport there may also be 11 certuin filtering effect produced by the membrane it elf; a partial retention or a formation of a "tail" may result in the case of particularly larg or asymmetric molecules. Such an effect, even in the ca e of agar gel of very low dry weight, has been em'isaged (47), but in thi~ laboratory it ha not yet been obsen'ed, even with macroglobulin~ of great molecular weight. Their obRerv d relative mobilitie in the agar gel seem to be identical to those in liquid media, but it. is not impossible that a certain "tailing" effect is actually taking place. A retention in the agar gel has been obsen'ed in the case of native fibrinogen (36) and the lysozyme of egg white. In the latter cn e it is probable that the retention is due to a sort of combination of this enzyme with the agar, which is a polysaccharide, because, when larger amounts of lysozyme are u ed, a certain mobility can be ob. rved (1 ). With the exception of these two in tance , the agar gel appear to be superior to the other substance tudied so far. Agar gives ufficiently rigid gel still containing 99% of liquid and the relative mobilities of many different sub tance are the same in them as in free electrophoresis. These gels are transparent and can be easily transformed into thin, dry elastic films which may be pre erved for an indefinite time, and in which various substances may be made apparent with certain dyes.

G

PIERRE GRABAR

2. Purification of Agar

Commercial agar preparations differ in origin and state of purity. A few brand of highly purified agar can be used without further purification, but their properties are not always constant from one batch to another and should be controlled. Others contain many impuritie (dust, salts, low molecular weight components, and nitrogen-containing sub tances) which must be eliminated. In order to obtain convenient gels with a low concentration of agar, it is important to avoid its degradation by hydrolysi which is easily provoked by frequent or prolonged heating. At a concentration from 1 to 1.5%, a suitable preparation of agar will form an elastic gel and not a friable paste which will break up when compressed between the fingers. Two methods of purification will be described, both applicable to agar of good quality, obtained either in fibers or in powder. A. PURIFICATION BY HEA.TING AND WASHING

The agar is introduced into gauze sacks and washed for at least 24 hours in running water, or, better, in repeated changes of distilled water. For this purpose it is important to use neutral distilled water containing no detergents or traces of impurities such a are frequently encountered in water purified on resin columns when not correctly operated. After this preliminary washing, the agar is rapidly melted in distilled water to give an approximate concentration of 7%. As soon as the agar is completely melted, the solution is poured into a cuvette or large cylindrical beaker. When the gel has solidified, it is cut into small cubes of about 0.5-1 cm. These cubes are washed for 3 days in distilled water, which is changed twice a day; at the end of this process they become white. These cubes are then melted in their own water. This material can be preserved in well-stoppered bottles at refrigerator temperatures for 1 or 2 weeks or more. Anti eptic or antibiotic substances may be added for prolonged storage. The dry weight of this preparation is determined in order to know its exact concentration. All melting or heating must be kept as short as possible and carried out in a water bath, in order to avoid hydrolysis and caramelization of the agar.

IMMUNOELECTROPHORETIC ANALYSIS

7

B. PURIFICATION BY FREEZING AND ALCOHOLIC PRECIPITATION

The following unpublished technique has been worked out by C1. Peaud-Leno I, following uggestions by A. Cote. It cODsi t: of eliminating insoluble constituents by centrifugation, and nitrogencontaining substances by freezing and thawiug (30,31 ). The agar i then fractionated by alcohol (22,26) to provide primarily a high molecular weigh t agar preparation. High quality Japane e agar fibers (250-500 g.) are carefully separated without being broken and wa hed at 50°C. in a large ve el (at lea t 25 liters capacity) with running tap water for 1 hour. The washing is repeated with distill d water, a1. ·o at 50°C., for 1 hour. The washed fiber are dissolved to give a concentration of approximately 3% in di tilled water at the 10weHt po sible temperature (such a rapid m Iting in an autoclave). Addition of 1 g. of activated charcoal per Hter of solution and a 5 minute centrifugation at 60°C. eliminate insoluble particles. The supernu!K'1,nt , hould now be clear, and nearly colorle. s. After cooling, the solidified gel is cut into pieces about 5 cm. square, which are kept at _10° . until completely frozen. The. piece are allowed to thaw at room temperature and the liquid is squeezed out through gauze heets. The fibers thus obtained are washed once in distilled water, and again compre.. ed on gauze. They are then suspended in distilled water, the total volume being the same a that of the initial olution, 'aCI i added to provide a 0.8% solution, and the agar i di olved by heating. The solution obtained i cooled to 65°C. and absolute alcohol at the same temperature i carefully added until precipitation begin (about].l volume). Thi pr cipitate i centrifug d at 6o-65°C. and discarded. To the supernatant is added with tirring 0.2-0.3 volume of hot absolute alcohol until a !mow-like flo 'clllution appear.. The precipitate i allowed to settle overnight at 37° ., the. omewhat cloudy supernatant is . iphollf'd away, und th precipitate i. centrifuged, wa hed with absolute al ohol, and dried at 3rC. The ground white precipitate can be preserved in well-stoppered fla k. It forms a very rigid gel at room temperatur at a concentration of 1% and its nitrog n content does not exc ed 0.013-O.0l5%. 3. Buffered Agar Gels A small pI' liminary t -. t wiII suffice to e tabli ·h the preci e concenLru.tion of the agar giviug a r slstant and ela tic gel; usually 1-1.5%

PIEHHl!: GHAllA.H

will be satisfactory. A conv nient quantity of agar purified by one of the methods described or a highly purified commercial agar is rapidly di olved in a buffer to give the final concentration establi 'h d in the preliminary experiment and the final concentration of the buffer chosen for electrophoresis (see Section II.2). If the resultant solution i not perfectly clear, it can be rapidly filtered by light suction on a Buchner funnel through a double layer of filter paper. After addition of an anti eptic substance (for example, merthiolate1:10,ooO), the liquid i' di tributed in mall flask containing the volum just nece 'sary for the preparation of one plate, or of any desir d number of plates, of a cho en dimension, thu avoiding unnecessary supplementary heating. 4. Preparation of Plates The electrophoresis is preformed in a thin layer of gel formed 011 It photographic gla's plate. The adhe ion of the agar to the giu::;s i ' improved if the plate are carefully washed; it can be increased if a few drops of melted agar (1 %) are fir t pread on the surface and dried at 80°0., as is done for tubes (29), or in open air. A uniform layer of agar gel on the e plate is then formed. The simplest way to obtain a perfectly horizontal base for this operation i to pour a layer of crude melted agar into a photographic tray firmly fixed on a table and allow it to harden. The prepar d glass plate is placed on this surface; two strip of chromatographic filter paper 4 cm. wide and as long as the glaSJ plate are placed 011 the ends of the plate, the paper' extending about 3 cm. beyond the glass (Fig. 2). The purified melted agar solution in buffer i' poured into the tray; it covers the gla and paper strip which later ",rill 'erve a the electrical connection' of the gel. The volume of the agar solution used for one plate i calculated to form a layer of 4 mm. When the gel has hardened, the plates are lifted from their base. For this purpose, the gel around the gla s plates and the filter paper is fir't circum cribed with a patula. A light inci ion is made in the gel over the border of the plate where it i covered by the paper strips (line l-l' in Fig. 2), and the strips are bent to a 90 degree angle. Melted agar is poured on the cut CD in Fig. 3) to fill it and form a continuous agar layer (Fig. 3). Either before or after this last operation (i.e., the lifting of the

9

JMMUNOF.r... l~CTROPHORl';'rrc A AJ...Y fA

plate), one or more holes are made in the gel. These holes erve a reservoirs for th ample to be analyzed. The forms and dimensions of the holes may vary a. a function of the volume of this sample.

-!

I I I

-,-1 I I

0

I I

I I I

I

I,

I

:

B

A

I11~0 ,

B

I

I

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II I

I

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0

I

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II :, I :

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__ .J_..t

'-- -30--";', o~

10- --- ______ - - - - -

~IO~ --30 - 11>

-180 - - - - - -- _______ ..; I'

Fig. 2. Preparation of an agar plate for the lEA. A, plate; B, filter paper trip. Dimensions in rum.

r--- - - - --- --- - 1 8 0 - - - - - - - - - - - - - . . , : ,

C

A

: I

4

B

E

Fig. 3. chematic reprr entation of the apparatus for lEA. The glas plat> (A) with filter paper strip (B) is covered with the agar gel (C) i the incisions in D ar filled with m Ited agar. E, buffer solution in the electrode tray . F, platinum wire electrode fixed on a pIa tic upport. G, DinleD ions inmm.

An elongated trough, perpendicular to th migration axis, allows the use of a larger volume than a round hole of the same width on thi axi. Simple punch s, easily manufactured from copper tub ,are u ed to make the. e holes (Fig. 4).

10

rIERRE GRADAR

At the end of the el ctrophore i ,long wells parall I to th migration axis ar cut out of the gel with a dcntnll'lpatula. The 'e well. serve as reservoil's for the immun 'era. They urc in mo~t iUl'it...'\ n 'es 5 nun.

~' 0-' J q I

I

28

14

I

rJ

I-

-l3!--

""13:'

'-8 ol

Fig. 4. Different forms of reservoir for the olution to he anniyzrd. sions in mm.

Dimen-

wide and occupy nearly the whole length of the plate. The holes should be sealed at the bottom with a drop of melted agar. The placement of the reservoirs will depend upon the purpose of the - "l

I

I

2'S

I

I

[

I

_ _t IS

--2 0--' -

I I I

)' .~

I

,I I

130

1::5

....

: IT]

C~

I

I

I I

I

I

2~

: . . . - - - - - - - - - - - - - - 180--- -

I

I

I I

---------...,1

--+

Fig. 5. Example of placement of reservoirs in an agar plate. Dimensions in mm.

analysis. In most ca. es, the hole for the sample is made equidi tant from the cathodic and anodic ends of the gel, in order to compensate the electroendosmotic flow (see ection II.3). Figuro 5 illu trate. the

IMMUNOELBC'J'ROPHORI!:'rlC A ALnHS

11

location of the reRervoirs in a ] 3 X 18 cm. plate. The distances (see Fig. 5) between the central hole and the immune serum wells may al 0 vary with the concentration of antibodies in the particular immune s rum and with the free diffusion rate of the antigens ( ee ection III.2), but in mo t instance it is 5-10 mm. In order to facilitate the preparation of the. e hole, a drawing of their disposition can be mad on the oppo. ite Ride of the glass plate with glass ink; one can also prepare a drawing on a graph paper and place it under the agar plate. The 'ides of the well' mu ·t be regular and clear-cut; the use of a ruler is recommended. The solut.ionR to be analyzed, mixed with melted agar of such concentration that the final mixture possef'."e the Rllme aga!" concentration as the rest of the plate, are introduced in the ccntml reF:ervoir. The agar RolutiotJ is maintained in u melted I'wte in a water bath at 42-4:3° .; a meu:o:ured volume of the solution to be a nalyzed is rapidly mixed with a known volume of thiR melted agH and the mixture i placed in the reoen·oir. When this sample i, solidified, a few drops of pure melted agar olution are poured over it to con._olidate it. junction with the agar gel on the platt' . ThiR technique usually allows one to obtain uniform results. With some substance, however, even this brief period of heating mUHt be avoic..led. In the, e ca. es the olution to be analyzed can be rnLxed with a ~u pension of mall particle of an agar gel. Then this mixtUl'e, which must have a consistency of a pa te, is placed in the reservoir and covel' d with a few drops of melted agar. Another posFibility has heen proposed by Bu tamente and Wunderly (cited in 5]), who URe a narrow filter paper strip saturated vl'ith the liquid to be analyzed, which i' placed on the surface of the gel. ASter] 5 minutes, it i. taken off und the vurfllce of the gel is washed with saline. This technique allows one to avoid heating, but the diffusion rate of the constituentR of the liquid may not be equal and therefore a certain disparity of the quantitntive relationship among the constitUllllts may result. Tn some case it may be desirable to low down the diffusion of the antibodies; fOl' this purpose the immune serum j . also mix d with melted agar before being intl'odllc -d illto th lateral welL G neraJIy, however, the immun . ('rum is poured into the. e wells in liquid form, so that diffusion is mor rapid and the appearance of th precipitation i ' accelerated.

12

PIERRE GRAllAR

II. ELECTROPHORE IS IN A GELIFIED MEDIUM The idea of using g 1s to support electrophore i i not new. Various method have been de cribed, among which the technique of Gordon et al. (7) is based on the use of an agar gel. More recently, several authors have published technique analogou to the one described here, but devoid of the use of specific precipitation for detection of the con tituents of the analyzed solution. These variou technique , which may be called "simple electrophore i in gel " in contrast to the "immunoelectrophoretic analy is," furni h re ult imilar to tho e given by paper electro ph ore is. They pos ess, however, the advantage that the tructure of a good agar gel is more uniform than that of filter paper; therefore, the colored spots are better defined and more regular than those on filter paper. The spot obtained in imple electrophore is in agar can be e timated quantitatively by photometric measurements; thi evaluation is facilitated by the tran parent nature of the film obtained by drying the gel (ee ection lV.2). By performing two parallel experiments, i.e., immunoelectrophoretic analysi , which will give primarily qualitative but detailed information, and simple electrophoresis, which allow :1 quantitative estimation of the main fractions of the mixture tudied (39), it i possible to obtain a very complete picture. The technical details of the electrophoresis in agar which are decribed below, although established for lEA, can be used:1 well in simple electrophore is. 1. Electric Current

Since the de ired re ult i maximal electrophoretic disp rtiion of the constituents of a mixture, even when the mobilities of some of them are similar, it is evident that the greate t po ible voltag s ·hould b u ed. Moreover, high current, in hortening the time of electrophoreis, will dimini h the free diffu ion of the components of the mixture, thus improving the separation. However, the evolution of heat pI" duced by the current tend· to dry the gel and cau e it to crack, which limjts the use of high voltage. It has been tabli hed that differences in potential of the order of 3-6 volt Icm. of gel, when the salt concentration i. not too high

IMMUNOELECTROPHORETIC ANALYSIS

13

(see ection 11.2), usually produce good separation without prohibitive drying of the gel and without requiring the u e of special instaUatiom; such as refrigeration. The rate of evaporation can be lowed somewhat by performing the eleciropboreHis in a cold room or refrigerator, but in most case' it can be carried out at room temperature, using the voltage mentioned, and the slight drying of the gel has no ill effects. It is recommended that the gel be protected again tail' drafts during electrophoresis and that the work be don in a room where the air is not too dry. The voltage must b measur d inside the gel becau e of the 10 of current at the filter paper junction. , even when they are covered with the agar gel. 2. Buffer Solutions

A choice mu t be made between two contradictory considerations. On the one hand , it i n ee ary to have a sufficiently high concentration of the buffer to avoid change in the pH, and on the other hand, as stated above, in order to diminiEh the heating of the gel it i preferable to use dilute solutions. It ha been experimentally establish d that wi th buffer. 'olutions of an ionic strength r / 2 = 0.025-0.05 these two aims can be reconciled. In most case the ionic trength of the gel is of the order of r / 2 = 0.025-0.030, wherea in the buffer olution used in the elE'ctrode vessel it is 0.05. A drop by drop flow of the latter solution through these ve sel prevents change of the pH during the run. When the olution to be analyzed is particularly rich in electrolytes, it i preferable to diminish their concentration by a preliminary dialy. is or 'imply by dilution. If thi i not possible, e.g., becau e of the solubility prop rties of the con tituents, the lectrophoresis may be performed in the presence of more concentrated buffer solution, but then it is n ce sary to work in the cold and use lower voltage, thu requiring an increase in operating time if t,he same electrophoI' tic dispel', ion is sought. Th pH range in which agar gels can be used lie betwpen 6 a.nd 9. Various buffers can he u d, good r sults having b en obtained with barbital and with borate buffers. Any alteration or denaturation of a protein may modify it: reaction with the cotTe, ponding antibody. Therefor, u. e of alkaline media

14

PlERRF. GRAUAR

(e.g., pH > 8.2) for electrophoresi involve a certain l'V k, unle 1'\ preliminary experiments haye , hown that this prec< ution is untlPC(\sary. 3. Electroendosmosis

The agar i not an utral sub tance. It has some polar groups and thus a certain charge and a tendency to move in all pjectl'ic field, but the molecules of agar are immobilized in the gel and therefore the liquid part of the gel will move in the opposite direction; i.e., un electroendosmotic flow i observed. Thi movement i very regular, the whole liquid being displaced; no disturbances in the relative distribution of the constituents of the analyzed mixture will take place. The displaced liquid is not replaced from OUU ide the gel and a unilateral diminution of liquid results. The final position of each ubstance at the end of the electrophorpis will be determined by the interplay of two movements: an individual transport by the electric field proportional to it. charge and n. general transport by the electroendo motic flow. If the fir. t is smaller than the second, a negative po ition will be found. ubstances who e mobility i equal to the electroendo mosi. would seem not to move at all, wherea substances po sessing a high mobility will show an apparent displacement less thun their real electrophoretic transport. The importance of the electroendo motic tran. port can be checked by control experiment. ,,,ith various substances devoid of any electrophoretic mobility, such a the glycogen. , dextrans, or Jevans; the e experiment allow the determ.ination of the "point of zero migration," and thus the real mobilities of the constituents of the analyzed liquid (see Section VI.2). The electroendo mosis dep nd upon the origin of the agar and the method used for its purification, as well as upon the nature and the concentration of the buffer solution. It is thu useful, when determinations of mobilities are desired, to perform a simultaneous evaluation of the electroendosmosis. When the analyzed liquid contains some constituents possessing low mobilities, it is nece sary to position the re el'voir for this liquid in the middle of the plate; otherwise, the e substances may be moved off the plate by the electroendosmotic flow. In orne CnSE'R, on t,he other hand, when the aim is to separate either only very rapid or very

lMMUNOELECTROPHORETIC A ALYSlS

15

slow constituents, the analyzed liquid is better placed at the anodic or the cathodic limit of the plate. The electrophoretic transport of macromolecular substances is paralleled by the transport of mall molecules and ions, which produce. a lowering of the electrolyte concentration in the gel. This can be en. 'ily appreciated by mea uring the conductivity across the gel, perpendicular to the axi of migration. Under standard conditions the impoverishment in the salt concentration, which is analogous to au clectrodialysi ,ha no vi ible consequences, but when the electrophoresis is continued for longer periods of time (e.g., more than 5 hours under standard conditions), the migration may slow down. In order to provoke further migration, it is necessary to reestablish the COllcentration in the gel. Tbi can be done by placing the gel in the buffer solution for a short time (20 or 30 minute). In thi way it i po 'sible to perform a more complete el ctrophoretic disper!< ture enriched in the particular antigens for the immunization. When the aim is the preparation of an immune serum containing only one antibody, it is necessary to inject the antigen in a highly purified form. Even then, frequently the animal will form antibodies toward other substance " pre ent only in trace amount in the injected product, which can be d tected only when very large amounts are subjected to analysis or 'when impure preparations are used to detect this antibody. In any ca e, for controlling the pw·jty of an isolated substance it is preferable to utilize an immune serum obtained by injecting the initial impure mixture from which thi substance hc'\s been extracted, or a relatively impure product, and not, a i erroneou ly done by some inve tigators, an immune erum obtained by injecting the highly purified product. It is evident that with this last erum there is less chance of detecting an impurity. The animal species utilized for the production of immune sera has a certain importance. On the one hand, differences in the capacity to form precipitating antibodies depending on the animal species are recognized. On the other, it i generally admitted that an animal of a species related to that from which the antigens originate produces antibodies which permit more specific differentiation than does an animal of a less closely related pecies. In most ca es rabbits are used for the production of immtme sera, but very good yields of precipitating antibodies can be obtained in chickens, ducks, goats, donkeys, and, particularly, horses. Whereas rabbits are generally immunized for some 6-8 weeks, goats and horses can be maintained under immunization for many months, thus producing large amount of highly potent immune sera. It is recommended that some preliminary tests on a trial hleedjng of

IMMUNOELEC'l'ROPHORETIC ANALYI::HS

19

the immunized animal be performed in order to see whether a large amount of bleeding is warranted, and to continue the immunization if this erwn is not sufficiently rich in antibodie .

2. Use of Immune Sera When the electrophore'i i finished, thc immune serum i poured into the lateral wcll cut out of the gel. The antibodies diffusing into thc gel form in oluble complexes when they meet th homologou ' antigen '. The antibodies diffus from the well in a front parallel to the axis of electrophorctic migration, \ hereas the antigen diffuse in all dircctiOIl' from the cllip 'oidal or round 'pot to which they wcre transported by the CWTtmt. Thus they meet with each other on a linc which forms a more or Ie eHip oidal arc (see Fig. I), The antigen-antibody complexes are insoluble only when they arc in definite proportion. An es 'ential difference exists between rabbit antibodie . (and antibodies form d by all the other animal u d for production of immune ra) und tho e present in the serum of hoI' e hyperimmunized with protein antigens. Wherea the rabbit gi\"es insoluble eomplexe ' even with smn.ll amounts of the conoe pondiug antigen, and forms 'oluble complexes only in the pre ence of an exces of l1ntigen, hoI' e l1ntiprotein antibodie flocculate only in a narrow rang of antigen-antibody ratio. Thus the formation of a pecific precipitate in the agar gel will depend upon the relative proportion of antigen and antibody used in the experiment. If the equivalence zone (i.e., the quantity of antigen which precipitates optimn.lly the amount of antibody in a given volume of the immune erum) i known for l1t least ome of the antigen exi ting in the analyzed liquid, the volume of the immune serum to be used for l1 given amount of thi liquid can be estimated. But even in this cas ,a in all tho e in which no preliminary quantitu.tive relation has been e tu.bli hed, it i nec sary to perform a erie of experiments with varying amount of the reactu.nts (antigen olution to be analyzed and immune serum). In fact, depending upon th se amount, the relative proportion of the different con titu n of the mixture can corre. pond for some of them to l1n antibody exce ,for oth rs to the cquivalenc zone, and for still other to an antig u exc 1£, in the fir 't cas , for example, hor e antibodie are u ed, soluble complexes may be formed, as they will be with any immune rum in

20

PUlRRE GRABAn

the la t in tance, i.e., antigen excc . Thus no vi ible precipitate wm appear. Preliminary experiment performed with varying amounts of the reactants are neces ary to avoid thi drawback. Another intance must not be overlooked. Wh n a sub tance i ' pre ent in the analyzed liquid in a very low concentration (e.g., an impurity in a pUl'ified pr paration), it presence can be detected only when relatively large quantiti s of the liquid are used. If it can be admitted

~[rloU'O[fon

a

_, 10

v_..---_ _ _ _ _ _ __ ---. __, S

""!, - - - - - -- -----!I '+- - - - - - - 60- - - - - - ...

b

Fig. 7. Placement of re ervoir in an agar gel for preliminary experiment. Dimensions in rom.

that the limit of visibility of a pecific precipitate in a gel i of the order of few p.g., an impurity of about 0.1 % can be detected when 1 mg. of the sub tance is ubmitted to the analysis. It has been tated above (Section 1.4) that the distance between the hole for the analyzed liquid and the well for the immune serum must be cho en as a function of the concentration of the antibodies in the immune erum and th diffusion rate of the antigen. Thu, if thi distance is mu.1I, the diffusion rate rapid, and t,he quantity of antibody insufficient, no pr cipitation arc may be ob. erved. On the other hand, with the same proportion of the same ubstan'e 't must be po. ible to ee a precipitate, even if it disappears later by

JMMUNOF:L~:

:1'ROPHORETI

ANALYSI

21

di olution in antigen exc ss, if th distance between the reservoirs is orne what Larger. However, too great distance ' arc not desirable becl1u the formation of the precipitin arc ' is slowed down and the nec s ary quanti tie of reactant are often to be increa 'ed. In order to e timate the value of an immune. erum and e tabli. h convenient distances between the reo ervoirs, preliminary experiments everal without electrophoresi can be performed on agar plates. small trough are cut out in a gel, as shown in Figure 7a; the immune rum, .g., 1 mI., i pour d into the elongated well, wherea. different quantities of the analyzed liquid ar placed in the small troughs. This experiment permits one to e tabli h approximately the mo t convenient concentration of the antigenic solution to be used with the same immune serum in lEA. Figure 7b shows another y tern which may be u ed to establish the distances between the reo ervoir which should allow a good vi ibility of all the precipitation bands and thu. be applicable in lEA . uch plates, prepared with a 1-2% agar solution containing 0.8% of NaCl, are maintained during the diffu ion of the reactant in a moi t chamber, as the plates of lEA (see Section III. 3). 3. Formation of Specific Precipitate

The sp cific precipitation is gen rally optimal at neutral pH and at physiological salt concentration. * When the electrophore i i performed at another pH or It concentration, convenient condition can be established in the agar plate by placing it in a cuvette containing a suitable buffered olutioll after the electrophore is; the diffusion of the salt i rapid and in some 20- 30 minute the proper pH and salt concentration can be obtained in the gel; during uch a limited time the free diffu ion of the macl'omole ule eparated by the electrophoresi i not significant. However, it ha been e tabli hed experimentally that in rno t ca es these precaution are unnece sary becau e the alts and buffer of th immlll erum which diffuse from the lateral well are generally suffici nt to establi h conditions uitable for the specific precipitation. The rate of diffusion bing a function of th prevailing temperature, it is evident that the appearanc of Rp cific PI' cipitin arc will be more or Ie. mpid, dep nding upon the temperatul'. On the other hand, it * Chi ken immune scra giv more abundant precipitation in the presence of 10% Na. 1 (50).

22

l'IF.RRF. aRABAR

is kno"\\'1l that the quantity of specific precipitate depends al 0 upon the temperature; in most ca es, but not in all, it is gl' ater at than at 37°C. In thi labomtory, when the lateral well, :11'0 filled with the immune erum the agar plate are preserved in a room at 1°C. con w.nt temperature. udd n variation of temp ratUl'e mURt be avoided. In order to slow dow'll drying of th gel, the plate are maintained in "moi t chambers," i.e., plastic boxe , the bottom of which 0.1' covered with several layers of filter paper soaked with water; a small quantity of CUS0 4 i di olved in thi water to protect again, t the development of mold . The agar plate must 0.10 be protected again t microorganisms. In mo t cases it is ufficient to incorporate an anti, eptic substance in the gel (e.g., 1: 5,000 merthiolate); 1 or 2 drop of a 0.1 % solution of merthiolate are al 0 di tributed with the finger on the surface of the gel before the plates are placed in the "moi t chamb r." "When the relative proportion of antigens and antibodies, through their diffu ion in the gel, corre pond to the zone of precipit.'Ltion, arcs of pecific precipitate will appear. The time of their appearan e depends upon the concentration of the reactant, the diffusion, peed, and the di tan e between the reservoirs. Therefore, the arcs corrender "'ponding to different antigens will not appear simultaneously. tandard conditions ( ee Section V.1) the first arc generally appear after about 24 hours and their development i complete after 4-5 days, wherea. in the micro technique (see ection V.2) it i complete after 24 hour. It is sugge ted that the development of the arcs be noted and recorded, since some of them may disappear if an antig n is in exce s.

°

IV. RECORDING OF RE ULT Two procedure can be used: the direct photographic r cord of the precipitation arcs in the gel, and staining of the arcs after washing and drying the gel. 1. Direct Photographic Record

The gel being trallsparent, the precipitin arcs are c1 arly visible and caD be photographed on a film or on high oontrast photogmphic

Il\fMUNOEJ.ECTROPTIORETlC ANAJ.ysrs

pap r. The glas plate with the gel i. placed directly on the photogl'aphic paper in a tray and cover d with saline solution, thereby avoiding the l'eproduct,ion of any iJ'regularitie. of the surface of the geL uch photJogl'aphs cun be taken during the development of the arc' to follow theil' moclifi 'I1tions and check po sible di .olution of arc in antigen exces. ·, doubling of bands, or the late appearance of arcs. However, it is wi 'e: (a) not to prolong the sojourn of the gel in the saline, (b) to avoid sudden changes of temperatur during the e manipulations, and (c) to re-cover the uJ'face of the gel with an anti. eptic aft.er the completion of photography, 2, Washing and Drying of Gel When the development of the arc is b lieved to be fini hed (easily checked by direct observation or by compari on with photographs), the gel is ubmitted to 11 \\'a. hing with saline solution for 2- 3 days with frequent change. of the liquid. This washing eliminates from the gel all ub tance -' which have not participated in the immune reaction. The, pccifie precipitate, being nearly in oluble in aline, should be the only substance left in the gel. Occa ionally other relatively in 01uble sub:tances r main, and particularly on the edges of the immune . erum wells. ert.'"I.in mncrornoleculnr components and soluble antigen-antibody complexes which have a very low diffu. ion rate may also remain in the gel. A wet., . mooth . heet of filter paper is applied tightly on the gel, care being taken to avoid any air bubbles between the paper and the SU1'fac of the gel. mall holes are made in the paper over the re ervoir with a needle and the gel i allowed to dry at 37°C, or at room temp l'atul'e, a ventilator being utilized if an accelerated drying is desired. The use of filter paper (43,45) allows tra.nsformation of the gel into :1 perfectly tran parent film, whereas, if the gel is dried without thi paper, cracking and cry tallization of salts generally spoil it, When the gel i dry, the filter paper eparates from its surface and the film is firmly attached to the gIn s plate, A light rinsing with di tilled water is sufficient to make it entirely transparent, In order to increa e the insolubility of the specific precipitate, the g I before drying or the film after this operation can be treated with a 2% olution of acetic acid or with a 5% solution of trichloroacetic acid and then carefully wa h d with di tilled water to eliminate completely the acid: which may eventually hydrolyze the agar.

24

PIERRE GRABAR

The same technique of drying can be applied in simple electrophoresis in agar, but in thi inst..'Ulce, after the end of the electrophor si , the gel i first placed in a fixative (such as 1% acetic acid in th ca e of protein ) and then after a careful washing it can be dried a stated above and tamed as usual (45). 3. Colorations

Various dyes can be u ed to characterize different substance present in the gel as such (imple electrophore is) or a part of the specific precipitate in lEA. But thi precipitate, being particularly rich in protein (the antibodie are the mo t abundant part of it), may rna k orne of the colorations which are characteri tic for an antigen. A. STAlNlNG FOR PROTEINS

The gla plate with the agar film on it is placed for 5 hours in amido black solution or for 3 hours in axocarminc olution; then it is washed in the "washing olution" until the background becomes colorless. Other dyes such as the colored pH indicators can also be u ed. It has been observed experimentally that in lEA., when the immune serum used was from a horse, the azocarmine gave better re ults, whereas with rabbit immune sera amido black or indigo carmine wa~ more suitable. The reagents are prepared in the following manner: the dye (1 g. of amido black or 0.5 g. of azocarmine) is di olved in 900 m!. of acetate buffer (equaL volumes M i lO acetic acid and sodium acetate) and 100 mI. of pure glycerol. The "washing solution" consists of 20 ml. of acetic acid and 150 m!. of pure glycerol, made up to I liter with distilled water. B. STAINING FOR LIPOPROTEINS

The glass plate is placed on piece of glass or wood a few mm. thick in a tray, the film side turned down. A satw·ated solution of udan Black (or Oil Red 0) in 60% alcohol is poured in the tray, care being taken to avoid air bubbles on the film. The tray is hermetically covered (plasticine can be used to make a tight joint between the border of the tray and a glass covering plate) and left for 2 hours.

IMMUNOELECTROPHORETIC ANALYSIS

25

The plate is then washed in two change of 50% alcohol for 15 minutes and dried for a few minutes at 37°C. Just before use, 0.2 ml. of 25% NaOH is added to 100 ml. of dye olution. C. STAINING OF GLYCOPROTEINS OR POLYSACCHARIDES

The plates a1' fir t treated for 15 minutes by a 1% solution of periodic acid in 50% a lcohol, and then wa hed for 15 minutes in distilled water and placed for 5 minute in the following solution: 50 ml. of a 0.01 1\1 aqueous olution of a-naphthol plus 50 ml. of a 0.01 M aqueous solution of p-ph nylenediamine plu 10 ml. of a 10 volume % H 20 2. After a 10 minu washing in CUlTent water, the plates are dried in the incubator (40).

... * *

om p cial technique of taining for the detection of hemoglobin (6), ceruloplasmin (41), p roxida e (3 ), and copper (42) have been de cribed.

* * ...

When staining is finished, the agar film can be separated from the glass plate. In order to facilitate thi operation and to make the film ela tic, the plate i treated for at lea t 5 hour in a 15% glycerol solution (thi tr atment is not nece snry after staining of protein since the "wa bing olution" contain glycerol). When tripped from the gla ' , the film ha the appearance of a cellophan heet ; it i perfectly transparent and ela tic. It can be pre erved for an indefinite time and 'an be u. ed for photom tric mea urement of colored 'pots obtained by 'imple el ctrophore '. or for photographic recording in lEA.

V.

TA DARD TE HNIQ E A TD MICRO VARIANT

rEA i susceptible to many application ; in certain en e , at lea t, the technique mu t b adapt d to the sub tance to be tudied. Many variants of the method may be fore e n. Therefore, in the preceding ections the general aspects of the method have been de cribed and discussed to facilitate po ible adaptations. In thi ction, a hort re ume will b given of the tandard technique and of a micro variant which hav been employed suc e sfuIly for obtaining much u eful information; thi de cription may rYe as an exampl of certain technical detail .

26

PI ERRE GRABAn

1. Standard Technique

This technique has been only slightly, modified since 1953 (17) and has been particularly u eful in studies on serum con tituents (e.g., refs. 2,3,5,9,12,13,34,35,42,44,46,49). Gla plate 18 cm. long and of varying widths (from 6 to 24 cm.), depending on the number of samples to be analyzed, are covered with an agar gel (1- 1.2%) in a barbital buffer, pH 8.2, 0.025-0.03 ionic trength. With 4-5 volt j cm. in the gel, the con tituent of normal erum are di persed in 4-5 hoUl.. over a total length of 12- 14 cm. The volume of the re ervoir for the ample to be analyzed is 0.1 or 0.3 m!.; the hole is either elongated (e.g., 14 X 3 or 2 X 3 mm.) and the end are rounded or it is circular (diameter 8 mm. ) (see Fig. 4). The lateral reservoir for the immune erum is ] 5 X 0.5 em. The distance between the re ervoirs is generally 10 mm., but with ome immune sera it had to be reduced to 8 mm. (Fig. 5) or even to 6 mm. The quantity of protein in the sample ta.ken for analysi varies from 0.1 mg. (in the ca e of a purified sub tanee) to veral mg. (in the case of a mixture or for the detection of an impurity in a purified product). In studie on serum, 0.02 and 0.1 m!. sample are used in order to detect various constituents, since some of them can be observed only in a larger sample. Various volume of immune serum have to be utilized, depending on the concentration of antibodies. In most ca es, with a good immune ~erum 0.5 or 1 ml. is more than sufficient, but with some era it i necessary to u e a larger volume. If the reservoir i too mali, this volume can be added in several portion, but the total mu t be added in a relatively short time (not exceeding 2 hour ), and care must be taken to add each portion before the preceding one has been completely absorbed by the gel, which occurs relatively rapidly. Mo t of the studies on human serum have been performed with antisera from horse , * this animal producing many different antibodies and at a relatively high concentration. The development of the precipitin arcs takes place in plastic boxes ("moist chambers") in a room at 18°C. constant temperature; 3-4 days are generally sufficient for development; the plate. are then wa hed and the precipitin arcs stained. • Such antisera can be obtained from Pa.ris (XVO), France.

rcastcur, 30 rue du Docteur Raux,

IMMUNOELECTnOPHORl~TIC

27

AN AJ~ YSlS

2. Micro Techniques Micro techniques have th advuntage. of giving rapid re. ults and requiring less immun erurn and a smaller quantity of liquid to be ann.lyz d. However, omotimes their us is limited by the difficulty in detecting a minor component and distingui. hing between neighboring arcs, although photographic enlargement of the records can facilitate interpretation of the result . lides (76 X 26 mm.) on cheidegg I' (33) u.'es micro copic gla which i poured 2 rn1 . of a 2% ag[Lr in the arne buffer a in the standard technique. The hot olution covers the slide and tay on it, fOr ming a meni cu at the edg . ; the olidified gel i about 1 rom. thick. With a 'imple apparatu con tructed of shortened hypodermic injection ne die. aod two razor blade, two small hole and a long slit

,

~--------------------------~- I

3 _4,; - ------ -· - - ' -2

~

,I

2'6 t::==========:::1~ 2 o : ____________________ -Li' 4 ---- - 40 -----.

- - - - - - - - - - 76 - - - - - - - - - -

Fig. R. Plll('rmrllt of rr. rl'voil's in Illl nglll' plat,(> for the mirro immunoelectrophon'tic wehniCju(' of ,ehricirggrr. DimensiOIlS in mm.

ar made in the gel (Fig. ). Th two holes crv a ' reservoir for the sample to be analyz d. The contact betw en the gel and the electrode ve sels is ffected by filter paper strip. With a 6 volts/ cm. potential, the protein of erum are di per ed on nearly the whole length of the plate in 45 minute. The agar i taken out of the long lit and immune erurn i poured into the elongated re ervoir thu formed. Th precipitate begin to appear after 30 minute and the total development i generally finished in 24 hours. The washing, drying, and taining are the same a in the tandard technique.

* * *

Many other variant can be or have b en u' ed. Thus Wieme and Rabaey (4 ) utilized even smaller g I form d on cover slips, wherea in this .laboratory intermediate dimen ion between the tandard techniqu and cheidegg r' variant are u d when the quantity of immune s rum or of the liquid to be analyzed i re tricted, or when a rapid I' suit it'< d sired (e.g., for clinical use) .

28

PIERRE GRABAR

VI. INTERPRETATIO

AND DISCU

10

OF RE ULT

At the beginning of thi article it wa tated that lEA can be u ed (a) to determine the number of con tituent in a mixture and (b) to identify them by two or even more differ nt characteri tic : their electrophoretic mobility, their immunological pecificity, and ometimes their chemical nature or particular activity. These possibilities will now be enlarged upon and the advantages and disadvantages of the method will be di cussed. 1. Determination of Number of Constituents in a Mixture The use of specific precipitation for the detection of th constituents in a mixture offer the advantage of the specificity and the sensitivity of immunochemical methods. The limit of the sen itivity of the precipitin reaction have been mention d above, and it i certainly superfluous to enlarge on it well-known pecificity. Becau e of this property, every constituent of a mixture reacts only with it homologou. antibody. In lEA two substance po sessing similar or even identical mobilities (and in this case they cannot be differentiated by other electrophoretic methods) can be di tinguished becau e, if their chemical configuration i not identical, their antigenic properties will differ. Thu they will react with different antibodies and form two independent arcs of specific precipitation. The number of independent arcs or bands of specific precipitation allows one to determine the minimal number of constituent in the analyzed mixture, as it doe ' in the other, purely immunochemical methods using specific precipitation in gelified media (27-29). In these method, when a mixture is analyzed, bands of specific precipitation can be superimposed, wherea in lEA the chances of such mixed band occurring are con iderably reduced because the constituents of the mixture are separated from one another by electrophoresis. A coalescence of arcs can take place only if two constituents have the same electrophoretic mobility and if, moreover, the rate of their free diffusion in the agar gel and their antigen-antibody ratios are such that the two arcs will appear in the 'ame place. The probability of a simultaneous coincidence of all the e factors is very low. Moreover, it is always po sible to perform control experiments by using two different immune sera. If a coalescence of two lines has taken plo.ce

IMMUNOELECTROPHORETIC ANALYSIS

29

by use of one serum, it is improbable that it will also occur with another one which will generally contain different propOJ:tions of antibodies ( ection III.l) (8,11,12). Th detection of the con tituents of a mixture by lEA is based on a double diffu ion in ' a gel, as in the method of Ouchterlony (28). This method, when compared with the other techniques of precipitation in gel, ha ' the advantage of allowing compari on of several antigens. When orne of the comitituents of a mixture are available in a purified state, it is po. sible to define the bands formed by them in the mixture. lEA al 0 allow ' compari on of the constituents of the mixturc because after the electrophoresis they will be situated, in relation to the re 'Cl'voir of immune serum, in the same po ition as in the conventional double diffusion method ( e Fig. 7). Moreover, in lEA it i ' not necessary to u 'e previou Iy isolated con tituents becau c they arc separatcd from on another by electrophoresi and are defined by their electrophoretic mobility. In contra t to these advantage ' lEA also ha. orne inconveniences: 1. The mo t importnnt, but olle which is common to aU immunological methods, is the principal reagent, the immune erum. The variation in t he animal respon e to immunization and the differences in antigenicity of variou ' protein have been stres ed in Section III. The immune era are thus always quantitatively and often quaHtatively different, even when they have been produced by animals of the same sp cie which were 'ubmitted to the same schedule of prolonged immWlization. One i therefore obliged to examine every individual immune sel"Um and eventually choo e tho e which how the best re ·ults. Ncverthele , a certain doubt may per i t about the total number of constituent in the mixture studied, because it i alway po.'sible that none of the animals u ed has formed antibodies against a substance which may be a particularly poor antigen (as iE', for example, hemoglobin). 2. The chemical natme of the gel may om time interfere; complexes betw en a component of the mixture studied and the sub ~nce providing the gel ub trate may b formed. This que tion ha been discus. ed in ection 1.1. 3. It was shown that in the precipitin reaction in gelified media in the pre ence of an exce of one of the reactants, the precipitin band enlarge and can, in ca e of large exce s, di ociate into two or more lines, even when the reaction i due to only one pure and homoge-'

30

PIERRE GRABAR

neous antigenic ubstance (1,19,32). uch a phenomenon can also be ob erved in lEA. In the case of diffusion in gelified media, without electrophore i it is often difficult to distinguish uch a doubling of the precipitin band from the pre ence of two independent line due to two different antigens. uch a di tinction i much easi r in lEA (11). Three in'tance can be envisaged: (a) if the liquid tudied contain two con tituent po e inlT imilar but not identical mobilities, the cre t of the precipitin arcs will Dot coincide (Fig. 9a): (b) if the mobilities of the two ub tance' are identical, two independent and appro}'.'imately parallel arcs will b formed (Fig. 9b); and (c) if an arc ::orre ronding to one substance i dissociating into two or more lines, a

...-:::::=---.

b~

d ____

~

Fig. 9. Different forms of precipitin arc (sec the text).

one observes first an enJargment of the arc and then its di '''ociation, in which ca e the crests of the arcs are ituated at the same place and the line join either at the end (Fig. 9c) or at the center (Fig. 9d), depending on whether the antigen or the antibody is present in large excess. This la t image is particularly characteri tic with horse antibodies. Such pictures are not alway sufficiently clear and in some cases they are difficult to interpret; in order to undersLand them it is sometime' neces ary to repeat these experiment , using varying quanti tie and different immune sera. 2. Definition and Identification of Constituents of a Mixture Several means of identification of the COD tituent of a liquid can be employed. They can be grouped in three categories: definition by the position of the precipitin arc; identification by immunol gical specificity; and characterization by special reactions such as particular staining or enzymic reactions, etc.

JMMUNOELECTROPHORP.1'TC ANM,YSI8

31

A. APPEARAN E AND POSITION OF THE ARC

The appearance, th form, and the po ition of the precipitin arc, as well a the speed of its formation, may fUl'nish useful information. Each will be discu .. 'ed eparn.tely. The appearance of a precipitill arc may vary; it may be morc or less sharp. It depends, on the one hand, upon the origin of the antibodie. The antipl'otein formed by the horse precipitate in a narrower zone of antigen-antibody ratios than the antibodies of other animals; therefore the arc formed by horse antibodi s are generally very fine. On the other hand, thi depends also on the antigen-antibody ratios: the lines formed when the two reactant are pre ent in proportions corre ponding to the equivalence zone are finer than tho e formed in the presence of an excess of either one of them. In the latter case, as m ntioned above, the arc may enlarge or dis ociate into evera! lines, and even disappear completely as a con quence of the formation of soluble antigen-antibody complex when a large exce s of horse antiprotein antibody or a large exce of antigen i prO! nt (. ee ection III.2). rphe curoalure of t,he arc and itR . ituation in I' lation t.o the ource of antibodies, i. ., parallel t.o the axi of electrophoretic migration, depend both upon the diffu ion rates of the two reactant , and thereby upon their molecular weight. and form , and upon their relative proportion. If the arc i. markedly curved and is situ,'l.ted near the I1xis of migration, this can be int.erpret/ed ac an indication that the antigen diffuRe very. lowly (or in some cas, that it is re ined by the g 1) , whereas if the precipitin line i nearly horizontal, i.e. only slightly curved, it can be inferred that the antigen diffuse rapidly. But the effects can b , at lea t partially, balanced by the relative proportions of the antigens and antibodies. When the e two reactant po e similar diffusion rates, the arc will be ituated nearer the immune serum \vell, in ca e of antigen exce ,and nearer the migration axis if the antibodie are relatively abundant. The form of the arc mayal 0 be influenced to a certain degr e by these proportions. When the precipitin line is particularly elongated, this may be due to two different reasons. It can be the con equence of a partial retention of a ub tance by the gel, thu. forming It "tail." It may also corre po e res nce of component po essing different electrophor ic mqqilDi~ .)u .."ture without any previou treatment. Conditions can be chosen (pH, temperature, etc.) in such a way as to avoid 10 of some particular biological activity of the ubstance to be tudied. Very small amount are necessary to perform a complete analysis. 2. The gels are particularly rich in liquid (98-99%). The electrophoresis i thu similar to free electrophoresis in liquid and there are fewer chances of encountering effects of liquid-solid interfaces as in the case of electrophoresis on paper (35,44), starch, cellulose, etc. On the other hand, the electrophoresis in a gel has the advantage over the electrophore is in a liquid medium of slowing down the free diffuion of the macromolecular sub tance at the end of the electrophoresis, which facilitate. their detection. 3. The u e of the specific precipitation reaction allows fine and specific identification of components of a liquid. Sub tance po. sessing even identical mobilities can be distinguished and impUl'itie of les than 0.1% can be detected. 4. Enumeration of the constituents of a mixture by the precipitin reaction is greatly facilitated by their preliminary di. persion during the electrophoresis.

IMMUNOELECTltOP HOnE'riC AN AI, YSH:i

37

5. A double and sometimes triple definition or identificatiou of the constituents of a liquid i po sible. They may be defined by their electrophoretic mobility, by their immunological pecificity, and by certain other characteristics. The only serious disadvantage of thi method, which is common to all immunochemical procedures, i the fact that the immune. era are biological products and are therefore difficult to tandardize. The present method il:> mainly qualitative and can give only very rough quantitative information' by dilution methods. But the simplicity of its usc, installation, and equipment, a' well as the ea of developing various modifications, makes it pO ' 'ible to envisage a large spectrum of applications. The results alJ'eady obtained have 'hown the advances which it may help to accompli h. The author wi 'hes to rxprr. s his thanks to Dr. Tocl Ro e for the correction of the English text of this article.

References 1. Burtin, P., Bull. soc. chim. biol., 36, 1021 (1954).

2. Burtin, P., P. Grabar, G. Boussier, and M. Jayle, Bull. soc. chim. bioi., 36, 1029 (1954). 3. Burtin, P., L. Hartmann, J. Heremans, J. J. cheidegger, F. WestendorpBoerma., R Wirme, C. Wunderly, and P. Grabar, Rev. franf. etudes din. et biol. , 2,161 (1957). 4. Durieux, J., and M. Kaminski, Bull. soc. chim. biol., 38, 1145 (1956). 5. Fauvert, R, P. Burtin, L. Hartmann, and P. Grabar, Rev. franf. etudes ·lin. et bioi., 1, 17, 175 (HI56). 6. Fine, J. M., J. Uriel, and R Faure, Bull. soc. ('him. bioi., 3 ,649 (1956). 7. Gordon, A. H., B. Reil, K. 'be ta, O. Kn 's'l, and F. arm, Collection Czech Chem. Communs, 15, 1 (1950) . . Grabar, P., Ann. N. Y. Acad. Sci., 60, 591 (1957). 9. Grabal', P., Arch. sci. bioi., 39, 5 9 (1955). 10. Grabar, P., Bull. soc. chim. biol., 36, 65 (1954); TMrapie, 9 163 (1954). 11. Gralo.l', P., Colloq. Diffusion, Montpcllier (1955). Pubis sci. ministcre air (France), 59, 3 (1956). ]2. Gl'II.bo.r, P., Repts Int rn. Congr. Biochem., Brusscls, 37 (1956). ]3. GrabaT, P. and P. Burtin, Bull. soc. chim. bioi., 37, 797 (1955). ]4. Grabar, P., J. Cour 'Oil, and J . . riel, UnpubH hed r ults. 15. Grabar, P., W. W. Nowinski, and B. D. Generefl,ux, Nature, 178, 430 (1956).

38

PIERRE GRAllAR

16. Gru.bar, P., a.nd K. B. Tan, Unpublished results. 17. Grabar, P., and C. A. Williams, Jr., Biochim. et biophys. acta, 10, 193 (1953); 17,67 (1955). 18. Kamin lei, M., J. Immunol., 75, 377 (1955). 19. Kaminski, M., Colloq. Diffusion, M ontpellier (1955); Pubis. sci. mini8tere air (France), 59, 69 (1956). 20. Kaminski, M., and J. Duri ux, Bull. soc. chim. bioi., 36, 1037 (1954). 21. Karjala, S. A., Personal communication. 22. Kizevetter, I. V., Bull. Far East Branch Acad. Sci. U.S.S.R., 21, 85 (1936). 23. Knedel, M., 0" Coll. Hopit. St. Jean, Bru.ge8, 72 (1957). 24. Kohn, J., Clin. chim. acta, 2, 297 (1957). 25. Kohn, J., Nature, 180, 986 (1957) 181,839 (1958). 26. Kruyt, H. R., and H. G. B. de Jong, Kolloidchem. Beih., 28, 1 (1928). 27. Oakley, C. L., and A. J. Fulthorpe, J. Pathol. Bacteriol., 65, 49 (1953). 2 . Ouchterlony, 0 ., Acta Pathol. Microbiol. Scand., 25, 1 6 (1948). 29. Oudin, J., Ann. inst. Pastwr, 75, 30 (1948). 30. Pavlov, P. N., and R. Volska, Ukrain. Khem. Zhur., 10, 485 (1935); cited in Brit. Chern. Abstr., B429 (1936). 31. Ross-Robertson, G., cited in Endeavour, 4, 69 (1945). 32. Salvinien, J ., and M. Kaminski, Compt. rend. Acad. Sci., 240, 377 (1955). 33. Scheidegger, J . J., Intern. Arch. Allergy Appl. Immunol., 7, 103 (1955). 34. Scheidegger, J. J., Bull. soc. chim. biol., 39, Suppl. 1, 45 (1957). 35. Schultze, H. K, Clin. chim. acta, 3, 24 (1958). 36. Seligmann, M., B. Goudernand, A. Janin, J. Bernard, and P. Grabar, Rev. HematoZ., 12, 302 (1957). 37. Slater, R. J ., Arch. Biochem. Biophys., 59, 33 (1955). 38. Uriel, J., Bull. soc. chim. bioZ., 40, 277 (1958). 39. Uriel, J., Clin. chim. acta, 3, 234, 384 (1958). 40. Uriel, J ., Clinica y Laboratorio, 60, 7 (1958). 41. Uriel, J., Nature, 181, 999 (1958). 42. Uriel, J., H. Gotz, and P. Grabar, J. suisse med., 87, 431 (1957). 43. Uricl, J., and P. Grabar, Ann. inst. Pasteur, 90,427 (1956). 44. Uricl, J., and P . Grabar, Bull. soc. chim. biol., 38, 1253 (1956). 45. Uriel, J., and J. J. Scheidegger, Bull. soc. chim. bioi., 37, 165 (1955). 46. de Vaux St. Cyr, C., J. Courcon, and P . Grabar, Bull. 80C. chim. biol., 40, 579 (195 ). 47. Wierne, R. J., Klin. Woch8chr., 34, 1264 (1956). 48. Wierne, R. J., and M. Rabaey, Naturwissenschaften, #, 112 (1957). 49. Williams, C. A., Jr., and P. Grahar, J.lmmunol., 74,158,397,404 (1955). 50. Wolfe, H. R., M. Goodman, and S. Norton, J . Immunol., 66, 225 (1951). 51. Wunderly, C., Experientia, 13, 421 (1957).

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME VII

The Analysis of Basic Nitrogenous Compounds of Toxicological Importance A. S.

CURRY,

Home Office, Forensic Science Laboratory, Harrogate, Yorkshire

1. Introduction . . ... . . . . . . . . . . . . . . . . . . . . .. '" .................... Isolation ... . .. .... . . . . . . . . . . . . .. ........... . .. . . .... ......... 1. Initial Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Methods Using Ethyl Alcohol .. . ... . . . ... ... ..... . ... .. .... B. Methods Using Aqueous Extraction Processes . . . . . . . . . . . . . . . .. C. Other Methods . . . . . . . . . . . . . . . . . . . .. ....... ...... . ....... 2. Extraction into Immiscible Solvent . . . .. ....................... 3. Isolation of Vola.tile Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Ill. Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1. Introduction .......... .. ........... . . . . . . . . . . . . . . . . . . . . .. 2. Paper Chromatography . . . . .. .. . ............... . . ..... A. Solvent Systems Using n-Butanol and Unbuffered Paper . . . .. . . . B. Solvent Systems Using Pa.per Impregnated with Buffer olution . . C. Reversed Phase Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. D . Other Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3. Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. A. Methods of Concentrating All Alkaloids . . . . . . . . . . . . . . . . . . . . .. B. Methods eparating One Alkaloid from Another. . . . . . . . . . . . . .. IV. Identification ....... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. 1. Elution.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2. Tests Made on Paper hromatograms. . . . . . . . . . . . . . . . . . . . . . . . . .. A. Color Tests . . .. ........... ... . ... ....... .. ......... . . . . 3. Oth r Methods ......... .. ..... ... .................... A. Ultraviolet pectrophotometry . . ........................... B. Ooservation.e on Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. C. Biological Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. QUantitative Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . .. 1. Introduction. . . .. . . . . . . . . . . . ......... . . , . . . . . . . . . . . . . . . . .. A. General Methods . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . .. B. Colorimetric Met,hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .... 2. Methods for Particula.r Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. VI. Summnry .. .................................................. . . Refer 'nces . . . . . . . . . . . . . ............. .. ..... . ............... . .. ,

n.

39

40 41 41 41 42 44 46 47 47 47 48 49 50 50 51 51 51 52 53 54 55 62 62 62 63 65 65 66 66 67 69 70

40

A. S. CURRY

I. INTRODUCTION Up to about ten years ago the analy i' of vi, cera for alkaloids was onfined mainly to an examination for evidence of su pected criminal or accidental poisoning. The foren ic expert in these ca es et a v ry high standard of analytical technique: indeed, the succes ful detections of aconitine in the Lamson ca e in 1 81 and of hyo cine in Mrs. Crippen, five months after death, in 1910, are eloquent example of their successes. Two changes in the approach to these analy es have occurred in r cent years: first, there ha been a considerable increase in the number of alkaloid and basic sub tances of synthetic origin introduced into clinical practice, and, econdly, the analysis is now often directed toward di covering the concentrations throughout the body of drugs admini tered in therapeutic quantities. The e two changes have meant that the tests for the detection and e. timation of such compounds have had to be re-examined for incren. 'ed specificity and ensitivity. It is the intention of this chapter to examine orne of these methods. There is no descriptive noun that includes both the naturally occurring nitrogen-containing compounds long defined as alkaloids and the new ynthetic compounds. Becau e both type can be extracted from aqueous alkaline solution by an organic solvent and are not analytically distinguishable in the extraction, it is propo ed henceforth in this chapter to use the word "alkaloid" as a collective noun for both classes of compound. It is not proposed to discu . the analysis of the quaternary ammonium compound and other water-soluble compounds such a adrenaline and hi tamine which are not extracted by immiscible solvents. There are three main groups of workers who are concerned with analyses of this type: (a) research chemists concerned either with the chemistry of natural products or the development of new synthetic drugs, (b) clinical biochemists concerned with the determination of body concentrations of known drugs, and (c) toxicologi ts who have first to unravel the problems of identification before as aying the poison. Becau e these differing approaches involve extremes of analytical techniques, a closer definition of the scope of this chapter is necessary. It is intended to act as a guide to the analysi of alkaloid that have been chemically characterized: it is also intended

NITROGENOUS COMPOlTNDS

41

to envi age thn,t the total available weight of alkaloids is Ie s than 3 mg. Thi automatically implie that not only would there be great difficulty in obtaining the clas, 'ical chemical data such as the per ·ent.ages of carbon, hydrog n, and nitrogen if a single crystalline alkaloid or its dcrivative could be isolated, but 'uch determination. would destroy the xtract. This wcight limit means also that an infrared absorption pectrum could be obtained only under favorable circumstance and that the potential value of the X-ray diffraction camera i unlikely to be fully realized. Although the techniques described for the isolation can be used regardles of the quantity of alkaloid that i pre ent, in later sections it is as umed that the presence or absence of metabolites is to be demonstrated, and that in many cases the identities of the alkaloids have to be established. Only when purification and identification are complete can the techniques for the quantitative assay follow.

II. ISOLATION From the descriptive point of view, the isolation stage of the analysis is the one that po s the mo t difficult problems in a review of this type. There are several "general" methods for isolating alkaloids but the choice of the particular method depends to some extent on the type of material from which the alkaloid is to be isolated. Notwithstanding this reservation, all the 'e methods can be subdivided into similar groups. In the order in which they are performed the e are (1) defatting and deproteinizatioll in the initial isolation stage leading to an aqueou acidic solution, and (2) extraction of the alkaloid into an organic solvent and concentration into small bulk. 1. Initial Extraction

There are many varied methods of accomplishing thi stage of the isolation. These will be considered separateliY. A. METHODS USING ETHYL ALCOHOL

Alkaloids and the salts of alkaloids with weak organio acid are generally soluble in a100hol. The tissue, animal, or plant, is mace1'-

42

A.S. CURRY

ated with warm ethyl alcohol acidulated with tartaric, citric, or acetic acid, and is then allowed to stand for several hours. PI"·ipit..'l.tion of the protein occur: and flltmtion is usually fairly callY. Removal of the alcohol and water is accomplished next by cith r low temperature evaporation over several days or di tillation under reduced pressure. There is often serious frothing at thi. stag but this should present no difficulty if modern antifroth heads are used. In all these extraction methods there is the possibility of the los. of the volatile amines inlN the distillate. Acetic acid, for example, i not a sufficiently strong acid to prevent the 10 s of amphetamine. If, however, a strong acid is used, the possibility of the hydrolysi of many alkaloids containing an ester group, or one of the other similar acid-labile groups, is a real hazard. After the removal of the aqueous alcohol by distillation or low temperature evaporation, boiling absolute alcohol is poured on the residue. Solution of the alkaloid salts occurs and a further precipitate, granular in appearance, can be easily removed by filtration. Evaporation of this alcohol leaves the crude alkaloid extract. The precipitates obtained at both stages of the extraction should be well washed with warm acid alcohol and the washings evaporated. This reoidue i combined with the main alkaloid extract. This outline of the well-known Stas-Otto process is most incomrl~tc j indeed, it is not possible to convey more than an indication of the method. Experience enables the worker to adapt his procedure to the type, quantity, and degree of putrefaction of the tissue being processed. The uninitiated should either watch the process being performed by an experienced worker 01, if this is not possible, should study the relevant section in Bamford's book on poisons (6) and Umberger's description of the method (151). This latter work include several variations, one of the most important of which concerns continuous extraction of tissue with alcohol. This, the most theoretically sati fying of all the methods, is unfortunately too complicated to detail here and the reader is referred to the original work. B. METHODS USING AQUEOUS EXTRACTION PROCESSES

The many methods of precipitating protein used in clinical biochemistry have been applied to toxicological analyRis. The simplest

Nl'fnOGENOUS COMPOUNDS

43

method is to heat aqueous solutions with dilute acid. The following procedures are typical. Method 1: Daraway and Tompsett (41). Homogenize 50 g. of liver with 100 ml. of 0.85% aqueous sodium chloride solution. Place the mixture in a boiling water bath for 5 minutes, add 50 ml. of 2 N hydrochloric acid, and heat the mixture for an additional 10 minutes. After cooling, dilute to 250 ml. and filter. Method 2: Feldstein and Klendshoj (55). Grind a portion of tissue, about 500 g., in a Waring Blendor and transfer to a 2 liter flask. team-distill from acid solution to remove the volatile poisons. Filter the residue in the distilling flask. Suspend the insoluble precipitate in 500 ml. of boiling water to extract any further toxic materials and filter. Combine the filtrates. 1t should be noted that both these methods will involve the loss by hydrolysis of labile alkaloids such as ergometrine and atropine. Although plant tis ue and urine are amenable to this treatment, \'lsceral extracts are often extremely difficult to filter and many methods hM"e been tried to overcome thi difficulty. The e mu t be COl idered with respect to their theoretical soundnes if they are to be applied as general methods of isolation. It is tempting to assume that methods which give good recoverie with a number of alkaloid might be suitable as general methods. This is not necessarily the ca ·e. Becau'5e it is obviously impossible to demon trate their over-all soundness, the chemist, being faced with a choice of s veral methods, usually choo es one, perfects his technique in using that method, and tru t to luck. The theoretical and practical soundness of the alcohol extraction method has resulted in its being the method favored by mo t workers but, because of the large volumes of alcohol that have to be u ed and the time required for an extraction, there has been a search for a quicker and ea ier alternative method. The ODe that has proved its worth to the writer is the use of ammonium sulfate as the protein precipitant. The application of this method to toxicology was first published by Daubney and Nickolls twenty years ago (42,43) and it has become one of the chosen m thod in British laboratori . Method 3. The material to be treated (500 g. of liver, brain, or aliquots of alimentary tract contents) i macerated 'with approximately 400 mi. of dilute (5%) acetic acid, and sufficient ammonium sulfate is added to maintain a saturated solution when the beaker is

44

A.S. CURRY

placed in a boiling water bath for 30 minutes. After this time the precipitated protein is filtered off through a paper pulp pad on a sintered glass Buchner type funnel. Filtration i easy and mpid. The solid on the pad is then washed well (3 X 200 ml.) with hot dilute acetic acid. Minimum uction should be used. These filtration stages are slower than the first one but are essential. Nickolls has recently modified the method (115) by using hydrochloric acid instead of acetic acid, the cidity being kept between pH 2 and 3 to avoid losses by hydrolysis. Two other protein precipitants which can be used are tungstic acid and trichloroacetic acid. Both these methods are extremely well known and are uitable for the precipitation of protein from blood or macerated tissue. A typical scale of working for the former method is a follow. Method 4. 100 g. of liver is macerated with 120 ml. of 10% aqueous sodium tung tate and 200 ml. of water. Dilute, approximately normal, sulfuric aoid is then added to pH 3 with rapid (preferably electric) stirring. Precipitated protein can then be removed either by filtration or by centrifugation. Coagulation of the protein occurs and filtration is much easier if the liquid is heated in a boiling water bath for 30 minutes. Althougb the u e of trichloroacetic acid in the isolation of adrenaline is well known, complex formation has been observed (136) and therefore may occur between it and other alkaloids. Loss of many alkaloids also occurs in the tung tic acid method becau e they are coprecipitated with the protein. There are often occa ions in clinical biochemistry, however, when these methods may be of use. Both methods are very easy and quick; they are capable of dealing with as little as 1 m!. of blood but can also be adapted to treat 500 g. qUftntities. If it is shown that th alkaloid appear quantitatively in the filtrate, they are of great value. C. OTHER METHODS

Alkaloid can be extracted from plant tissue by Soxhlet extraction on the dried material. Large quantities can be handl d, but for visceral extraction there is such a relatively large volume of water to be removed that the method is of limited usefuhle s. One procedure is to dry out the macerated tissue with anhydrous sodium sulfate. It

NITROGENOUS COMPOUNDS

45

seems probable that freeze-drying will be used increasingly to dehydrate the ti sue, followed by direct extraction with immiscible solvent. Stewart and his co-workers (138,140) have described other method whereby alkaloid are adsorbed onto kaolin or the Rynthetic magnesium silicate ]'lorisil and are subsequently eluted by aqueous acidic or organic olvent. Decalso has also been u ed (18,19). The Edinburgh school has u ed mainly trichloroacetic acid coupled with the tas-Otto method prior to adsorption but urine and pIa rna need no treatment other than dilution. The optimum pH for ad orption onto F lori. il is between 7 and 8.5 for tis ue extract, 8 and 9 for blood, and 5 and 6 for urine. The reason for this is not clear. Procedure (41). A column 15 X 1 cm. is packed ,,-ith 60-100 me h of Florisil (Floridin Co., Inc., Warren, Penna.) and is wa hed with 100 mI. of 10% ammonia solution, followed by 200 mI. of water. The alkaloid is adsorbed from the aqueous deproteinized extract, the column washed with 100 mI. of 0.5% ammonia, and then with 100 ml. of water and 50 m!. of 30% acetone. The alkaloid can be eluted from the column with a solution of 80 parts ethanol, 15 parts ammonia, and 5 parts water. This size of column is sufficient for 250 m}. of urine or for 100 g. of tissue containing up to 5 mg. of alkaloid. 109. of kaolin i ufficient for the 800 m!. of fluid obtained from 400 g. of tissue. The alkaloid is obtained by Soxhlet extraction of the dry powder of kaolin, sodium carbonate, and odium sulfate, or more imply by shaking the kaolin with sodium carbonate solution and ohloroform (6). Detail of a method utilizing alumina for the i olation of morphine from opium are as follows (6]): A repre entative sample of opium is powdered and approximately] g. is accurately weighed into a mall porcelain dish. It is triturated with 4 mI. of 3: 1 mixture of 95% ethanol and a dilute olution of ammonia B.P. Aluminum oxide i added gradually and triturating continued until a free-flowing powder i obtained. Transfer the powder to a dry chromatographic tube of about 1.5 cm. diameter and 40 cm. long previou ly plugged lightly above the tap with ootton wool. Remove any adhering powder from the di h and pestle with ootton wool moi tened with alcohol, I1nd add to the tube. Insert the lower end of the tube through a hung fitting into the n ck of a 250 rol. separator and elute the morphine with 100 ml. of chloroform- isopropyl alcohol (3: 1), adjusting the rate of elution to about 1.5 m!. per minute, using slight positive pressure if necessary.

40

.•. CURRY

In this outline of the main methods for isolating alkaloids, it ha not been po, ible to s::ly which meth d i the be t or inde d which one is to be used for a particular purpo e. In the literature the reader will find not only the methods ue crib d above but many combinations of them. It is hoped that a sufficient description has been given to indicate the complexity of the problem and some of its olutions.

2. Extraction into Immiscible Solvent In nearly all these extraction proce e. the final solution is an aqueous one at a pH between 2 and 3. In toxicological analy is, acidic poisons are fir t extracted from this solution by ether or chloroform. In many cases an alkaloid salt may also be coextracted. Papaverine, for example, is extracted from the ammonium sullate acetic acid dige t by ether. Becau e many alkaloid sa.lt , particularly the salts formed between the alkaloids and organic acid' or hydrochloric acid, are 01uble in organic solvents, all the alkaloid salt will be extracted from the acidic solution if continuou extraction i used. Although details of the solubilities of the alkaloid and their salt in various solvents are readily available (e.g., Merck' Index), they are, in practice, only a rough guide of the partition to be expected between tbe aqueous phase and the immiscible solvent. This i becau e appreciable quantities of inorganic salts, fat or fatty acid, may al 0 be present, which complicate not only the extraction from acidic solution but also subsequent extractions from alkaline solution. The extraction of morphine is but one example. It is commonly suppo ed that morphine i not extracted to any appreciable extent from neutral aqueous solutions by ether or chloroform. When the olution is saturated with ammonium uifate, however, morphine is partitioned in such a wny that appreciable ammmt are extracted by ether or chloroform. The techniques of extraction can be very important. EmulsiolJs in this type of work are troublesome if straightforward shaking in a separator is used. Two techniques that help in preventing the formation of emulsions are rolling and continuous extraction. The former can be used with ether as a solvent while the latter is more often used with chloroform or mixture of chloroform and one of t.he alcohols, e.g., chlorofonn/propauoI5: 1 (by volume) (55). After removing the solvent by evaporation, the alkaloid cun be further purified by dissolving it in dilute sulfuric acid, filtering, adding

NITROGENOUS COMPOUNDS

47

ammonia to pH 10, and re-extracting into the appropriate organic solvent. A solution of lead acetate added dropwise to an aqueous solution of the alkaloid in acetic acid precipitate many other impurities. The alkaloid is recovered from the filtrate, after lead ions are removed from the solution by hydrogen sulfide or ammonium sulfate, by adding ammonia, and extracting into immiscible solvent.

3. Isolation of Volatile Alkaloids These compounds represent a pecial case of i olation. Although they can be e}.'tracted by mo t of the methods described above, removal of solvent by evaporation from other than fairly strongly acidic 'olution leads to loss. This can be seriou because the pre ence of a volatile alkaloid may not have been suspected. The u ual method of extracting such bases is to steam distill from alkaline olution. Great difficulty because of frothing can be experienced, but a large air space above the liquid, an antifroth head, and the addition of a little silicone grease will minimize the difficulty. The di tillate is collected in dilute hydrochloric acid and evaporated on a steam bath. If the alkaloid i acid-labile, it can be extracted into ether from alkaline solution, dried with sodium sulfate, and precipitated as the hydrochloride by passing in Hel ga . Another means of concentrating the alkaloid is to precipitate a complex salt, using a general alkaloid reagent uch as one of the double iodides or a selective reagent such as picric acid for nicotine. The volatile base, although forming a discrete group a far as isolation is concerned, are included in the follo,""ing sections which are concerned with the pUl'ification, identification, and quantitative assay.

III. PURIFICATIO 1. Introduction

The crude extract left by evaporation of the ether or chloroform in the final stage of the isolation process may contain the following basic compound: (a) the required alkaloid, or mixture of alka loid, (b) metabolites of the required alkaloid or alkaloids, (c) compounds with

48

A. S. CURRY

similar solubility properties occurring naturally in fresh tissue, and Cd) products produced by putrefactive proces es. Before the purification stage can be fully discussed, it is necessary to discover the approximate proportion of required alkaloid or alkaloids in the total extract. The purification of 1 J.lg. of ergometrine in 1 mg. of crude extract requires a different technique from that of ] mg. of morphine in 1.5 mg. of crude extract. A "screening" method of inve tigating the e:\.'tract is therefore desirable. 2. Paper Chromatography One solution to the problem is the use of paper chromatography. If aliquots of extract are examined in this way, a separation of the compounds can be obtained and an immediate approximate e timate of the relative concentrations of each component is possible from densitometry measurements on the pots. It is not necessary to identify the alkaloids at this stage because general spray reagents are used. Careful consideration of the aliquot to be put on the chromatograms is recommended. For general purposes the following reasoning seems applicable. 10-20 J.lg. of alkaloid i a quantity which should be detectable if the spray reagents described below are used. If 1% of the aliquot fails to reveal a spot, it is likely that less than mg. quantities of alkaloid are present in the total extract. If 10% i now put onto the chromatogram and no spot is obtained, then there is less than a few hundred~. of alkaloid in the total extract. Only at this stage is it necessary to put the whole of the remainder of the extract on another chromatogram and so make the function of the chromatogram not only one of "screening" for alkaloids but al 0 one of purification. Whatever the ultimate outcome, paper chromatography is a suitable technique for this stage of the analysis. A solvent system or combination of systems which can guarantee to separate all the permutations and combinations of compounds that are covered in this review is a practical impo ibility. It is not urprising that many different systems have been propo ed. All have the aim of providing a high degree of resolution and it is not possible to do more than review some of them. General principles may indicate the olution of a particular problem if one has not already appeared in the literature. No attempt has been made to provide a complete bibliography of the application of paper chromatography to the separation of alkaloids.

49

NTTROGENOUS COMPOUNDS

Three main types of solvent sy terns have been used. discussed below.

These are

A. SOLVEN'f SYSTEMS USING n-BUTANOL AND UNBUFFERED PAPER

This is undoubtedly one of the most widely u ed solvents. Table I shows the combinations that have been u d by various authors. TABLE I Solvent ystems with n-Butanol (Unbuifered Paper) (Parts by volume used with 100 parts n-butanol) Solvent 2

olvent(s) 3

Compounds investigated"

Ref.

aturated ",ith 14% acetic acid Saturated with 20% acetic acid Glacial acetic 4 Saturated with ethanol 100, acetic acid 10 Glacial acetic 20

Water 50 Wat,er 50

Cocaine and its metabolites 28 General alkaloids 115

Water ]00

Glacial acetic 25

Water 125

Glacial acetic 6 .7 Glacialncetic 10 Glacial acetic 20 Glacial acetic 3] 0

Water 26 Water 30 Water 53 . 5 Water 177, but;yl acetate 520

Morphine, scopolamine, ephedrine Ergot alkaloids; general alkaloids Opium alkaloids Opium alkaloids Morphine and strychnine Papaverine and narcotine

aturated with 4% HCI Saturated with 20% HCl 0 . 1 N HCI]OO HCIlOO Formic acid 10 Formic acid 16. 5 Formic acid 8 . 5 Saturated with water Propionic acid 10 Ethanol 100 Ammonia 2

Water 100 Water 100 Water 58 Water 60 Ethanol 25 Water 100 Water 100 Water to cloudiness

General alkaloid

111

General alkaloids

111,113

General alkaloids General alkaloids Morphine al.kaloid , leptazol, coramine, and atropine Aconitine and brucine General alkaloids Morphine and strychJline Drugs of addiction Ephedrine General alka.loids Ephedrine Nicotine derivatives

143 58,64,110 46 46 132 147

111 III 77 11 64 '

132 154

26 64 26 4

" "General alkaloids" implies that many different alkaloids have been investigated by us of these systems.

50

A. S. CURRY

B. SOLVENT

SYSTEMS

USING

PAPER

IMPHEG ATED

WITH

BUFFER

SOLUTION

In this technique the filter paper is impregnated prior to development with an aqueous solution of a buffer solution and dried under conditions that are reproducible. It is undesirable to dry at a high temperature (above 70°C.) unless the paper is to be equilibrated over water or solvent before use. In the majority of publi hed papers nbutanol i used as the developing olvent, although it is not unusual to saturate the butanol with buffer solution before use. The lower alcohols have also been u ed (142), a has ether- water. Although some workers have used potassium chloride or sodium acetate (112), potassium and sodium mono- and dihydrogen phosphate or citrate buffers are now favored. The strength of buffer is usually between MI5 and M / 15 (109,148). Many different groups of alkaloids have been investigated and these types of systems have also been favored in toxicological analysis in which all alkaloids have to be considered. Many authors have observed that the R, of a particular alkaloid is highly dependent on the pH of the buffer. This is rno t useful, for it is possible to change, easily and quickly, the R, of an alkaloid by altering the pH of the buffer and so obtain better conditions for the separation, and hence purification, of a particular alkaloid. Papers that demonstrate this are those by Curless and Woodhead (27), Mannering and his co-workers (97), Buchi and Soliva (23), and Bettschart and Fluck (14). Goldbaum and Kazyak (63) have, by observing the variation in R, of twenty-eight alkaloids at four pH's, used the method to assist in identification. Schmall and his co-workers (J 30) pave varied the pH of the paper in zones to separate mixtures of alkaloid . In this paper chloroform was the developing solvent and double strength MacIlvaine buffers between pH 4.2 and 6.4 were used in 2 cm. lengths with 1 cm. unbuffered paper zones in between. For general purposes, with use of one system, an intermediate pH is desirable and an extension of Curry and Powell's report (40) of a citrate buffer is described below (see Tables IV and V, Section IV.2). C. REVERSED PHASE SYSTEMS

Table II indicates the type of systems that have been used.

51

Nl'l'110GJ~

'"

;.,

:C-;~

15

~

:.a

ci

~

~

~

0

..d

~

or; d 0

0.3

.

::I

Qj

>

-.

~

...

4>

> .....

'~" II)

~

~C

.,~c CJ II) 4>

...

4> 0 ..d ::I ~c=

~

'0

60

A. S. C{ffiRY

reagent. Warming at 90° for 10 minutes completes the methylation." A spray of similar general specificity i~ potas ium iodoplatinate, which many authoTR prefer to Dragendorff's reagent. There is little to choo e between them as far as sen itivity is concerned. The iodoplatinate spray is prepared thus (63): Mix 45 mI. of 10% potassium iodide, 5 ml. of 5% platinic chloride, and 100 m!. of di tilled water. Iodine sprays have their advocates a a general method of revealing alkaloids; although the sensitivity is relatively low, different colors are obtained and so selectivity is increased. A typical pray is made by dissolving 2 g. of iodine in a solution of 4 g. of pota. sium iodide in 94 rol. of water. Another general spray which can be used is 0.5% bromocresol green in alcohol. The sensitivity is high and can be increased by gently blowing ammonia vapor over the surface of the paper after spraying. This spray is particularly useful for detecting the volatile secondary amines. Not only do these alkaloids give blue spots on a yellow background but, on subsequent spraying with Dragendorff's reagent, the alkaloid-dye complexes react. Depending on the reaction to Dragendorff's reagent, positive or negative, the schemes shown in Tables IV and V are followed. The other spray used are: TABLE V Chromat.ographic Identification of Alkaloids Giving Negative Reaetion to Dragendorff Spray ReagentG 0.1 % KMnO. spray

I

t

t

Positive

Negative

I

p-Dimethylaminobenzaldehyde spray

t

I

t

Positive Negative "Ergometrine .26 Benzocaine .86

G

I

p-Dimethylaminobenzaldehyde spray

I Positive None

Neaative Caffeine .70 Amphetamine .40 bReserpine .84

The numbers are R, values in butanol-citric acid system.

~ Significant ultraviolet fiuorellCellCe.

NITROGENOUS COMPOUNDS

61

4. Aqueou8 cobalt thiocyanate, 2%. This gives a pale pink background. Many alkaloids give blue colors with it either immediately or within a few hours. 5. Aqueou8 KMnO., 0.1%. Some alkaloids give an immediate reaction, showing as yellow spot on a pink background. 6. p-Dimethylaminobenzaldehyde. 50 mg. of p-dimethylaminobenzaldehyde in 10 ml. of ab olute alcohol plu 2 ml. of concentrated sulfuric acid. The paper, after spraying, is heated at 60°C. for a few second. This reagent mu t be freshly prepared. Although it is possible to superimpose certain sprays, the arrangement in thi description as 'umes that the sprays are used on separate chromatograms. The complexes formed on the chromatogram between the alkaloids and the double iodides can be cut out, decomposed with strong ammonia and a trace of sulfite, and the purified alkaloid extracted into chloroform. All these sprays are relatively non pecific but the p-dimethylaminobenzaldehyde reagent, by rea 011 of the different colors it produces, acts as a bridge between the many published color reaction for alkaloids, i.e., the "classical" color tests, and the later techoiques such as paper chromatography. There is a vast literature on the colors produced by a number of these reagents (Marquis, Mandelin's Froehde's etc.) on the alkaloids, and they are usually applied as spot tests on a white tile. Bamford (7) u ed these systematically for a number of alkaloids and more recently Umberger and his co-workers (152) have given a comprehensive list. Although many of them are based on concentrated sulfuric acid, thi i no bar to their being poured on citrated paper chromatograms. Because mixtures of alkaloids have been separated into individual members 011 the chromatogram, the e tests are considerably more reliable when performed in this way. In toxicological work they are invaluable because, from the R/value and the behavior to the sprays in Table IV, it is often po sible to pinpoint the probable alkaloid and so choo e the color tests to confirm the identification. If identification has not been achieved, data as to structure can also be obtained by using reagents which are designed to attack certain functional groups in the molecule. The complex iodides generally react with tertiary amines but do not form spots with primary and secondary amin s. Ninhydrin i a u eful reagent for such secondary amines as ephedrine i and Macek and his co-workers (96) drew attention to Feigl's nitroprusside-acetaldehyde reagent

62

A.S. CURRY

(54) as a spray to detect secondary amines. This reagent is prepared as follows: 5 g. of sodium nitroprusside is dissolved in 100 m!. of 10% aqueous acetaldehyde with 2 g. of sodium carbonate added. Many authors have published systematic chemes similar to that described above for separating and identifying alkaloids on paper chromatograms (12,54,96,109,131). It is not possible to do more than give references becau e the many combinations of alkaloids, olvent systems, and detecting sprays make a full description an impossible task. The basic approaches are all similar; the reader must choose that which is mo t suited to his own purpose.

3. Other Methods A. ULTRAVIOLET SPECTROPHOTOMETRY

It is possible to measure the ultraviolet absorption curve of the alkaloid on a chromatogram after cutting out the spot and fixing the paper in a cardboard cell holder in a spectrophotometer. Elution from the paper prior to measurement is also used. The absorption of many alkaloids varies significantly with pH and it is a good practice either to take measurements over a range of pH or to read the alkaloid in absolute alcohol and 0.1 or 1.0 N sulfuric acid. It is not possible here to give complete curves of all alkaloids. Reference can be made to collections of curves and data (80,116). The practicing toxicologist builds up his own collection, and those concerned with a particular alkaloid are able to find by experiment whether its absorption is high enough to be of u. e in the analysis. Submicrogram quantities of alkaloid can be conclusively identified by this technique. Papaverine, for example, bas E~ ~m. = 1800 for the major peak and the variation of the wavelength of the absorption maximum with pH is of great value in identification (57). The correlation of ultraviolet (116) and infrared ab orption (93, 98) with structure has been discu "'ed at length in an important series of articles in the Bulletin of Narcotics. The X-ray diffraction patterns (10) and collected physical data (52,53) in the same series represent one of the most important collections published in recent years. B. OBSERVATIONS ON CRYSfl'ALS

One of the oldest methods of identification is the observation of crystal form. Bamford described the production of complexes utiliz-

NITROGENOUS COMPOUNDS

G3

ing a flattened capillary tube as the container (17). The ends are sealed after the reagents have been introduced and mixed. Recently Clarke and Williams have introduced (36) a method of conveying micro drops of reagent and alkaloid solutions to the surface of a cover slip which is then upturned over the cavity of a cavity microscope slide. Ringing with gum arabic delays evaporation, and crystals form slowly over several hours. A 5 mm. diameter glass rod, 12 cm. long, heated in the middle and pulled out until its length is about 20 em., is used. It is then broken at its thinnest point, which should have a diameter rather less than 1 nun. The narrow end are ground flat and thoroughly washed to remove glass splinters. The surface of a solution of the alkaloid, in 1% acetic or hydrochloric acid, is touched with the end of a microrod and the adhering drop of liquid is transferred to a cover slip. Volumes of approximately 0.1 c.mm. (0.1 ~l.) can be conveniently handled. The reagent is similarly transferred to the cover slip before mixing with the alkaloid. Crystals can be obtained with extremely small quantities of alkaloid; the authors (36) claim sensitivities for most of the common alkaloids in the 0.01-0.25 ~g. region, and say that 1 drop (0.05 ml.) of a 0.1 % solution provides material for no less than 500 different tests. This method of handling small volumes of solution is used not only for crystal tests but also to transfer solutions to white tiles for color te8ts. There is no doubt that crystal tests performed in this way are a valuable method of confirming an identification. It is important, however, that pure solutions of alkaloids in approximately known concentrations are u ed. The elution of alkaloids from paper chromatograms into very. mall volumes as described above is a convenient way of preparing the solutions necessary for Clarke's method. 20 ~g. of alkaloid can be eluted from the chromatogram into 0.02 ml. and, in the absence of characteristic ultraviolet absorption of fluorescence data, confirmation of identity must be sought in crystal and color tests. C. BIOLOGICAL ACTIVITY

Many alkaloids have a characterist.ic biological activity which can be used as a criterion of identity. It can be shown that the isolated extract contains material with similar activity. One of the most sensitive tests for aconitine is its ability to kill mice. This test, coupled with another showing the characteristic action of aconitine on the

64

A. S. CURRY

tongue, is used in many toxicologicallabol'atories. The fact that the ob erved biological activity occurs at a particular R, in a chromatographic system of high re olution is good chemical evidence of positive identification. There are many examples of this type of test; the mouse tail test for methadone (3) and t he well-known action of the ergot alkaloids are only examples. Identification is undoubtedly the most difficult task for a forensic toxicologist. and the clinical biochemist may feel that there has been an excessive discussion of the problem involved in such an analysis. NeverthelesR, a knowledge of the purity of the extract is es,ent,in.} beTABLE VI Identification of Alkaloids Compounds Aconitine Selected alkaloids Selected alkaloids Less ommon alk3loids Aliphatic amines Aromatic amines Local anesthetics Local anesthetic' Local anesthetics Antihistamines Antihistamines Antihistamines Antihistamines Antihistamines Antispasmodics Atropine and hyoscine Benactyzine Cocaine Dromoran Heroine Methadone Methadone Morphine and nalorphine Morphine Nicotine Opiates Opium alkaloids Pethidine Reserpine Scopolamine

Method

Ref.

Paper chromatograph~' 101 157 Flavianic acid crystals General crystal formation 36,160 General crystal formation 33 13,164 Color reactions Color reactions 9,164 Crystals 32,163 Vacuum micro sublimation 22 Color reactions, paper chromatograghy 70,159 Chloroplatinic acid crystnls 50 Color reactions 66,114,118 Picrate crystals 25 Ultraviolet data 83,114 Crystal tests 34 Color reactions and crystals 67 Infrared 21 Color reactions 86 Color reactions 122a Chloroplatinic acid crystals 90 158 Color test 160 Color tests and crystals 3 Biological tests and color reactions X-ray diffraction and crystals 119 Crystal complexes 89,!)1 Color test 94 Color test 155 Crystal tests 35 Styphnate crystals 117 Color tests 38 Crystrus 162

NI'fROGENOU8 COMPOUNDS

65

fore quantitative determination can begin; its importance cannot be overestimated. Table VI may be of a si tance when confirmation of identit,y of a particular alkaloid is required.

v.

QUANTITATIVE ASSAY 1. Introduction

The identification sk'tge of the analysis should have provided an indication of the quantity of alkaloid in the total extract. If paper chromatography has been u ed, the methods of revealing the po ition of the alkaloid spots will also have given an estimate of the amounts on the chromatogram. It i nece sary to con ider isolation procedures before methods designed for quantitative analysis are discu ed. The recovery of an alkaloid from plant or animal tissue depends not only on the method but al 0 on the alkaloid. Anthisan, for example, can be recovered quantitatively by both the ammonium sulfate and the tung tic acid methods from human vi cera but, if ergometrine is added to animal tissue, recoveries of only 40-50% are obtained with both the ammonium sulfate method and continuous extraction of odium sulfate-dried ti sue with chloroform (85). There is ome difference of opinion about the efficiency of the StasOtto alcohol extraction process. Many workers are entirely satisfied, but the method is criticized by others who allege low recoverie . The quantitative aspect of the analysis must be put into perspective. It is tempting, and probably ju tifiable, to assume that a constant proportion of the alkaloid is recovered over a range of concentrations, for a particular alkaloid utilizing a given method. The only method of discovering the recovery is by experiment; whether the alkaloid occurs in the ti sue in the free or metabolically altered form is a complication that can be solved only by further exten ive experiments. For these reasons, in medico-legal work, it is u ual to report only the identity and quantity of alkaloid actually isolated. In clinical chemistry, becau e it is required to relate clinical effQot of a given drug with a figure for its concentration in the blood or the urine, less specifio methods of analysis are acceptable. Chemical method of assay need not be specific, provided that it i known that only one drug has been admini tered to the patient. Colorimetric tests on blood serum and urine requiring no complicated isolation procedures are common

66

A.S. CUnRY

in this type of work. Such tests are not amenable to general discussion; they are again particular to the alkaloid and to the technique. It is permissible in this class of test to work to a higb degree of precision. Such an approach is also possible in medicolegal work. provided that the analysis has shown that no interfering compounds are present. The purity of the solvents used in the extraction proce es must be considered. It is possible that reactions of the type ob erved by Caw and Fo ter (29,30) between strychnine and the impurities in B.P. chloroform may be duplicated by other alkaloids. This could be a serious factor in this type of analysis, especially when continuous extraction is used. The decompoRition of many alkaloids on alumina columns is well known and peroxide in ether are another source of trouble. With the exception of the "direct" tests u~ ed in cliuical work, the accuracy that it is profitable to pursue in a quantitative analysis mu. t be viewed in relation to the i olation procedure. In many medicolegal cases it is the pre ence of the alkaloid that is important; the effect is either determined by clinical evidence or inferred by the presence of large quantities in vomit or the alimentary tract. A. GENERAL METHODS

Two of the main methods for the assay of all alkaloids are the gravimetric assay of the alkaloid, its salt or complex, and the titration of the free base witb standard acid. Ion-exchange resins are used for the separation of the ions of alkaloid salts and this is a technique which, coupled with an automatic titration apparatus, can be used for rapid routine analysis. Levi and Farmilo (92) determined twelve common alkaloids with a precision of 1.6%, using Amberlite IR-4B; the scale of working was of the order of 75 mg. Other workers have used ion-exchange for specific problems, often on a mg. scale. Because the choice of resin depends on the alkaloid involved in the analysis, full details cannot be given in such a general review. D. COLORIME'l'RIC METHODS

For general colorimetric analysis the stoichiometric complexes between alkaloids and many dyes are extensively u ed.

NITROGENOUS COMPOUNDS

67

Daraway and Tomp, ett (41) have recently investigated fourteen alkaloids, six dyes, and three immiscible solvents. The best combiJlation for general u,'e was found to hc hromothymol bluc-benzene. All the alkaloids reacted except morphine, henzocaine, aJld yohimbine. A typical determination is de, crib cd below. Procedure. Adjust the pH of 10 ml. of urine to between 8 and 8.5 by adding 0.1 N sodium hydroxide; add 25 mi. of benzene and shake the mixture for 10 minutes. Spin it in a centrifuge and shake 20 mi. of the separated benzene layer with 0.5 ml. of buffered (0.1 % in M / 15 phosphate buffer. pH 7.4) bromothymol blue solution for 5 minutes. Spin the mixture in a centrifuge, then remove 15 mi. of the benzene layer and shake with 4 mI. of 0.1 N sodium hydroxide solution. Take readings for the colored aqueous solution against a blank in a spectrophotometer at 510 mJ,L. In eight experiments, the recovery of 50 J,Lg. of strychnine added to 10 ml. of urine ranged from 89 to 102%. Other compounds which have been investigated by similar techniques include methadone, pethidine, reserpine (123), and amphetamine (81). The colors obtained on the chromatograms with the general spray reagents discussed above can be estimated with a den itometer to give an assay of the alkaloid. Daraway and Tompsett used paper chromatography to separate brucine from strychnine before eluting and determining each eparately by the bromothymol blue-benzene method described above. Excellent recoveries and assays were obtained.

2. Methods for Particular Alkaloids Ultraviolet spectrophotometry is undoubtedly one of the most useful quantitative techniques for those alkaloids that have sufficiently high absorption maxima to bring them within its scope. Accurate control of pH is essential. Mixtures oan also be determined by using measurements over a range of wavelengths. Quinine and strychnine and strychnine and brucine mixtures are typical examples. References in Table VII bring together some of the more recent work on the quantitative determination of particular alkaloids and groups of alkaloids by various methods.

A. S. CURRY TABLE VII Quantitative Determination of Alkaloids Compounds Some alkaloids Alkaloids Local &nestheticB Analgesics Amphetamine Antihistamines Antihistamines Belladonna Tropine alkaloids Benactyzine Brucine Caffeine and theobromine Cocaine Chlorpromazine Chlorpromazine Codeine and morphine Daptazole Ephedrine Ergot Hyoscine and hyoscyamine Methadone Methadone Methorpbinan Morphine Morphine Nalorphine Phenadoxone Pilocarpine Procaine Quinine Quinine and strychnine Reserpine Reserpine Strychnine Strychnine Strychnine and brucine Veratrum

Method used

References

Methylorang reaction 41,62 Chemical reactions, colorimetric 24,120 Ion-exchange and titmtion 76 Reinecke salt 156 Colorimetric and general 103, T04,122b Ultraviolet 8,83 20 Ion exchange 167 Colorimetric 59,73 Colorimetric 72 Ultraviolet 17 Ultraviolet Ultraviolet 106 Ultraviolet 5 48,49,87 Colorimetric 126 Ultraviolet 102 Ultraviolet Reinecke salt 95 31 Colorimetric 56 Review, ultraviolet, etc. 47 Paper chromatograph~' and colorimetric 156 Reinecke salt 51 Colorimetric 156 Reinecke salt 60,121,144,166 Colorimetric 17 Ultraviolet 134 Ultraviolet ]35 Ultraviolet 161 Colorimetric 17,p.479 Ultraviolet 16 Ultraviolet 16 Ultraviolet Electrophoresis and ultraviolet ]25 68,123,168 Colorimetric 15 Ultraviolet 37 Colorimetric 15,17,p.547,45 Ultraviolet 39 Infrared

NITROGENOUS COMPOUNDS

69

VI. SUMMARY This review has attempted to describe some of the techniques used in analyses for alkaloids. The success or failure of these techniques must be shown by experimental results and not by academic argument. Because poisoning by alkaloids, with a few exceptions, is relatively rare, reports of successful analyses are correspondingly small in number: in addition, many toxicologists are hesitant to publish reports of single cases. The two cases described below, therefore, act as a summary for this chapter. Case History. No.1. A 30-year-old woman was found drowned in bel' bath. Because of evidence of vomiting, sweating, and dilated eye pupils, a chemical search for poisoning was undertaken. Insulin wa i olated from slices of buttock tissue taken from around hypodermic needle puncture marks. The husband of the deceased, who was subsequently convicted of the murder of his wife, alleged that the hypodermic mark were from injections of 0.5 mg. of ergometrine maleate that he had given in an attempt at abortion. Because a hypodermic syringe at the scene of the crime contained traces of procaine penicillin, the analysis of pieces of the buttock tissue and the viscera of the decea ed for ergometrine and procaine was undertaken. Becau e ergometrine and procaine both react with the p-dimethylamino benzaldehyde reagent described above, this reagent, combining high sensitivity and specificity, was cho en as the method of detection. Paper chromatography was u ed a the purification method; butanol- citric acid, on buffered paper, was the solvent. The limit of detection for both alkaloids was approximately O.l/.4g. Ultraviolet spectrophotometric measurement, coupled with paper chromatography, were u ed to show quantitatively the stability of ergometrine in heated acetic acid olutions and also to investigate the partition of this alkaloid between aqueous alkaline solutions and immi cible solvents. Ergometrine was found to be relatively insoluble in immiscible solvents. However, if ammonium ulfate was added to the aqueous phase so as to make a aturated olution, and ether was used as the immi cible solvent, the partition ratio could be raised to approximately 3: 1 for ether-aqueous ammoniacal ulfate solution. Further experiment showed that 1 /.4g. of ergometrine maleate added to 200 g. of buttock tissue could be isolated by using the ammoruum ulfate-acetic acid method de cribed above with a reoovery

70

A.S. CURRY

of 40-50% (compare (85)). Procaine recovery was even higher. Further experiments showed that ergometrine could easily be detected in 10 ml. of urine passed by a woman who had been given 11 0.5 mg. injection in the previous few hours. Patients who had received injections of procaine penicillin excreted relatively large amounts of unchanged procaine, easily detectable in a volume as small as 1 ml. of urine. The success of these experiments meant that the negative results for ergometrine and procaine in buttock and urine samples from the deceased were of significance. Case History No.2. An 18-month-old child died five days after the ingestion of 10 mg. of morphine sulfate. Treatment included injections totaling 20 mg. of N-allyl normorphine (lethidrone, nalorphine). These two alkaloids are chemically closely related; their ultraviolet absorption is low, and not di tinctive; in addition, color tests on each are identical (119). Notwith tanding these difficulties and the very long time interval between ingestion and death, positive tests for both alkaloids were obtained on extracts from the intestine contents. The method of isolation was that using ammonium sulfate and hydrochloric acid. Paper chromatography, as described above, separated the two alkaloids, which were detected on the paper by the Marquis reagent (concentrated sulfuric acid and formaldehyde). Both give violet colors with this reagent. 10 J.l.g. of either alkaloid give excellent spots. Acknowledgments I wish to express my thanks to Hui Weng Chee, Department of Chemistry, Kuala. Lumpur, for technical help during the preparation of Tables III, IV, and V, and to Dr. F. G. Tryhorn for his helpful comments and criticisms.

References 1. 2. 3. 4. 5.

Achor, A. B., and E. M. K. Geilling, Anal. Chern., 26, 1061 (1954). Aldin, 0., and J. DUrst, Pharm. Acta Flelv., 81, 457 (1956). Alba, A. R., and K. Ohela., Acta Pharmacol. Toxicol., 11, 156 (1955). AUouf, R., and R. Munier, Bull. soc. chim. biol., 84, 196 (1952). Ampuero, F. M., a.nd M. J. Echea, Rev. fac. farm. y bioquim: 14, 7 (1952); through Chern. Abstr., 47, 9560 (1953).

NITROGENOUS COMPOUNDS

71

6. Bamford, F., Poisons-Their Isolation and Identification, 3rd ed., Churchill, London, 1951, pp. 151--Q2. 7. Bamford, F., Poisons-Th.eir Isolation and Identification, 3rd ed., Churchill, London, 1951, pp.247-70. 8. Banes, D., J. Assoc. Offic. Agr. Chemists, 34, 703 (1951). 9. Barakat, M. Z., N. Wahba, and M. M. EI- adr, AnalY8t, 79, 715 (1954). 10. Barnes, W. H., and H. M. Sheppard, Bull. Narcotic3 U.N. Dept. Social Affairs, 6, No.2, 27 (1954). 11. Beguiristain, J. M. B., Arch. med. exptl. (Madrid), 18, 279 (1955); through Chem. Abstract8, 50, 12153 (1956). 12. Belles, Q. C., and H. W. Sievert, J. Lab. Clin. Med., 46, 628 (1955). 13. Bertetti, J., Arch. chim. Roma, 43,351 (1953). 14. Bettschart, A., and H. Fluck, Pharm. Acta Helv., 31, 260 (1956). 15. Bhattacharya, R. N., and A. K. Ganguly, J. Pharm. and Pharmacol., 4, 485 (1952). ]6. Bhattacharya, R. N., and A. K. Ganguly, J. Pharm. and Pharmacol., 6, 191 (1954). 17. Biggs, A. I., J. Pharm. and Pharmacol. , 4,479,547 (1952). 18. Bjorling, C. 0., and A. Berggren, J. Pharm. and Pharmacol., 5, 169 (1953). 19. Bjorling, C. 0., and A. Berggren, J. Pharm. and Pharmacol., 5, 615 (1953). 20. Blaug, S. M., and L. C. Zopf, J. Am. Pharm. A8soc., 45, 9 (1956). 21. Browning, R. S., S. E. Wiberley, and F. C. Nachod, Anal. Chem., 27, 7 (1955). 22. Buchi, J., X. Perlia, and A. Strebel, Pharm" Acta Helv., 28, 109 (1953). 23. Buchi, J., and M. Soliva, Pharm. Acta Helv., 3D, 195 (1955). 24. Budesinsky, B., Ceskoslov. Farm., 5, 579 (1956) . 25. Burgin, A., J. pharm. Belg., 8, 12 (1953). 26. Capone, A., Boll. chim. farm., 90,465 (1951). 27. Carles, J. E., and H. B. Woodhead, Nature, 168, 203 (1951). 2 . Ca taneda, V., and J. Chiriboga, Bol. soc. quim. Peru, 22, 214 (1956); through Anal. Abstr., 3097 (1957). 29. Caws, A. C., and G. E. Foster, J. Pharm. and Pharmacal., 8, 790 (1956). 30. Caws, A. C., and G. E. Fa ter, J. Pharm. and Pharmacol., 9, 824 (1957). 31. Chatten, L. G., and L. I. Pugsley, J. Am. Pharm. Assoc., 41,108 (1952). 32. Clarke, E. G. C., J. Pharm. and Pharmacol., 8,.202 (1956). 33. Clarke, E. G . C., J. Pharm. and Pharmacol., 9, 1 7 (1957). 34. Clarke, Eo G. C., J. Phar?n. and Pharmacol., 9, 752 (1957). 35. Clarke, E. G. C., and M. Williams, Bull. Narcotic3, U.N. Dept. Social Affairs, 7, Nos. 3, 4, 33 (1955). 36. Clarke, E. G. C., and M. Williams, J. Pharm. and Pharmacal., 7, 255 (1955).

72

A. S. CURRy

37. Cole, E. R, J. Proc. Roy. Soc. N. S. Wales, 81, 276 (1947). 38. Cortesi, R, and H. Laubie, Bull. soc. pharm. Bordeaux, 93, 116 (1955) i through Chem. Abstr., 50, 10337 (1956). 39. Couture, G. P., and R A. Burley, Anal. Chem., £4, 1918 (1952). 40. Curry, A. S., and R . Powell, Nature, 173, 1143 (1954). 41. Daraway, Z. I., and S. L. Tompsett, Analyst, 81, 601 (1956). 42. Daubney, C. G., and L. C. Nickolls, Analyst, 6£, 851 (1937) . 43. Daubney, C. G., and L. C. Nickolls, Analyst, 63, 560 (193 ). 44. Deckers, W., Naturwissen8chajten, 40, 553 (1953). 45. Demoen, P ., and P. Janssen, J. pharm. Belg., 7, 80 (1952) . 46. Dobro, M. S., and S. Kusafuka, J. Criminal Law Criminol., #, 247 (1953). 47. Drey, R. E. A., and G. E. Foster, J. Pharm. and Pharmacol., 6, 839 (1953). 48. Dubost, P., and S. Pascal, Ann. pharm.jranran ion.

v.

CHEMICAL METHOD

Although a great many principles have been brought into UKC for the chemical determination of alcohol, nearly all those methods which have found extensive application involve the oxidation of alcohol and determination of the amount of oxidant required, either volumetrically or colorimetrically. 1. Oximetric Methods

The most frequently used oxidant is dichromate in sulfuric acid, but permanganate and vanadium pentoxide have also been found to be efficient, and others such a;s bromine, iodine pentoxide, and osmic acid have been used. The chief difficulty with these methods i obtaining conditions in which the oxidation proceeds stoichiometricaUy. Many of the methods utilize empirically determined factors for converting the measurements into alcohol concentration. A. CHROMIC ACID METHODS

Chromic acid is probably the first oxidant used for quantitative alcohol determination, as it was employed in 1852 by Cotte (16). Bodlal1der (5) in 1883 developed a method in which a fixed amount of dichromate is titrated with the unknown alcohol solution. Nicloux (55) used a number of dilute standanl alcohol solutions which are

ET.H YL ALCOHOT, TN DLOOD A.NT) TISSUE

223

titrated with chromic acid and used for comparison in the analysis. This procedure was, for many year , the accepted method for alcohol determination in forensic cases, but it is somewhat doubtful whether the oxidation take place toichiometrically under the e conditions in which a 'urplus of oxidant i not present. Moreover, the titration end point is sharp only with relatively concentrated solutions of dichromate and a lcohol. The method, therefore, requires liberal amounts of blood or other sample. Benedict and Norris (3) modified the procedure by allowing the alcohol solution to react with a surplus of dichromate-sulfuric acid. The excess was determined by titration with ferrous ammonium sulfate, while potassium permanganate was used a indicator. A great number of variations of the chromic acid oxidation method were published subsequently, differing in the principle used for estimation of the excess dichromate and in the apparatus used for distillation or diffusion. Casier and Delaunois listed about fifty methods of this kind. Only a few of these will be mentioned here as most of them involve minor technical differences. They may conveniently be treated in two group, those involving distillation as a step in the analysis, and tho, e involving orne kind of diffusion-desiccatioD. Methods in the latter group are used because diQtillation procedures are not easily adapted to routine analysis and are apt to introduce errors due to contamination with material which may react with the oxidant. In special cases, methods in which distillation proces es are used as steps in the analysis may still be useful, but in the e it is advisable to use other than oximetric principles for the estimation proper. Distillation methods. (a) Volumetric Methods. Determination of the excess chromic acid is frequently performed by titration. Hansen (29) in 1925 employed, probably for the first time, addition of KI and titration of the iodine liberated by means of thiosulfate. Tuovinen (80) used a similar method. Newman (51) removed alcohol from 1 mI. of blood or urine in a 50 mI. Erlenmeyer flask, containing a thin layer of anhydrous sodium sulfate, through distillation in vacuo. The distillation is performed at 40-45°C. and the distillate i collected in dichromate-sulfuric acid. The excess dichromate is determined iodometrically. Harger (30) added tartaric acid to tis ue minced with 1 volume of water and performed a steam distillation which was stopped when a

224

FRANK L UNDQUJST

volume corre ponding to the original tissue volume had been collected. For analysis of blood, tung tic acid filtrates are used. An aliquot of the distillate is oxidized by means of dichromate-sulfuric acid, and the exce sis e timated by titration with ferrous sulfate containing methyl orange a indicator. Ferrous sulfate titration was also adopted by Nicloux (54) and by Nicloux, LeBreton, and Dontcheff (56), whose method has been used rather extensively. Blood (0.2-0.5 g.) is distilled with addition of picric acid (6 m!.) through a micro-Vigreux column. 2.5 m!. of distillate is collected and oxidized with dichromate in sulfuric acid. An excess of ferrous ammonium sulfate is added and the remaining ferrou s ion is titrated with dilute potassium permanganate. Nineteen test analy es gave the correct result within ± 2%. Rochat (69) modified this method slightly. (b) Colorimetric and Photometric Methods. The dichromate concentration may also be determined by colorimetric or photometric techniques. Bogen (6) performs a steam distillation in a stream of air which is drawn through a chromic acid solution. The color i directly compared with a number of standard mixtures. Heise (31) use a similar procedure after distillation with a mixture of picric and tartaric acids. Varying amounts of di tillate are boiled with a fixed amount of chromic acid and the color is compared with a number of standards. An accuracy of 0.01 % alcohol is claimed. Newman and Abramson (52) measured the decrease in optical density at 480 mJ.l in a photoelectric colorimeter. The distillation is performed in vacuo at 50-55°C. The optical density is measured after dilution to 25 ml. and appears not to be proportional to the concentration of dichromate. A calibration curve is therefore used. The chromic salt formed in the reaction may also be measured colorimetrically, and this principle is superior because the accuracy of the analysis is independent of the exact amount of dichromate employed. Shupe and Dubowski (77) measure the optical density at 600 m,u after suitable dilution. Distillates of blood and urine treated with a mixture of picric and tartaric acids are used. The optical density, corre ponding to alcohol concentrations below 1 mg. per ml. , is rather small and the accuracy accordingly not very high. Diffusion methods. The chief advantage of the numerous diffusion methods is that the whole analysis can be performed in one apparatus of simple design. This principle was introduoed by

ETHYL ALCOHOL IN BLOOD AND TISSUE

225

Widmark in 1922 (86). The apparatus designed by him is extremely simple and ingenious, as shown in Figure 3 (Section V. 4.A). It consists of an Erlenmeyer flask, originally 50 ml., but the 100 ml. size has proved easier to manipulate. The flask has a ground glass joint (no. 19) and a stopper on which a small glass cup is suspended about 1 cm. from the bottom of the flask, as seen in Figure 3. The sample to be examined is placed in this cup and the volatile substances of the blood are allowed to diffu e from the cup at 60°C. until completely desiccated into a 1 ml. dichromate-sulfuric acid mixture in the botton of the flask. After removal of the stopper, the excess dichromate i determined by iodometric titration. Another advance introduced by Widmark i the use of glass capillary tubes for the transport and storing of blood samples to be analyzed for ethyl alcohol. In this method the mat,erial for analysis is weighed on a torsion balance. This is the first published micro method for alcohol determination requiring only around 100 mg. of blood or other sample. A complete description of the procedure is given in Section VA. Widmark's original paper (86) was criticized by Nicloux (54) by LeBreton (42), and by Nicloux, LeBreton, and Dontcheff (56) because the accuracy wa found to be rather poor. This was due to uncertainty in pipetting the viscous sulfuric acid-dichromate mixture. In the final form, a published in Widmark's monograph (87), the addition of the sulfuric acid-dichromate mixture is made by mean, of an all-glas yringe pipet. This modification is essential and ensures a high degree of accuracy. Many modifications of the Widmark method have been published. orne authors have employed ferrous salts for the titration. Cavett (15) titrated with ferrou sulfate containing methyl orange a originally propo ed by Harger (30). As long a dichromate is pr ent, the indicator is oxidized and colorless. The end point is pink. The accuracy is not stated. Pfeil and Goldbach (64) employed a titration in strongly acid solution with 0.01 N FeSO., and used diphenylamine as indicator. They claim that, when this titration is used, the theoretical factor, 0.115, for conversion of ml. of 0.01 N thiosulfate to mg. of alcohol is found instead of the empirical factor, 0.113, which i constantly ob erved in the iodometric procedure. The accuracy is presumably imilar to that of the original Wid mark method. Other author. have measured the dichromate excess colorimetrically after suitable dilution. In Abels' method (1) 0.2-0.5 mI. of blood is

226

}'RANK LUNDQUIST

absorbed on a roll of filter paper, which is inserted between the neck and the stopper of an ordinary conical fla k containing dichromate and sulfuric acid. Only 90% of the alcohol is recovered. The color i compared with standard solutions. A similar procedure is described by Sheftel (76), who measured the color in a colorimeter. A more indirect method for the colorimetric estimation of the excess dichromate was used by Pfeil and Goldbach (65), who employed the color reaction between chromic acid and s..ruphenylcarbazide, introduced for alcohol determinations by Williams and Darwin-Reese (88). A very strong red color is formed which may be measured accurately at 550 m~ . As the Rensitivity of this reaction is great, the contents of the Widmark fla k have to be diluted to 1000 ml. to obtain a suitable color. This method is undoubtedly superior to the direct colorimetry of either chromic acid or the cbromic salt formed in the reaction, becaURe the optical densities in the latter cases are very low. A number of diffusion method have been worked out with reaction chambers similar to the Conway diffusion unit. The first of thei'e seems to be that of Winnick (89), who u ed conventional Conway units of the standard size. The units are left at 50°C. for 2 bours, to achieve transfer of alcohol from the outer to the inner chamber and oxidation by the dichromatErSulfuric acid mixture. About three times as much material is required as in the Widmark procedure. Titration is performed iodometrically and the accuracy ill probably good. Some authors have constructed elaborate modifications of the diffusion unit for this purpose. Thus McConnell (49) employed a diffusion chamber which can be operated at reduced pres ure. The analysis is performed at 85°C. and the time is reduced to 30 minutes. Titration is performed with 0.2 N thiosulfate. An even more complicated apparatus is described by Scandrett (72), who combined diffusion and distillation, by submerging part of the equipment in boiling water, and keeping the receiver in which the oxidation taker, place at 50°C. by means of circulating water. The analysis takes 30 minutes. The apparatus is apparently effective but unsuited to routine analysis. Newman and Newman (53) increa ed the height of the normal Conway unit so that the center well can hold nearly 10 ml. One ml. of blood is required. Diffusion is facilitated by addition of saturated pota.'3sium carbonate, as recommended by Ryan, Nolan. and Conway (70). The unit is kept at 55°C. for 2 hours or at room temperature for 12 hours. Iodometric titration is employed. The accuracy is very good. standard deviation =0.01 mg. per m!.

ETHYL ALCOHOL IN BLOOD AND TISSUE

227

B. OXIDATION METHODS EMPLOYING OXIDANTS OTHER THAN CHROMICACID

A number of differeut oxidants have been tried, but only those which have proved of some value will be mentioned. Permanganate was found by Friedemann and Ritchie (24) and Friedemann and Klaa (23) to oxidize alcohol under alkaline conditions to oxalate. After acidification, complete oxidation of the oxalate formed to CO2 is brought about. Deproteinized blood is distilled twice, the second time with mercury sulfate and calcium hydroxide. To the distillate iR added an excess of potru: iwn permanganate in approximately 1 N NaOH, and the mixture is kept at 100°C. After 30 minutes, exces, acid is added to the cooled solution and the remaining permanganate is determined iodometrically. The maximal error is given a. ±3%. The oxidation of ethyl alcohol does not proceed quantitatively to CO 2 and water, but the yield is claimed to be very constant. The method of Friedemann and Klaas has been used extensively but requires the utmost care to avoid contamination which may cause erroneous results. Permanganate has also been used for analy es with the diffusion technique. MacLeod (47) used alkaline KMnO. in conven. tional Conway units. Under these conditions permanganate is reduced to manganate. The excess of permanganate is determined by titration with thiourea. The method i quite empirical and not too acourate. Haggard and Greenberg (28) used iodine pentoxide for oxidat.ion in a procedure which utilizes a different principle. The sample is placed in the bottom of a U tube which is connected to another U tube packed with alternate layers of iodine pentoxide and glass wool. The tube containing the sample is heated to 100°C. and a stream of air is passed over the blood and through the tube with iodine pentoxide which is heated to 150-180°C. All the alcohol is driven off within 5 minutes and reacts with the iodine pentoxide with formation of hydrogen iodide and free iodine. which are collected in two wash bottles. the first with water and the second with KI. The reaction is not stoichiometric. The amounts of iodine and hydriodic acid are determined separately by titration with sodium sulfite, and, from the amount of each and their ratio, the alcohol is calculated. The method is rapid, but the accuracy is far from satisfactory, especially in the micro modification in which 0.1 ml. of blood is used. Vidic (83,84) made a most interesting attempt to use vanadic acid instead of chromic acid for the oxidation of alcohol. In this process

228

FRANK LUNDQUIST

the red vanadic acid is reduced to the tetravalent blue vanadyl sulfate. The blue color is determined after suitable dilution in a photometer at 650-700 mp.. Under these conditions the pre ence of vanadic acid does not disturb the measurement. As Beer's law seems to hold, a calibration curve is unnecessary. Analysis is peIformed in ordinary Widmark flasks charged with odium metavanadate di solved in sulfuric acid. A larger amount of blood is used, however, than in the Widmark method. About 0.5 ml. is absorbed on a piece of .filter paper, and placed in the cup. As the reaction does not go to completion, a fixed reaction time has to be cho, en (150 ::I: 5 minutes), and the temperature must be higher than for chromic acid oxidation (85-90°0.) and must be kept very constant. The results seem to be nicely reproducible and this has been confirmed by Pa ulus and Mallach (63). The advantage of the method is that the reduced substance is measured directly. The amount of vanadic acid applied is therefore not critical. The heating time and temperature have to be adhered to strictly, however, and unfortunately the color is not strong and, accordingly, the accuracy is limited. A implified version of the method by which the color i compared directly with a number of . tandard solutions i also described. 2. Other Chemical Methods A. DETERMI!NATlON OF THE OXIDATION PROD CTS OF ETHYL ALCOHOL

Acetaldehyde. Several methods make use of the reactivity of acetaldehyde formed by partial oxidation of ethanol. Henry et ai. (33) distilled the material under alkaline conditions and oxidized with dichromate in approximately 30% sulfuric acid. The aldehyde formed is collected by distillation and allowed to react with p-hydroxydiphenyl in strong sulfuric acid at 30°0. The violet color is mea ured at 560 mp.. The analytical error may be very large. Schmidt and Manz (74) utilized the absorption maximum at 261 m,u of the thiosemicarbazone of acetaldehyde. A modified Oonway difTu ion unit made of perspex, with a dichromate-chromic alt-sulfuric acid mixture as oxidant is employed. Interference from ketones (acetone) is circumvented by employing a blank analysis with sulfuric acid, but without oxidant. Any ketones or aldehydes present in the sample will diffuse into the center well containing thiosemicarbazide. Provided that further oxidation of these substances does not take place in the

Wl'llYL ALC0110L IN BLOOD A L> 'l'lSSUE

229

presence of dichromate, this procedure should furnish a good correction. Acetone was found to be practically without influence on the determination of alcohol. Less than one-tenth of the ethyl alcohol is, however, caught as aldehyde. The accuracy and reproducibility seem, nevertheless, to be satisfactory. The same authors have al 0 published a method (75) which employs the same equipment, but in which the aldehyde is measured by polarography directly in the center well of the modified Conway unit. Thi procedure secure a high degree of specificity. Formaldehyde may thus be distinguished from acetaldehyde, while higher members of the series will be determined as acetaldehyde. Pfeil and Goldbach (66) performed catalytic oxidation of alcohol vapor which is passed through a small glass tube with an electrically heated copper-plated wire. The acetaldehyde is caught in a solution of sodium nitroprussate and morpholine in 0.1 N HCI. The red color produced i measured colorimetrically. Acetic acid. Determination of the acetic acid formed by oxidation of ethyl alcohol with chromic acid has the advantage that the yield is approximately stoichiometri '. Acetic acid is stable in chromic acid under the conditions usually employed. Gettler and Tiber (26) worked out a method especially for alcohol determination in large amount of tissue. The distillate is oxidized with the chromic acid-sulfuric acid mixture and the acetic acid formed is collected by distillation. The distillate is titrated with standard NaOH. The yield of acetic acid is only 85% but is claimed to be constant. Ethyl alcohol may be determined by this method in the presence of methanol because methanol is completely oxidized to CO 2• Beeman (2) modified Gettler's method for use with 10 ml. of blood. B. DETERMINATION OF ETHYL AL OROL AS ETHYLENE

An entirely different principle was adopted by SchifTerli (73) in an attempt to work out a new method for determination of ethanol. Alcohol is catalytically dehydrated to ethylene, which reacts with bromine to form dibromoethane. The excess br:omine is determined, after addition of potassium iodide, by titration with thiosulfate. The dehydration i performed at 300°C. by pa sing the alcohol vapor through a tube with pumice stone treated with pyrophosphoric acid. This catalyst has to be renewed after ten analyses. Schifferli claims that 95-100% of the alcohol i tl'an formed to ethylene by thi treat-

230

FRANK LUNDQUIST

ment. Paulus and Mallach (62), however, found only 75-90% COllversion. M, the method requires a large amount of blood (10 m!.) and the accuracy is small, owing to both systematic and other errorR, this interesting innovation is of little u e for alcohol determination in biological material, at least in its present form. C. METHODS INVOLVING ESTERIFICATION OF ETHYL ALCOHOL

Fischer and Schmidt (20,21) used esterification of ethyl alcohol with nitrous acid to form ethyl nitrite. which is determined iodometrically. Gettler and Tiber (26) tried this method. but were not satisfied with its accuracy. Esterification with hydriodic acid as performed ill the methoxyl determination of Zeisel has been found to give reliable results in the examination of putrefied material (Bonnich en et al. (8». The apparatus of Stritar (78) is convenient for this analysis, but a large sample of material is required. Several micro modifications of the Zeizel method have been published. such as that of Nicolai (57), but even here 5 m!. of blood is used. Niederl and Whitman (58) used an apparatus imilar to Pregl's micro-Zeisel equipment. Alcohol is transferred to the Zeisel Bask from aqueous solution by bubbling CO 2 through it at 100°C. Though biological material has not been examined directly, a calculation shows that about 1 m!. of blood should be sufficient for an analysis. About 0.03 mg. of alcohol is constantly lost in the analytical proce s. In all Zeisel methods, the material' (distillates from blood or tissue) is boiled cautiously in a stream of carbon dioxide with about 3 volumes of concentrated hydriodic acid (1.9 p. gr.). The ethyl iodide which distils off is freed from traces of iodine and hydrogen iodide by bubbling through a suspension of red pho phorus in water at 60°C. The vapor is caught in a solution of silver nitrate in 90% ethyl alcohol. After heating with nitric acid and water, the silver iodide formed is measured by weighing, or the excess of silver nitrate is determined volumetrically. Treatment with concentrated hydriodic acid will give rise to formation of volatile iodidetl from other alcohols such as glycerol, which forms isopropyl iodide (Zeisel and Fanto (92», When purified distillates, as described in Section III, are used, the method should be specific for volatile alcohols and alkoxy compounds. Both methyl

ETHYL ALCOHOL IN BLOOD AND TISSUE

231

alcohol and ethyl ether will thus be sources of error in this determination. The method is not suitable for routine analysis, but may be a valuable supplement to the enzymic method in pecial ca es. The iodoform reaction has also been employed for quantitative e timation of ethyl alcohol (59), but it is not specific.

3. Specificity and Accuracy of Chemical Methods for Determination of Ethyl Alcohol All the oximetric methods have in common a serious lack of pecificity, a all oxidants used so far are also able to react with volatile substances other than ethyl alcohol. It is therefore important to consider to what extent such substances may occur in the material for analysis. In the case of blood, urine, or other samples from living persons, it is of course absolutely necessary for medico-legal purpORes that the figure arrived at expresses the concentration of alcohol within certain limits of accuracy. Therefore, we may briefly consider the sources of error and how serious they are. The substance which has most often been suspected of interference in alcohol determinations is acetone. It has been observed that the oxidation of thi sub tance by chromic acid is dependent on temperature. At 80°0. the reduction equals that cau ed by the same weight of alcohol, whereas at 50°C. acetone does not seem to be attacked at all (64). If, therefore, chromic acid is used at a suitable temperature, as in most of the diffusion methods, acetone will not be a source of error, even at high concentrations. Bjerver et al. (4) examined the Widmark method 'with regard to the influence of ketonemia in 583 diabetic ubjects including many with ketonuria. No interference wa ob erved; in all cases the reduction measured a alcohol was less than 0.1 mg. per g. of blood. Paulus (60) found that by Widmark analysis aqueous olutions of acetone gave ri 'e to a reduction expl'e.'sed as ethanol, corresponding to one-tenth its concentration. Only in cases of diabetic coma (Paulus and Mallach (61)) will serious errors arise from this somce. The presence in blood of other volatile reducing substances may occur during anesthesia; ethyl ether e pecially will give ris to false reaction. In ether anesthesia, a u ed in smgical operations, a reduction cOl'l'esponding to nearly 1 mg. of alcohol per m!. of blood is frequently observed. Other commonly us dane thetics, e pecially the chlorinated hydrocarbons, interfere much Ie s.

232

FRANK LUNDQUIST

Claim that the breathing of vapors from various industrial solvents may cau e measurable interference with Widmu,l'k analy e eem to be unwarranted (19). The pre ence of methanol in the blood will cau 'e reduction in the oximetric methods, but several independent possibilities for detecting methanol poi oning exi t. If a sui tab I margin is allowed to take into account the po. sibilities mentioned above, any of the diffu ion methods employing chromic acid at a reasonably low temperature (60°C. or less) would 'eem to be suitable for forensic determination of alcohol in blood and urine from living person. All the methods involving other oxidant suffer from the uncertainty that the oxidation is not stoichiometric. The degree of re.'l.ction will depend on conditions uch as time, temperature, and acidity. The necessity to keep these parameters ab olutely constant make the methods Ie s valuable for routine ul:ie. The same is true of all the methods based on the determination of acetaldehyde formed through partial oxidation of ethyl alcohol. These method, on the other hand, have the advantage of greater specificity. For alcohol determination in material which may contain a number of unknown substances such a ' putrefied tissue or blo d, di tillation with mercury ~alts in acid and alkaline solution, a discus, ed in Section III. 1, hould precede the analysis proper. For a purely chemical determination of alcohol in such a ea e the Zei. el analysi , combined with te ts to ensure the ab ence of methanol and ethyl ether, will probably be the safest choice. If in rare case the pre ence of propanol or butanol is suspected, the acids formed on oxidation with chromic acid may be identified by determination of the partition coefficient between ethyl ether and water (Friedemann (22)), or gas chromatograpny may be employed (Wolthers (90)). 4. Widmark Method for Large-Scale Determinations of Ethyl Alcohol in Blood and Urine

The Widmark method offers the following advantages: (1) It is a micro method requiring only about 100 mg. of material fo]' one analysis. A ufficient amount of blood for triplicate analysis may be obtained easily from an ear lobe or finger tip, an evident advantage in foren 'ic ca. es. (2) The apparatus i simple, easily cleaned, and well

ETHYL ALCOHOl, I

BLOOD ANn TISSUE

233

suited for routine analyses on a large scale. (3) The method is very rapid. One technician can perform about ]00 analy es within it workiug day, including the cleanillg of glassware. (-1) The accuracy i. sati. factory compa/'ed wjth all other method" even those that use 1 m!. of blood. Range of concentrations measured. With an amount of material of 100 mg., the presence of 0.1 ;mg. of alcohol per g. can be detected with certainty, and concentrations up to 5 mg. per g. can be measured. In order to obtain completely reliable results, at least 10% of the chromic acid should remain after oxidation. A. APPARATUS

Galvanized steel wire trays. Such trays are conveniently made to hold thirty to fifty fia ks; larger trays are too heavy to handle when loaded (see Fig. 1). Water baths. An electrically heated water bath with thermostat control kept at 60° ± 1°C. is placed under a good hood. An ordinary

Fig. 1. Steel wire tray with Widmark flasks. orne of the flasks are furnished with brass rings to weigh them down in the wa.ter bath.

234:

FRANK L UNT>QUIST

air thermostat may be u ed, in which case the fla 'ks are allowed to st.and 20 minutes longer because temperature equilihration is slower. Krogh syringe pipets. The pipet, i. ' adju:,;if-d t,u approxim,ately 1 m!. The tip is bellt., w ' :,;howll iJlInk, E., 126 {reI. 6 ,69), 13 (ref. 70), 1 ~O K.leschick, A., 129, 130 (ref. 124), 142 Kline, L., 125, 140 Knedel, M., 32 (ref. 23), 38 Knessel, 0., 12 (ref. 7), 37 Koga, S., 102, 108

320

AUTHOR INDEX

Kohn, J., 5, 38 Kok, B., 78 (ref. 38), 108 Kolthoff, I. M., 2 1 (rd. 71a), 307 Koppe, J. 1,.,65 (ref. 5), 70 (n·r. R5), 73

Kom, K D., 145-192 Kornberg, A., 242, 249 Korngold, L., 150 (ref. 104), 189 Kpstir, J. V., 194 (ref•. 42, 43), 196, 21J,. Kbzelka, F. L., 220, 249 Kratzing, C. C., 212 (ref. 44), 214 Kream, J., 132, 140 Kreb , H. A., 193,214 Kretcmnar, A. L., 126,140 Krippahl, G., 105 (refs. 71, 72), 109 Krise, G. 1., 196 (ref. 3), £lS Kruyt, H. R, 7 (ref. 26), S8 Kubelka, P., 93, 108 Kuby, S. A., 194 (ref. 46), 21J,. Rudrnac, ., 51 (ref. 69), 73 Kuehl, F. A., Jr., 126 (ref. 73), 140 Kuhn, R, 159 (ref. 47), 187 Ruo, P. T., 154, 15 , 189 Kuppenheim, H. F., 78 (refs. 11, 31, 32, 34), 96, J07, J08 Kurozumi, T., 100 (ref. 63), 103 (ref. 63), 108

Kusafuka, ., 49 (ref. 46), 72 Kyker, G. C., 126, 140 L Lalla, O. F. de, 149, ]72 (ref. 41), 185, 187 Lambert, M., 179, 189 Langley, W. D., 195, 211,. Lardy, H. A., 194 (ref. 46), £14 Larsen, V., 64 (ref. 86), 7S Lasser, R. P., 159 (ref. 119), 190 Latimer, P. H., 103, 108 Laubie, H., 64 (ref. 38), 72 Lauenstein, K., 138 (ref. 70), 140

Laurell, C. B., 150 (ref. ]07), 151, 154 (I' f. 10 ), 189 Luuson, H. D., 195, fBi4 Lawry, K Y, 153 (ref. 29), 154 (ref. 29),156 (ref. 20) , 1 '7 Leu, C. H., 114 (ref. 75), 118 (ref. 114), 125 (r('L. 59, 76), 129, 130 (refs. 77, 114), 131, 132 (ref. 74), 136,140,1J,.1 Leach, H., 6 (ref. 7), 7S LeBaron, F. N., 113, 116, 117, 118 (ref. 44),122 (ref. 44), ]24 (ref. 43), 139,11,.0

LeBreton, E.,]4 (ref. 30), 187, 220, 224, 225, 249, 250 Lederer, K, 53, 7S, 116 (ref. 147), 124 (ref. 79), 132 (ref. 79), 140, 143 Lederer, M., 53, 7S, 124 (ref. 79), 132 (ref. 79), 140 LeDizet, L., 115 (ref. 39), lS9 Lees, M., 113, 124 (refs. 43, 45), IS9 Legallais, V., 79 (ref. 73), 109 Leitner, J. G., 297 (ref. 72), 807 Lens, J., 270 (ref. 53), 307 Lequire, V. S., 162 (ref. 194), 19£ Lerner, B., 132 (ref. 113), 141 Lever, W. F., 153 (ref. 112, 113), 15 ,188,189 Levi, L., 51, 62 (refs. 52, 53, 93,116), 64 (refs. 89- 91), 66, 72- 74, 159 (refs. 114, 149), 189, 191 Levine, C., 132 (refs. 19, 80), 135, 138, 140,298 (ref. 21), 306 Levine, H., 21 ,249 Levy, M ., 122 (ref. 34), 139 Levy, S. W., 155 (ref. 184), 167 (refs. 115, 116), 189, 192, 264 (r>£. (4), S07 Lewis, P. C., 103, 108 Lieb, H., 195, 215 Lillie, RD., 298 (ref. 73), S07 Linden, G., 284, 307 Lindgren, F. T., 149 (refs. 91, 11 ),

.A U'£HOIt INDEX

150 (ref. 118), 155, 164 (ref. 134a), 166, 169 (ref. 11 ),189, 190 Lipari, R., 150 (ref. 104), 189 Livingston, R., 97 (ref. 53), 108 Lloyd, C ..J., 45 (ref. 61), 72 Loew, E. R., 220, 250 Loewe, L., 159, 190 Long, C., 127 (ref. 81), 140 Long, D. A., 278, 307 Loomis, T. A., 160 (ref. 4),188 Lothian, G. F ., 103, 10 Lovern, J. A., 114, 116, 11 (ref. 83), 123 (ref. 3), 127, 129, 132, 136, 13~141

Lowry, O. H. , 12 (ref. 115),14.1 Luck, J. M., 130, 141 Ludovici, B. F., 9 (ref. 23) , 107 Luis, P., 64 (ref. 94) , 73 LundegArdh, H. , 79, S9, 97, 108 Lundgren, G., 240, 242, 248 Lundgren, P. , 6 (ref. 95) , 73 Lundquist, F., 217-251 Lynch, V. H., 90, 97, 108, 109 Lyon, T. P., ]49 (ref. 91), 153 (ref. 59) , ]59 (ref. 59), ]60 (I'd. 59),166 (ref. 59), 188, 189 Lyon, M. E., 15 (refs. 73, 110; J 11), 188 M

McBay, A. J., 6 (refs. 102, 161), 74, 75 McCamau , R. E., 12 (ref. 115),141 McConnell, W . B., 226, 250 McCormick, M. H., 122 (ref. 92), 141 McCoubrey, A., 6 (ref. 103), 74 McCurdy, D . H., 53 (ref. 145),75 MacDonald, J., 49 (I' f. 5 ), 62 (r f. 57), 72 Macek, K, 51 (ref. 96), 61, 62 (I' f. 96), 73 MacFadyen, D. A., 126 (ref. 143), 127 (ref. 144),142, 180,190

321

McGuire, T. A., 116, 123 (ref. 121), 142 Machebo W, M., 49 (ref. 111), 50 (ref. 112), 58 (ref. 111), 74 McIlwain, H., 212 (ref. 52), 215 McIndo , W. M., 119 (ref. 63), 140 MacIntosh, F. C., 291 (ref. 74), 307 McKeehan, C. W., 78 (ref. 11), 107 McKe han, W., 78 (refs. 31, 34), 108 Mackenzie, C. G., 112, 127, 139 McKibbin, J. M., 111-143, 148 (ref. 177), 192 MacLean, H., 112 (ref. 86), 141 Macleod, D. P., 53 (ref. 145), 75 Macleod, L. D., 227, 2150 Mcleod, N., 11 , 139 McNabb, A. R., 128,140 Mc rally, W. D ., 6 (ref. 104), 74 McVeigh. I., 118, 138 Mader, W. J., 6 (ref. 134), 75 Malta. anik, B., 117 (ref. 87), 141 Magid, E. B ., 160 (rd. 49),187 Maguire, M. F., 127 (ref. 1), 140 Majanen, ., 159 (I' ·f. 140), 190, 304 (ref. 4), 30 Mujor, R. H. , 210 (ref. 49), 214 Mula.ngeau, P., 116, ]19 (ref. 9), 139, 141 Malkin, T., 125 (ref. 90), 141 Mallach, H. J., 228, 230, 23], 250 Mallov, ., 112 (rei. 91) , 113 (ref. 91), 141 Man, E. B., 158 (ref. 1 ), 192 Mandel, E. E., 194 (ref. 50), 214 Mann, F. D., 159 (refs. 14·, 1 6),186, 192 Mann, G. V., 153 (ref. 29), 154 (ref. 29), 156 (ref. 29), 187 Mann, T., 132, 141 lanucring, G. L., 50, 73 Manning, J. B., 29 (ref. 53a), 307 Manning, J . M ., 62 (rei. 9 ), 73 Malllllueusel, L., 154 (ref. 197), 192

322

A U'l'HOR INDEX

Manz, R., 22 , 229 (ref. 75), 250 Marbet, R., 255 (ref. 75), 256 (ref. 75), 2 1 (ref. 10 ), 291 (ref. 76), 305, 307, 309 Mariani, A., 52 (ref. 99), 73 Mariani-Bettelo, G. B., 52 (ref. 100), 73 Marks, H. P., 290, 305 Marshall, E. K, Jr., 248, 250 Martin, A. E., 9 , 108 Martin, A. J. P., 298 (ref. 26) , 306 Martin, G. J., 53 (ref. 141),75 Martin, R. P., 194 (r·f. 72), 196 (ref. 72), 197 (ref. 72), 206 (ref. 72), 207 (ref. 72), 210 (ref. 72), 213 (ref. 72),215 Marx, L., 267, 271, 308 Marx, W., 267, 271, 306, S08 Mathis, C., 64 (ref. 101) , 74Matsumoto, M., 138 (ref. 155) , 14-3 Maurukas, J., 124 (ref. 7),138 Maw, G. A., 194 (ref. 51), 214Mayer, M. M., 17 Mead, J. F., 183, 187, 190 Meath, J. A., 113, 124 (ref. 43), 139 Meech, L. A., 127,139 Mehltretter, C. L., 53 (ref. 105), 74 Meisinger, M. A. P., 126 (ref. 73), 140 Meivilll', R. ., 206 (ref. 53), 215 Mendell, B., 167 (ref. 133), 190 Mende, C. B., 119 (ref. 63) , 14-0 Meneghini, C. L., 159 (refs. 114, 14.9), 189, 191 Meng, H. C., 156 (ref. 124), 160 (ref. 122), 162 (refs. 66, 78, 123), 167 (refs. 79, 81), 169 (ref. 80), 175, 188,190 Merrill, E. J., 68 (r f. 125), 74Mer ereau, W. A., Jr., 297 (ref. 78), 308 Messinger, W. J., 153 (ref. 126), ]56 (ref. 125), 190

Meyer, H., 131, 141 Middleton, E., 150 (ref. 127) ,190 Milch, L. J., 160 (ref. 128), 190 Miles, J. W., 68 (ref. 106), 74Miller, B. F., 195, 196,215 Miller, H. 1., 156 (ref. 1 0),1 92 Miller, Z., 196, 215 Milner, M., 90, 108 Mininni, G., 160 (ref. 5), 186 Mink, C., 124 (ref. 61) , 126 (ref. 61), 140

Mitchell, H. K., 124 (ref. 150), 125, 143 Mitchell, T. J., 298 (ref. 19), 306 Moeller, H. C., 154 (ref. 61), 156 (d. 62), 157 (ref. 129), 174 (ref. 61), 188, 190 Ms6lier, K. 0., 230 (ref. ), 248 Moerl08se, P. de, 51 (ref. 107) , 74Molho, D., 263, 308 Molho-Lacroix, L., 263, 308 Molle, L. , 52, 74 MommafTts, W. F. H. M., 212 (ref. 56),215 Monk, G. W., 100, 105, 107, 108 Monkhou8C, F. C., 267, 270, 273, 274, 307, 308 Moo're, P., 50 (ref. 109), 62 (r f 109),

74 Moore, S., 131, 141 Mora, R., 154 (ref. 130), 190 Mora, T. P., 29 (ref. 89), 308 Morais, E. de C. F., 49 (ref. 110), 74Morrison, J . F., 206 (ref. 61), 207 (ref. 61), 209 (ref. 61), 215 Moses, C., ]54, 156 (ref. 131), 160 (ref. 132), 190 Moubasher, R, 126, 141 Mould, D. L., 263 (ref. 82), 308 Moya, F., 53 (ref. 145), 75 Moyer, D., 118, 138 Munier, R., 49 (refs. 4, 111), 50 (ref. 112), 58 (ref. 111), 70, 74-

AUTHOR INDEX

Mushctt, C. W., 117 (ref. 54),118 (ref. 54), 140 Mussett, M. V., 282 (rd. 3), 308 Myers, D. K., ]67 (ref. 133), 190 Myers, V. C., 2]0 (refs. 4-6, 57), 213, 215 N Nachod, F. C., 64 (ref. 21), 71 Nadeau, G., 49 (ref. ] 13),74 Napke, E., 264 (ref. 64), 307 Narayanaswami, A., 212 (ref. 44),

e11,. Nason, H., 148 (ref. 134) , 190 Negelein, E., 240, 250 Neish, A. C., 179, 189 Neuhoff, E. W., 64 (ref. 11 4),74 Neuss, J. D., 6 (ref. 134), 75 Newman, E. J., 226, 250 Newman, H. W., 220, 223, 224, 226, 250 Nichols, A. V., 14.9 (refs. 91,11 ), ]50 (r f. 118), ] 55 (ref. 118), 164 (J'('fs. 1340.,175), ]66 (ref. 175),169 (ref. 118),189,190,192 Nickolls, L. C., 43, 44, 49 (ref. 115), 7e,74 NicloLLx, M., 220, 222, 224, 225, 250 Nicolai, H. W., 230, 250 Niederl, J. B., 221, 249 Niemi, T., 163, 190 Nihei, T., 102 (ref. 69), 109 Nikkila, E. A., ] 53, 154 (ref. 137), 159 (refs. 138, 140), 160 (ref. 137), 163, 164 (ref. 139), 169, 170, 190, 304 (ref. 84), 308 Nilsson, I. M., 269 (ref. 97), 272, 275, 308 Nixon, D. A., 118 (ref. ]7),138 Nobs, M. A., 91 (ref. 67), 109 Noda, L., 194 (ref. 46),214 Nojima, S., 128, 135, 137 (ref. ]00), 141

323

Nolan, J ., 226, 250 NorriR, F. W., 116, 11 8,139, 141 Norris, R. S., 223, 248 Northam, B. E., 11 ,141 Norton, S., 21 (ref. 50), 38 Nowinski, W. W., 4 (ref. 15),37 Nygaard, A. P., 241. (ref. 79). 250

o Oaklry, C. L., 2 (ref. 27), 38 O'Brien, P. F., 96, 108 Lisu.'rlind, S., 231 (ref. 59), £50 Oestreicher, P. M., 62 (refs. 53, ] 16), 72,74 Ohela, K., 64 (ref. 3), 70 'Keeffe , A. E ., 263 (ref. 87), 308 Olley, J., 114, 116, 117, 118 (ref. 83), ] 23 (ref. 83), 127, 129, 130 (ref. 103),132,136, 137,139-141 Oneley, J. L., 14 (refs. 142,143), 149 (ref. 144), 150 (ref. 144), 190 Opdyke, D. F., 160 (ref. ]45), 191 Opfer-Schaum, R., 64 (ref. 117), 74 Ormond, R. E ., ]26 (ref. 73), 1/,.0 Oser, B. L., 194 (r('f. 35), £14 Osol, A., G4 (refs. 3, 11 ), 68 (ref. 83), 73, 74 Ott, W. H., 160 (ref. 145), 191 Ouchterlony, 0., 28 (ref. 2 ),29,38 Oudin, J., 8 (ref. 29) , 2 (ref. 29) , 38 Overbeck, G. A., 167 (ref. 146) ,191 Oyama, V. 1., 122 (ref. 34), 139

o

p Page, I. R ., 112 (refs. 105, 145), 125, 140-14e, 167 (ref. 16 ),191 Pttllansch, M. J., 163 (ref. 150), 191 Palm, L., 154 (ref. 61), 156 (ref. 62), 157 (ref. 129), 166 (ref. 63), 174 (ref. 61), 188, 190 Palma, E. T. M., 49 (ref. 110),74

324

AUTHOR INDEX

Parker, K. P., 217 Partridge, S. M., 295 (ref. ), 298 (ref. 88), 308 Pascal, S., 6 (refs. 4 ,49), 72 Pascu, E., 298 (ref. 9), 308 Pasternack, L., 112 (ref. 105), 141 Pasternak, C. A., 298 (r f. 90),308 Paulus, W., 228, 230, 231, f50 Pavlov, P. N., 7 (ref. 30), 38 Payza, A. N., 16 (ref. 10], 147), 189, 191 Peaud-Lenoel, Cl., 7 Pedley, E., 64 (ref. 119),70 (ref. 119), 74 Perlia, X., 64 (ref. 22), 71 Perry, W. L. M., 278, 282 (ref. 83), 307,308

Peters, J. A., 156 (ref. 37), 157 (ref. 37),159 (ref. 37), 160 (ref. 40), 187 Peters, J. P., 158 (ref. 1 8), 192 Peters, R A., 27 Pfeil, E ., 225, 226, 229, 231 (ref. 64), £50

Pfiffner, J. J., 210 (ref. 57), £15 Phillips, J. D., 263 (ref. 91), 308 Phillips, J. I., 6 (ref. 72), 73 Phillips, R A., 195, 215 Phokas, G., 51 (ref. ]37), 75 Pierce, F. T., 160 (ref. 75), 168 (ref. 14 ), 188, 191 Pilgeram, L . 0., 124 (ref. 106), 141 Platt, B. S., 116, 141 Poethke, W., 68 (ref. 120), 74Pollard, A., 263 (ref. 91), 308 Pollard, A. L., 295 (ref. 3]), S06 Polli, J. F., 68 (ref. 104), 74 Polonovski, J., 124 (ref. 108), 141, 154 (ref. 130) , 190 Poole, A. G., 125 (ref. 90), 141 Poole, J. C. F., 162 (ref. 160), 163 (ref. 157), 191 Pope, O. B., 50 (ref. 97), 73

Porosowska, Y., 153 (ref. 126), 156 (ref. 125), 190 Porter, R R, 263 (ref. 92), S08 Posternak, T., 115 (ref. 109), ] 19 (ref. 110),141 Posthuma, R, 271 (ref. 42), S06 Potee, K. G., 153 (I' f. 29), 154 (ref. 29), 156 (ref. 29), 187 Powell, H., 50, 72 Pozzo, G., 159 (refs. 114,149). 189, 191 Prati, G., 159 (ref. 149),191 Price, T. D., 100, 108 Pride, R R A., 6 (ref. 121), 74 Pugsley, L. I., 68 (ref. 31), 71 Q Quick, A. J., 275 (ref. 93), S08 Quigley, T. W., 163 (refs. 102, 150), 164 (ref. 103), 165 (ref. 102), 166 (refs. 102, 103), 170 (refs. ]02, 103),189,191 Quivy, D., 2 1, 302, 305, 308

R Raaflaub, J., 196 (ref. 59),215 Rabaey, M., 27, 38 Rabek, V., 194 (r f. 42), £14Rabideau, G. .,78 (ref. 52), 105,108 Rabinowitch, E. I., 97, 107, 108 Racker, E ., 240, 241, 244, 250 Radin, N. S., 112 Rall, T. W., 160 (ref. 11), 186 Ramsay, W. N. M., 126, 141 Randolph, M. L., 171, 191 Rapport, M. M., 114 (ref. 112), 132 (ref. 113), 141 Rastgeldi, ., 269 (ref. 45), 306 Rathenasinkam, E., 64 (ref. 122), 68 (ref. 122), 74 Ratner, S., 194 (ref. 60), £15 Ray, R R, 172 (ref. 151), 191 Rebeyrotte, P., 154 (ref. 130), 190 Redetzki, H., 242, 243 (refs. 18, 6R), 248, £48-£50

3:25

AU'J'HOR INDEX

Redfield, H.., ISO (ref. 7), 186 Redmond , R F.: 160 (ref. 12 ),190 R eichelt, .J., 51 (ref. 123), 67 (d. 123) , 68 (ref. 123), 74 R eichenthal, J ., 12 , 129, 135, 138 Reicbl, D., ]75 (ref. 50), 187 R einert, M. , 2 6, 308 R eines, Z., 1.59 (ref. 152), 160 (ref. 152) , 191 R einhold, J . C., 154 (ref. 105), 158 (ref. 105), 189 R entz, J ., 68 (ref. 76), 73 Renwick, 93, 107 Re plandy, A., 51 (ref. 124),74 R euth('r, F. W., 49 (ref. 147), 5 , 75 Rhodes, D. K., 114 (ref. 75), 11 (ref. 114), 125 (ref. 76) , 129, ]30 (refr-. ii , 11 4), 131, 136, 140 141 Rhod('R, G. L. , 160 (ref. 13Z) , 190 Rieh, C., 273, 306 Richarz, G., 116, 158 Riclm10nd G., 159 (ref. 119),190 Rieker, A. f.,25 - 260 (ref. 61), 293, 291, 301 (r f. 65), 307 Rickett , . R , ]56 (r f. 15 ),191 Ritchie, E . H., 220, 227 , 249 Robb, J. ':'" 112 (ref. 91 ), 113 (ref. 9] ) ,11,.J Robins, E., 12 , 141 Robin ou, D. ., 147 (ref. 156) , 15 1 (ref. ] 53), 156 (ref. 15 ), 162, 163 (ref. 157), 1M (ref. 155) , 165 (ref. 155),166, 187, 191 Robinson , F. A. , 11 , 139 Rochat, J., 220, 224, 250 Rochovansky, 0 ., 194 (ref. 60), 215 Rodb il, M., IN> (r f. 161), 151 (ref. 162), 191 Roclnight, R., 118 (ref. 116), 142 Roe, C. E., 163 (ref. 150),191 Roland, P., 50 (ref. 14 ), 75 Rose, H . F., 101, 108

Roseman, ., 263 (ref. 96) , 308 Ros n, H. , 131,142 Rosenberg, A., 160 (ref. ]45), 191 Rosenberg, H., 206 (ref. 61), 207 (ref. 61), 209 (ref. 61) , 212 (ref. 23), 214, 215 Rosenberg, 1. N., 154 (ref. 163), 191 Rosenman , R H ., 15 (ref. 164) , 160 (refs. 165, 166) , 191 Rossiter, R J., ] 2 , 140 Ro s-Robertson, G., 7 (ref. 31), 38 Rothleder, E. E., 117, 140 Rubin, L., 164 (ref. ]34a) , 190 Rucker, P., 271. (r f. 77) , 308 Ruegg r, A. , 51 (ref. 139), 75 Ruggeri, L., 271 (ref. 77) , 308 Russell, J. A., J32, 137 (ref. 11 ),139,

142 o-Alesi, F. M., 263 (ref. 87) , 308 R yan, M. T., 226, 250 R yan, R R, 171,191

RUB

S Saar, H ., 248, 250 Sacks, J., 115, 142 Sakaguchi, ., 206, 207, 20 (I' f. 63) , 215 Salmon, W. D., 112 Salvani, L. , 160 (ref. 5), 186 alve ~ n , E., 64 (ref. 164), 76 Salvinicu, J., 30 (ref. 32), 38 alzman, . P. , 68 (ref. 126) , 74 SanD, I., 52, 74 aunder , L., 53 (ref. 128) , 74 avary, P., ] 3, 191 candrett, F. J., 226, 250 canu, A. , 167 -(ref. 16 ),191 chaffer, P . .,52 (ref. 133) , ?'5 cheidegger, J. J. , 3, 24 (ref. 45), 2A (refs., 3, 34-), 27, 37, 38 'h 'nck, R, 154 (ref. 197), 192 Schiffel'li, E., 229, 250

326

AUTHOR INDEX

Schindler, R., 51 (ref. 129), 74 chleyer, F., 218, 232 (ref. 19), 21,9 Schmall, M., 50, 74 chmidt, A., 230, 21,9 chmidt, 0., 228, 229 (ref. 75) , 250 Schmitz, A., 256 (ref. 36), 2 6, 292, 306 chneider, H., 125 (ref. 46), 139 ch¢nheyder, F., 286, 308 cholfield, C. R., 116, 118 (ref. 119), 123 (ref. 121), 127 (refs. 119, 120), 135, 1~ Schopfer, W. H., 115 (ref. 122), 119 (ref. 110), 141 , 142 Schotte, A., 167 (ref. 133), 190 Schotz, M. C., 167 (ref. 168), 191 chrade, W., 124 (ref. 123), 142 Schroeder, W., 105 (ref. 72) , 109 Schultz, O. E., 49 (ref. 132), 62 (ref. 131),74,75 Schultze, H. E., 26 (ref. 35), 36 (ref. 35), 38 chumaker, V. N., 160 (ref. 169), 191 Schwarting, A. E., 51 (ref. 150), 75 Schwartz, C. J., 156 (ref. 37), 157 (refs. 37- 39), 159 (ref. 37), 160 (ref. 40), 187 chwartz, L. R., 164 (ref. 170), 191 Schwarz, H. P., 129, 130 (ref. 124) , 142 Schwert, G. W., Jr., 98 (ref. 25), 107 cott, D. A., 254, 255, 264 (ref. 24), 267, 282, 283, 299 (ref. 22) , 308 cott, J. E., 269 (ref. 97), 308 Scott, W. E., 52 (ref. 133), 75 Scrivasta, R., 53 (ref. 128), 74 Seagers, N. J., 68 (ref. 134), 75 Sebesta, K, ]2 (ref. 7),37 Sebrell, W. H ., 115 (ref. 125), 142 Seifter, J., 162, 165 (ref. 172), 191 Sekal, E. H., 68 (ref. 125), 74 Seligmann, M., 5 (ref. 36), 38 Seljeskog, E., 194 (ref. 73),215

Semydarian, M. W., 272 (ref. 17), S08 eybold, A., 78 (ref. 55), 95 (ref. 55), 108 hafer, E. G. E., 50 (ref. 130),74 Shaw, W. H. C., 68 (ref. 135), 75 Sheftel, A. G., 226, 250 h pherd, D. M., 44 (ref. 136), 75 Sheppard, H. M., 62 (ref. 10), 71 Shibata, K, 77- 109 Shore, B., 150 (ref. 173), 164, 166 (refs. 174, 175), 192 hore, P. A., 68 (ref. 68), 73 horland, F . B., 114 (ref. 126), 142 • hupe, L . M., 224, 250 Sideri, C. N., 64 (ref. 11 ),74 iegel, H., 160 (ref. 145) , 191 ievert, H. W., 62 (ref. 12), 71 Silber, R. , 160 (r f. 145) , 191 imonton, J., 153 (ref. 59), ]59 (ref. 59),160 (ref. 59),166 (ref. 59), 188 Simpson, W. F., 272 (ref. 17),306 Sims, E. A. H., 206 (ref. (4), 215 ina, A., 126, 141 Sinclair, R. G., 113, 142 Sing her, H . 0., 164 (ref. 19 ),192 istrom, W. R., 90 (ref. 27),107 Slater, R. J. , 4, 38 linker, B. J., 158 (ref. 15), 186 Sloane tanl y, G. H., 113, 118 (ref. 128), 124 (r f. 45), 139, 142 t'imith, E. B., 154 (ref. 35), 187 mith, F., 263 (ref. 9 ), 308 mith, J. H. C., 91 (refs. 64- 67), 108, 109 mith, P . A. J., 153 (refs. 112, 113), 158 (refs. 112, 113), 189 Smith, R. H., 1 L3, 114, 118 (refs. 129, 130), 123, 142 mith, ., 45 (ref. 138), 75 Snell, E. E., 118, 142 uelbnan, 0., 270 (ref. 99), 308 SoliVl~, M., 50, 53, 71

327

AUTHOR INDEX

Solomon, B., 160 (ref. 166) , 191 Sonb, J., 194 (ref. 43) , 196,214 Sonoda, T. T., 125,140 Sordi, A., 159 (ref. 6), 160 (ref. 6), 186 Sorm, F., 12 (ref. 7), 37 Sperry, W. M., 112, 172 (ref. 176), 192 Spiro, M. J., 123 (refs. 133, 134), 133, 136, 137 (ref. 134), 138 (r f. 134), 142, 148 (ref. 177), 192 Spitzer, J. A., 161 (ref. 181), 192 Spitzer, J . J., 156 (ref. 180), 157 (ref. 178),161 (r f. 181), 162,192 Stadler, J., 166 (ref. 63), 188 Stanier, R. Y., 90 (ref. 27) , 107 Stauffer, J. F., 212 (r f. 67) , 215 tein, W. H., 131, 141 teinberg, D., 150 (ref. 7), 186 Steinegger, E., 51 (ref. 137), 75 teinman, M., 159 (r f. 47), 187 Stelgens, P., 196,215 Stenger, V. A., 281 (ref. 710.), 307 tern, E. S., 68 (ref. 121), 74 • tern, Y., 157 (ref. 183),19$ Stevens, . ., 277, 908 tewart, C. P., 45, 75, 126, 141, 158 (ref. 10), 172 (ref. 182),186,192 Stewart, M., 273, 907 Stiller, E. T., 263 (ref. 87), 308 Stocken, L. A., 196,212 (ref. 24), 214 Stoll, A., 51 (ref. 139), 75 Stolman, A., 45 (ref. 140), 75 Stolzenbach, F. E., 241 (ref. 38), 249 Storr, 93, 107 Strain, H. H., 295 (ref. 101), S08 Strassle, R., 281 (ref. 108), 909 Strauss, D., 49 (ref. 132), 62 (ref. 131), 74, 75 Strebel, A., 64 (ref. 22), 71 Strehler, B. L., 97, 107-109 Strisower, B., 149 (ref. 91), 189 Stritar, M. J., 230, 250

Strub, I. H., 156 (ref. 64), 188 Studer, A., 290, 308 Stiidemann, K. D., 263 (ref. 4), S05 Sullivan, M., 53 (ref. 141), 75 Summerson, W. H., 194 (ref. 35), 214 Suo, Y. T., 50 (ref. 142), 75 Sunshine, I., 68 (ref. 62), 72 Sutton, H. E., 298 (ref. 103) , 308 Suzuki, S., 138 (ref. 155), 143 Svendsen, A. B., 49 (ref. 143), 68 (ref. 144), 75 Swank, R. L., 155 (refs. 184, 185),167 (refs. 115, 116),189, 192 Sylv~n, B., 270 (ref. 99), 272, 306, S08 Synge, R. L. M., 124 (ref. 135), 142, 263, S08 Szasz, G., 160 (ref. 34), 187 Szerb, J . C., 53 (ref. 145), 75

T Tamiya, H., 102 (ref. 69), 109 Ta.n, K. B., 5 (ref. 16),38 Tatum, E. L., 119 (ref. 51), 139 Taussky, H. H., 195, 201 (ref. 66), $15 Taylor, W. E., 112 (ref. 96), 113, 115, 116, 122 (refs. 136, 137), 123 (ref. 137),141,142 Terren, A. J., 148 (ref. 134) , 190 Theorell, H., 230 (ref. 8), 231 (ref. 4), 239,241 , 242, 248,£50 Thiele, O. W., 126 (ref. 139), 142 Thies, H., 49 (ref. 147), 58, 75 Thomas, G., 50 (ref. 148), 75 Thomp on, A. R., 124 (ref. 140), 132 (ref. 140), 142 Thompson, E. O. P., 124 (ref. 140), 132 (ref. 140), 142 Thorne, C. B., 162 (ref. 66), 188 Tiber, A., 229, 230, 249 Tocantins, L. M., 267 (ref. 104), 280 (ref. 104), 285, S08

32

AUTHOR INDEX

Tolbert, N. E., 97 (ref. 53), 108 'i ". Tompsett, E., 43, 45 (ref. 41), 67, .68 (ref. 41', 72 '.', Tompsett, S. L., 43, 45 (ref. 41), 67; 68 (ref. 41), 72 Trabert, H., 68 (ref. 120), 74 Troll, W., 131, 142 -. Tropp, C., 194 (ref. 77), 215 Tryhorn, F. G., 55 (ref. 149), 75 Tuovinen, P. 1., 220, 223, 250 Turfitt, G. E., 64 (ref. 70), \ 73 C Turner, R. G., 22O, :S50 Tyler, V. E., 51 (ref. 150), 16 . 1

'J



u · Umberger, C. J., 42, 61, 75 Umbreit, W. W., 212 (ref. 67), 215 Uriel, J., 12 (ref. 39), 23 (ref. 43), 24 (ref. 45), 25 (refs. 6, 38, 40-42), 26 (refs. 42, 44), 33 (refs. 14, 39), 36, 37,38 Utsugi, N., 128, 13&, . 137 ~ref. 100)" 141 . I Utz, J. P., 159 (ref. 186), 192

w Wachsmuth, H., 64 (refs. 157, 15 ), 75 WadIhan, H., 295 (ref. 1 ), 306 Wadstrom, L. B., ]55, 160 (ref. 90), 165, 187, 189 Wagner, G., 64 (ref. 159), 75 Wahba, N., 64 (ref. 9), 71 Waldron, J. M., 156 (ref. 189), 160 (ref. 189), 192 Walker, J. B ., 194 (ref. 75), 215 Walpole, G. S., 209, 215 Walsh, J. W. T., 95 (ref. 70) , 1,0[} , Walton, K. W., 149 (ref. 144) , 150 (ref. 144), 190 Wang, C., 154 (rd. 74), 15 (ref. 74), 188 ' Warashima, E. , 119 (ref. 56.), 127, ]30 (ref. 56), 140 Warburg, 0:; 105, 109 Wassen, A., 241, 248 Waters, E. T., 255 (ref. (6), 256 (ref. 6{}), 285 (r f. 66), 307 Watson, R. C., 64 (ref. ]60), 75 Wattenberg, L. W., 272 (ref. 105), 309

v Vallee, B. L., 241, 2J,B, 250 Vallejo, F. G., 159 (ref.' 187), 192 . Van der Vies, J., 167 (ref'. 146), 191 Van Eck, W. F ..;'158 (ref. 188),192 : Van Etten, C. H:, 53 (ref. 153), 75 Van PilSUID, J. F., 193-215 ' ,10 ; Van Slyke, D. D., 112 (refs. 47,145);, 113, 115, 125, 126,127 (refs. 144), 139, 140, 142, 278 :' . Vaux St. Cyr, C. de, 26 (ref! 46),'38 Vidic, E., 49 (ref 'l54), 51 (ref; 154)~' 64 (ref. 155), 7iJ, "227; ' s5.} ~~.l Vilkas, E., ; 1~6'{'ref:I'i47)i, .11,.3 . r;' Vogt, H., 68 · (~f. 11li6)~~ , . 1 1, I' Volska:'R./7' (re1:::3Gjf. 38 ",,; " •... ".'!' Vries, A. de, 15i''(r'ei? 1~3j,' ~9S t", I I

Way) 'E. L., 6 (ref. 60) , 72 Webb, .J. W., .. 6 (ref. 161), 76 Webb, M., 172 (ref. 176), 192 ,. ,t, Weber, Oe IJ., 210 (ref. 49), 214 '!"'., Weekfey, F. B., 53 '(ref. 105),74 Weidlein, E : R., Jr., 115, 143 Weiner, N., 277 Weinig, E., 220, 251 . Weise, W., 194 (ref. 77), 215 Weiss;, F ., 52 (ref. 2), 64 (ref. 162); ':" ' !", 79 '.', Weiss, K. W;, 124 (ref:'ll), 132 '~et 11), 138 Weld, C. :a.~ 14t, ,i55 (ref. 19i), 156 (refs. 100,' 191), 161 (tef"I90), 19B Welin', 0., 163 (,ref. 83),·188,'.') Wells, I. C., 122, f{$: . r, '1'.." .~, ~:

.-

329

AUTHOR INDEX

Wenckert, A., 272, 275, S08 Werner, A., 156 (ref. 33), 160 (ref. 33), 187 West, G. B., 44 (ref. 13H), 75 Westl111, R G., 295 (ref. '),29 (ref. ), SO' We tendoJ'p-Boerrnn, R, 26 (ref. 3), 37 Westley, J ., 124 (r f. 150), 125,143 'Wheeldon, L. W., 12 , 143 White, ., 153 (ref. 59), 159 (ref. 59), 160 (ref. .5 9),166 (d. 59), 188 Whitehouse, :M. W., 29 (r('f. 49), 307 Vilhitman, B., 230, 250 Whitney, D. V., 291 (ref. 28), 306 Wibcrley, S. K, H4 (ref. 21), 71 Wickstrom, A., 64 (refs. 163,164),76 Widmark, E. M. P., 225,251 Wieme, R. J., 5 (ref. 47), 26 (ref. 3), 27,37,38 Wilande r, 0., 257 (ref. 106),263,309 Wilcox, A., 130,141 Wilh hni, A. E., 132, 13,9, 195 (ref. 27), 214 WilkinsoD, G. Ie, 157 (refs. 38, 39), 160 (ref. 40), J 7 Williams, . A., .Jr., 2 (ref. 17), 26 (refs. 17,49),32 (refs. 17,49),38 Williams, . M., 196 (ref. 3), 213 Williams, G. R., 167 (ref. 192), 192 Williams, M., 63, 64 (refs. 35, 36), 71 Williams, M. B., 226, 251 Williams, T . I., 53, 76, 295 (ref. 107), 309

Williams, T. N. W., 124 (ref. 154), 148 Wilmot, V., 155 (ref. 185), 192 Winnick, T., 226, e51 Winterstein, A., 255 (ref. 75) , 256 (ref. 75), 281 (ref. 108), 286, 290, 291 (ref. 76), 305, 307-309 Wislicki, L ., 157 (ref. 183), 192 Wissweiler, A., 78 (ref. 55), 95 (ref. 55), 108

Woldow, A., 159 (ref. 193), 192 Wolfe, H. R , 21 (ref. 50), 38 Wolin, E . A., 194 (refs. 70, 74),215 Wollish, E. G., 50 (ref. 130), 74 Wolthers, H ., 221, 232, 244, 247, 248 (ref. 46), 250, 251 Woodhead, H. B., 50, 71 Woods, L. A., 68 (ref. 166), 76 ' Yoolley, D. W., 115-117, 118 (ref. 153), 122, 143 Worley, L. M., 162 (ref. 194),192 Worrell, L., 68 (ref. 167), 76 "'ren, J . J., 124 (ref. 150), 125, 148 \\'ulf, H. J., 240, 250 Wunderlich, H ., 6 (ref. 16 ), 76 Wunderly, C., 11, 26 (ref. 3), 37, 38 Wurster, C. F., 91 (ref. 64, 67), 108, 109 Wynn, V., 124 (ref. 154), 143

Y Yamakawa, T., 138 (ref. 155),148 Yang, C., 79 (ref. 73), 109 Yankley, A., 153 (ref. 59), 159 (ref. 59), 160 (I' f. 59), 166 (ref. 59),188 Ycnun, E. W., 130, 131,139,143 Yownans, J . B., 156 (ref. 124), 190 Young, V. M. K, 79, 95 (ref. 24),107 Young, \V., 156 (I' f. 195), 192

Z Zacherl, M. K, 195,215 Zappaco ta, M., 210 (ref. 79), 215 Zeisel, ., 230, 251 Zilversmit, D . B., 156 (ref. 1), 186 Zimmer, U., 64 (ref. 159), 75 Zinn, W. J., 154 (ref. 196), 156 (ref. 196), 192 Zollner, N., 154 (ref. 197), 192 Zopf, L. C., 53 (ref. 20) , 68 (ref. 20), 71 Zuokerman, L., 164 (ref. 198), 192 Zweig, G., 124 (ref. 10), 132 (ref. 10). 138

UBJECT INDEX A Absorbance. See also Attenuance. of translucent materials, 78 Absorption spectra, azure A with h parin, 259, 260 Acetaldehyde, as measure of ethyl alcohol, 228-29 Acetic acid, as measure of ethyl a lcohol,229 Acetone, interference in alcohol determination, 231 Aconitine, determination, 49, 63, 64 Acrifiavin, heparin effect, 258, 259 Adenylic acid, 100 ADH (alcohol dehydrogenase), determination of ethyl nlcohol in blood, 240-4 Adrenaline, 44 Adsorption, of alkaloids, 45--46 in lipoprotein lipase prcparation, 169-70

Aerobacter suboxydans, 117 Agar. 156 gel for I ctrophor sis, 3-8 Alanine, 150 Alcohol dehydrogenase. See ADH. Algae, opal glass transmission spectra. 89, 90 Alginic acid, 156 Alizarin, heparin stain, 29 Alkaloid analysis, 39-75 assay, 65-68 identification, 53--65 biological activity, 63-64 color tests, 58-62 crystal forms, 62- 63, 64, 68

infrared, 62, 64, 68 spray reagents, 55-57 ultraviolet spectrophotometry, 58,62,64,67,68 ion-exchange methods, 52-53, 66, 6 isolation from tissue. 41-47 paper chromatography, 48-51, 59, 64.68 paper electrophoresis, 52 purification, 47-53 n-Allyl normorphine, 70 Alumina, adsorbent for alkaloids, 45 Amethocaine identification, 56, 59 Amino acids, in lipides, 124-38 of lipoproteins, ] 50 Ammonium carbonate, heparin purification, 267-68 Ammonium sulfate, ex1.raction of heparin , 267 protein precipitant in alkaloid ex1.raction, 43-44 Amphetamine determination, 56, 60, 67,68 Amylocaine identification, 56, 59 Amylopectin,156 Analgesics, assay, 68 Anesthetic , analysis, 64, 68 interference in alcohol determination,231 Anthisan de'termination, 56, 59, 65 Antibodies, antienzyme, 35 production. 17-19 for protein analysis, 2- 3 Anticoagulant activity of heparin, 261-62.283- 85 Antienzyme antibodies, 35

331

332

SUBJECT INDEX

Antigen-antibody complexes, in protein analysis, 2-3 Antigens, analysis, 2-3, 35 precipitin reactions. 3, 4, 17-22, 26, 29-30,31-33 Antibeparinoids, 157 Antihistamines, 53, 64 Antisera. See Immune sera. Antistine identification, 56, 59 Antithrombin heparin assay, 288, 290-91 Aquoprusside reagent, for canavanine determination, 211-12 Arginine determination, 200-10 Arlane identification, 56, 58 Aspartic acid, 150 Atherosclerosi , 159 Atropine, determination , 49, 53, 56, 59, 64 extraction from tissue, 43 Attenuance, 1,82-83,84, 94,96,98 integral, 105-6 R-corrected, 82, 103-5 rectilinear, 82,101-3 Automatic titration, 23 39 Azure A, absorption pectm. 259, 260 heparin a,'say, 292-93 chromatogram staining, 297- 9 metachromatic effect, 259, 260 Lovib'md color units, 260

B Bacteria, opal glass transmission spectra, 89 Barer method for spectra of translucent materials, 100 Barium ~alt of heparin, 250-57, 268. 299-300 Basophilic leucocytes, heparinrin, 265,273 Bean loaves, opal glass transmission spectra, 90 Beef, heparin in, 255, 264

BeJladonna assay, 68 Benactyzine d termination, 6'J, G8 Benadryl identification, 56, 59 Benzidine, heparin rea ·tion, 25 , 267-6 ,270- 71 Benzocaine identification, 56, 60 Berberin id ntili 'tition, 5G, 59 Bioassay of inositol, 1J 7-19 Bismarck brown, heparin effect, 259 Blood. e also Plasma, erum. ethyl alcohol analysi, , 217-51 guanidinium compounds determination, 193-211 heparin, anticoagulant efieet, 261 62 content, 264-65 determination , 2 5-90,301 - 5 extraction, 271-76 lipide inositol assay. 11 Brain, ethanolamine and erine COlltent, 129 extraction of alkaloids, 43-44 lipide analysis, 114-15 lipide ino itol content, 123 Brilliant cresyl blue, heparin effl'ct, 259 Brom cresyl blue, heparin effect, 259 Brucine determination , 49, 56, 59, 67, 68 heparin reaction, 258, 26 ,274 Buffers, in alkaloid cxtraction, 50 in protein electrophoresis, 7- 8, ]213 Butacaine identification, 56. 59 n-Butunol, solvent systems, 49-5], 69

c Caffeine determination , 56, ro, 6 Calcium ion s as llc('eptors for flltt.v u'id ,165 Calcium pho. pbatc gel, for lipoprotein lipase purification, 16970

SUBJECT 11'.1)EX

Canavanine determination, 211-12 Carotene, 90 Carotenoids, role in photosyntbesis, 90 Casein in heparin assay, 291, 292 Cations, inhibition of lipoprotein lipase, 166 Cell suspensions, opal glass spectrophotometry, 90 Cellulose, as heparinoid, 156 Cellulose acetate for electrophoresis, 5 Ceruloplasmin identification, 35 ChB.rles and Scott, heparin extraction rnethod,267-68,270 unit of measurement, 2 2 Chlorella, spectrophotometry, 83, 84, 9 ,100,102,103 Chlorocrclizine identification, 56. 59 p-Chloromercuribenzoate, inhibition of lipoprotein lipa c, 166 Chloropbylls, spectrophotometry, 98 Chloroplatinic acid in alkaloid analysis, 64 Cblorpromazine determination, 59,68 Cholinestera e content of lipoproteins, 150 Chondroitin sulfate(s), 255, 264, 273 Chromatography. See also Column chromatography, Gas chromatograpby, Pap r chromatography glycerides, 1 2- 85 heparin, 262-64, 297- 9 Chromic acid determination of ethyl alcohol in blood, 222-26 Widmark method, 232-40 bylomicrons, 151 , 164, 171 Cinchona alkaloids, separation, 51 Cinchonidine identification, 56, 59 Cinchonine identifi ation, 56, 59 Coagulation test systems for heparin assay, 2 6-91

333

Cobalt thiocyanate in alkaloid analysis, 56, 59, 61 Cocaine determination, 49, 56, 59, 64, 68 Codeine determination, 56, 59, 68 Coleus leaf, spectrophotometry, 104 Colorimetric analysis, alkaloids, 6667,68 guanidinium compounds, 198, 199201,203 heparin, 292- 93 lipides, 128-38 Column chromatography, alkaloid an.alysis, 53 beparin purification, 269 Complexing agents for alkaloids, 52 Coniine identification, 56, 59 Conway diffusion cell, 126, 127, 227, 228,229 Corarnine determination. 49. 56, 59 Cotarnine identification, 56, 59 Countercurrent distribution, heparin purification, 263 Creatine determination, 194-206 o-nitrobenzaldehyde method. 1979 picric acid methods, 199-200, 2016 iodine oxidation-ether extraction method. 201-6 Lloyd's reagent modification, 199-200 Creatine phosphate determination, 212-13 Creatinine determination. 194-206 o-nitrobenzaldehyde method 197-

9B picric acid methods, 199-206 iodine oxidation-ether e~1.raction method, 201-6 Lloyd's reagent modifications, 199-201 Cresyl violet, heparin effect. 259

334

SUBJECT INDEX

Cytochromes, spectrophotometry, 98,

100 Cytochrome systems, hydrogen transfer mechanism, 97 D

Daptazole determination, 68 Deproteinization in ethyl alcohol determination, 221 Deriva.tive spectra of translucent materials, 98 Detergents. inhibition of lipoprotein lipase, 166 Dextran sulfate, 156 Diacetyl, in arginine determination, 209-10 Dialysis in lipide purification, 113 Difference spectra of translucent materials, 97-98 Diffusion analysis, amino acids in lipides, 126, 127 ethyl alcohol in blood and tissue, 220,224-26,227,228,229 Diglycerides from lipoproteins, 165 Dihydroxyanthraquinone. See Alizarin. Diisopropy I fluoropbosphate, lipoprotein lipase inhibition, 166 p-Dimethylaminobenzaldebyde, in alkaloid analysis, 56, 59, 61, 69 Dinitrophenyl (DNP) derivatives, in colorimetric lipide analysis, 128-30 Diphospbopyridine nucleotide. See DPN and DPNH. Dipipanone identification, 56, 59 Distillation, removal of alcohol from tissues, 219-21 DNP. See Dinitrophenyl. Dog, heparin in, 255, 264-65. Dole procedure, fatty acid determination, 179

DPN (Diphosphopyridine nucleotide), 3-acetylpyridine analog, 241, 243 role in enzymic alcohol determination,241 DPNH (Diphosphopyridine nucleotide, reduced), in ethyl aJcohol determination, 241 Dragendorff's reagent. See Potassium bismuth iodide. Dromoran identification, 64 Dyclomine identification, 56, 59 Dye, heparin reaction, 258-61 E

Egg, lipide ethanolamine and serine content, 129 Egg phosphatides, ethanolamine and serine content, 128-29 inositol assay, 118 purification, 114 Ehrlich's reagent, staining in heparin assay, 298 Electroendosmosis, in gel electrophoresis, 14-15 Electrophoresis. See also Immunoelectrophoretic anal)" i , Paper electrophoresis. in gels, 12-17 heparin,263 lipoprotein lipase, 169, 185-86 lipoprotein patterns, 153 Emetine identification, 56, 59 Enzymes, beparin reaction, 257- 58 Emzymic methods, ethyl alcohol determination, 240-48 accuracY,247-48 procedures,242-43,245-47 reagents, 244-45 theoretical basis, 240-41 inositol assay, 117 Ephedrine determination, 49, 53, 56, 59,61,6

SUBJECT INDEX

335

Ergometrine determination, 43, 57, F 60,65,69-70 Ergot alkaloids determination, 49, 68 Fatty acids, determination in lipoE8cherichia coli, opal glass transmisprotein lipase assay, 177-79 from lipoproteins, 164 sion spectra, Ethanolamine in lipides, ] 24-3 FDNB. See Fluorodinitrobenzene. colorimetric methods, dinitroFerric chloride staining in heparin assay, 29 phenyl derivatives, 128-30 ninhydrin, 13(}-32 Ferrous sulfate, chromic acid titration , 225 sodium 1,2-naphthoquinone-4Filter paper, for opal glass transmissulfonate, 132-38 sion spectra, 86, 8 content in animal tissues, 129 Flavianic acid, 64 manometric methods, 125-26 Florisil, adsorbent for alkaloids, 45 separation from serine, 127, 134, Fluorescence, in alkaloid analysis, 56 137 Ethyl alcohol, in blood and tissues, Fluorodinitrobenzene (FD:N13), ethanolamine and serine 217-51 assay, 12 ,129 chemical methods, accuracy, Forensic analysis, ethyl aJ obol in 231-32 blood and tissues, 2] 7- 51 determination a ethylene, toxic alkaloids, 39-75 229-30 Fractionation, mucopolysaccharides, determination of oxidation 269-70 products, 228-29 Freezing, for spcctra of translucent esterification, 23(}-31 materials, 98-100 oximetric, 222-28 Fuchsin, heparin effect, 259 Widmark method, 224-25, 232-40 enzymic methods, 240-48 G physical methods, 221-22 removal of interfering substances, Gas chromatography, alcoholsepara219-21 tion, 232 extraction of alkaloids, 41-42 Gastric lipase, differentiation from solvent sy terns, 263-64 lipoprotein lipase, 167 Ethylene, as measure of ethyl alGel adsorption, of lipoprotein lipase, 169-70 cohol in blood, 229-30 Gelatin, heparin reaction, 258 Ethyl ether, interference in alcohol Gels for ele trophoresis, 3-8 determination, 231 Ethyl morphine identification, 57, 59 Glassware, silicone treatment, 303 Extraction, alkaloids from tissue, 41- Glutamic acid in lipoproteins, 150 Glycerides. See DigJycerides, Mono44 glycerides, Triglycerides. heparin from body fluids, 271-77 Glycerol determination in lipoprofrom tissues, 266-71 tein lipase assay, 179-82 lipide purification, 112-15

336

SUBJECT INDEX

Glycogen, 156 GJycoproteins, staining of, 25 Gordon method, fatty acid determination, 178 Guanidine determination, 210-11 Guanidinium compounds, 193- 215 arginine, 206--10 canavanine, 211- 12 creatin , 194--206 creatine phosphate, 212-13 creatinine, 194-206 guanidine, 210-11 guanidinoacetic acid, 206--10 methylguanidine, 210-11 role in metaboli m, 193-94 Guanidinoacetic acid determination, 206--10 Gum arabic. 156 H

extraction, from body fluid, 27177 micro methods, 270-71 from tissues, 266--71 hydrolysis products, 29 iden tification , 204, 294-300 metabolism, 264-65 prop rties, 255-65 purifi cation , 267-6 , 269 pecies difference, 256, 264-65 specific activities, 294 units of measurement, 2 2 ii-Heparin. &e Chondroitin sulfate. Heparinase, bacterial, 165 Heparinoids, effect on liPOP1'oteinS, 156--57 extraction from plasma, 275 Heparitin sulfate, resemblance to heparin ,2G4 Heroineidentifict\tion , 57, 59, 04 Hesperidin, phosphoD'lated, ] 56 Hexo amine in lipides, 138 Histamine, heparin reaction, 258 Histones, heparin reaction , 258 Hi tochemical reactions in antigen identification, 35 Homatropine identification, 57, 59 Horse antibodie ,19,31 Horse antisera, 26 Howell unit for heparin , 282 Hyaluronic acid , re emblanrc to heparin,204 p-Hydroxydiphen:r~ color reagent for ac taldehyde, 228 Hydriodic acid, determination of ethyl alcohol in blood. 230-

Haptens,3 Hartree. See Keilin-Hartree. Heart, ethanolamine and serine content, 129 lipide inositol content, 123 Heparin, 253-309 analytical data, 255 anticoagulant activity, 261-62 definition, 254 determination, 277- 300 anticoagulant methods, 283-85 in vivo, 285 in body fluids. 301- 5 chemical methods, 291- 94 coagulation test sy terns, 286--91 plasma, 286--88 31 antithrombin assay, 288, 290- 8-Hydroxyquinoline, in • akaguchl 91 color reaction, 206, 20 sulfated whole blood, 2 90 Hyoscine determination, 57, 59, 64, fundamental principles, 277-82 68 distribution, 264--65 Hyoscyamine determination, 68 effect on lipoproteins, 152-61 Hypercholesterolemia, 15

337

SUBJECT INDEX

Hyperlipemia, heparin effect, experimental, 15!}-60 normal, 153- 56 pathologi cal, 157- 59 Hyperten ion , 159 I

Idiopathic hyperlipemia, 157- 58 TEA. See Immunoelectrophoretic analysis. Immune sera, in immunoelectrophoretic analysis, 17- 22, 2!}-30 Inununochemical reactions in protein analysis, 17- 22 Immunoelectrophoretic analysis (IEA),1- 38 advantages, 36-37 disadvantages, 2!}-30, 37 elcctrophore is, 12-17 gelpr paration , 3- 11 identification of constituents, 3036 imm une sera p reparation , 17- 19 u 'e, 21- 22 micro t echnique, 27 precipitin reactions, 3, 4, 17- 22, 26, 2!}-30, 31- 33 recording results, 22-25 staini.ng techniques, 24-25 standard technique, 25-26 Immunological reactions, in antigen identification, 33-35 Infrared spectra, in alkaloid analysis, 62, 64, 68 Inositol in lipides, 115-23 ontent in tissues, 123 hydrolysi of e. tel'S, 115-16 microbiological assays, 117- 19 inhihitors, 121- 22 molds, 119 rf'producibility, 122- 23 yea t, 117- 19 periodatc oxirl::ttion , 115-16

Integral attenuance, 105-6 Integrating sphere transmi ion spectra, 95-96, 105-6 Interferometry, determination of ethyl alcohol in blood, 22122 Intestine, ethanolamine and serine content, 129 lipide inositol content, 123 Iodine, in alkaloid analysis, 60 in creatine, creatinine determination, 201- 6 heparin stain, 29 Iodine pentoxide, determination of ethyl alcohol in blood, 227 Iodometry, eth~'l alcohol determination , 223-24 Jon exchange methods, alkaloid analysis, 52- 53, 66, 68 arginin guanidinoacetic acid separation, 206 heparin purification , 263 inositol determination, 11 6 lipide analysis, 137 Isoelrc tric precipitation of lipoprotein lipa e, 169 Ivy I af, opal glass transmiSl ion . pectra , 92, 95, 103

K Kaolin, adsorbent for alkaloids, 45 Kei lin-Hartree Method of pectra of tr!l,n.lu 'ent materials, 9 100 . Kidn y ethanolamine and erine content, 129 lipide ino itol content, 123 Kirs 19uhr, for spectm of translucent materia.ls, 99

Kloeckera a'JYiculata, 119 Kloeckera brems, 11

338

UBJECT INDEX

L

Largactil identifications, 57 Leaves, spectrophotometry, 81, 87, 90, 92, 95,103, 104 Leptazol, 49 Lethidrone determination, 57, 59, 70 Leucocytes, heparin in, 265, 273 polysaccharide extraction, 276 Lipases. See also Lipoprotein lipase. differentiation from lipoprotein lipase, 167 Lipides, analysis, 112-43 ethanolamine and serine, 124-38 colorimetric methods, 128-38 manometric methods, 126-26 periodate oxidation, 126-27 inositol, 115--23 chemi almethods, 115--17 enzymic methods, 117 microbiological methods, 11723 extraction from plasma, 183 heparin effect, 154-55 hydrolysis, 136-36 purification of extracts, ] 12-15 Lipopeptides, 124-25 Lipoproteins. See also Lipoprotein lipase. Lipoproteins, 146-61 action of lipoprotein lipase, 161-68 chylormicrons, 151, 164, 171 classification, 149-51 compo ition, 150, 154-55 heparin effect, 152-61 identification, 35 occurrence, 146-49 preparation, 171- 72 staining, 24-25 Lipoprotein lipase, assay methods, 173-86 conditions, 176-76 electrophoretic, 185--86

glycerol determina,tion , 179- 2 unesterified fatty acids determination, 177-79 turbidimetric, 17G- 77 ultracentrifugal, l 5-86 differentiation from other lipases, 167 inhibition, 166 preparation, from po theparin plasma,168-70 BOur('e , 161-63 from tissue, ] 70 Liver, ethanolamine and serine content, 129 cxtraction of alkaloid', 43-44 heparin in , 264-65 lipide inositol assay, 118, 123 Lloyd's reagent, creatinine determination, 199-201 heparin purification, 267- 69 Lovibond tintometer for heparin assay, 292-93 Lung, ethanolamine and serine content, 129 lipide inositol content, 123 Lycopene, spectrophotometry, 90 M Manometric analysis, amino acids in lipides, 125-26 Marzine identification, 57, 59 Mayer's reagent, alkaloid complexes, 52 Mercury potassiwn iodides, alkaloid complexes, 52 Mesquite gwn, 156 Metabolism, of heparin, 264-65 role of guanidinium compounds, 193-94 Metachromatic activity of heparin, 258-61,292-93 Methadone determination, 57, 59,64, 67,6

339

SUBJECT INDEX

Methanol, interference in ethanol determination, 232 n-Methorphan identification, 56, 59 Methorphinan determination, 68 Methylguanidine determination, 21011 Microbiological assay of inositol, 11719 Molds for inositol assay, 119 Monoglycerides from lipoprokins, 165 Morphine determination, 45, 49, 5152, 53,57, 59, 64 , 68 Morpholine, ac>talclchyde indicator, 229 Mucicarmin e. 29 Mucoitin, 264 Mucopoly acchariclcR, 264, 269- 70 N

Naarsenite, inhibition of lipoprotein lipase, 166 Nalorphine determination, 64, 68, 70 Naphthoresorcinol,29 Narceine identification, 57, 59 Narcotic alkaloids, 51 Narcotine determination, 49, 57,59 Nephelometer for heparin determination, 305 Nephrosis, effect of heparin, 159 Neurosporacrassa "inositol-less," 119 Neutral red, heparin effect, 259 Nicotine, derivative ,49 identification, 57, 59, 64 Ninhydrin (Triketohydrindene hydrate), alkaloid analy is, 61 heparin stain, 298 serine assay, 126 o-Nitrobenzaldehyde, creatine and creatinine assay, 197-98 creatine phosphate determination, 213

Nitrogenous compounds, basic. See Alkaloids. itroprusside-acetaldehyde reagent, 61- 62 Nitrous acid, determination of ethyl alcohol in blood, 230 ethanolamine assay, 125-26 N upercaine identification, 57, 59

o Octylamine for heparin ell.'traction, 273- 74 Opal glas transmission spectra, 79, 3- 95 effect of reflection, 93-95 procedures, 83- 93 Opaque materials, optical characteristics, 81 Opiate, identification, 64 Opium, analysis, 45, 49, 53 Oxine. See 8-Hydroxy quinoline.

p Pancreas, lipide inositol content, 123 Pancreatic extract in heparin extraction, 269 Pancreatic lipase, differentiation from lipoprotein lipase, 167 Papaverine determination, 46, 49, 57, 59,62 Paper chromatography, alkaloid analysis, 48-51, 64, 69 amino acid determination, 132 heparin,262, 263, 294,295-99 ino itol determination. 116 Paper electrophoresis, alkaloid , 52 lipoproteins, 154 Pectic acid, 156 PectiD gels, 4 Pectin, sulfated, 156 Periodate oxidation, ethanolamine and serin assay, 126-27

340

SUBJECT INDEX

Permutit, ethanolamine and serine separation, 127 Petals, spectrophotometry, 81, 87 Pethidine determination, 57, 59, 64,

67 Phenadoxone determination, 6 Phenergan identification, 57, 59 Phenol in heparin extraction, 27475 Phloroglucinol, 29 Phosphatide , ethanolamine and serine content, 12 - 29 inositol a say, 118- 19 purification, 113, 114 Phospholipases, inhibition of lipoprotein lipase, 166 Phospholipides, analySIS, 131 content of lipoproteins, 150 Photometric ethyl alcohol determination, 224, 228 Photomultiplier tube for translucent materials, 96 Photosynthesis mechanism, 90 Photosynthetic organisms, 97 Physostigmine identification, 57, 59 Picric acid, alkaloid analysis, 64 creatine and creatinine determination, 199-206 Pilocarpine determination, 57, 59,

68 Plasma, creatine determination, 196 creatinin determination, 196,2046 ethanolamine and serine content, 129 heparin, assay, 301-4 coagulation test systems, 28688 extraction, 271-76 lipide analysis, 115 lipide inositol content, 118, 123 lipoprotein lipase in, 161-63. 16870,175

Polarographic acetaldehyde determination, 229 Polylysine, 157 PolYS8.ccharid ,extraction from white cells, 276 precipitin reactions, 3 staining, 25 Polystyrene latex for spectra of translucent materials, 100 Pontamine Fast pink B-L, 156 Pork, heparin assay, 255 Postbeparin pIa rna, lipoprotein lipa e, assay, 175 content, 161-63 preparation from , 1G8-70 Potassium bismuth iodide (Dmgendorff's reaITent) in alkaloid analysis, 52, 56, 58 Potassium iodoplatinate, 60 Potassium mercuric iodides (Mayer's reagen t) , 52 Potassiwn permanganate, alkaloid analysis, 56, 59, 61 determination of ethyl alcohol in blood, 227 staining in heparin a 'say, 298 Potassium platinic iodide, 52 Potassium thiocyanate in heparin extraction, 270-71 Precipitin reactions, antigens, 3, 4, 17-22,26,29-30,31-33 polysaccharides, 3 Procaine determination, 57, 59, 68, 69-70 Promazine identification, 57, 59 Propanol solvent systems, 263 Protamine, heparin reaction, 258 titration, 304-5 Protamine sulfate, as antibeparinoid, 157 inhibitor of lipoprotein lipase, 166 Proteins, content of lipoprotein, 150

SUBJECT INDEX

heparin reaction , 257-5 immunoelectrophoretic anal~'si " 1-38 separation from alkaloids, 41-47 staining, 24 Proteolipidp , 124 Proteolytic digestion for heparin e>..'traction, 269 Psoriasis, effect of heparin, 159 P)Tidine 3-nldeh~'de in enzymi(' alcohol determination , 243 Pyronine, hrparin effect, 259 Q Quasi-attenuBnce, 1, 2, Quinine, dpwrmination, 53, 57, 59, 67 6 hf'p!ll'in TE'a('tion , 25

R Rabbit an tibodies, 19 R-corrpcted uttenuance, 2,103- 5 Rectilinpar nttf'l1nan('p, 82, 101-3 Refif'ctanrr spectra of translUCf'nt materials, 103-5 Refractive index, determination of ethyl alcohol in blood , 221 22 Rrserpine ( rpasil), detemullation, 57,60,64,67,68 Reversed pha e solvent systems, alkaloid extractions, 50--51 R,Ynlues of alkaloids, 59 Rhodopseudomonas, opal glass transmis ion spectra, 89, 90

s Saccharomyces carlsbergensis, for lipide inositol assay, 11 , 119-23

341

Saccharomyce8 cereviBiae, for lipide inositol assay, 117-1 ,122 Sakaguchi color reactions, guanidinium compound assay, 19798, 206,207- 8,210--11 8-hydroxyquinoline modi1ication, 206,20 Schizosaccharomyces pombe, lipide inositol assay, 11 Scopalamine deterrnination, 49, 64 Scott. See Charle and cotto Seed , lipide inositol content, 123 t'mi-integral uttenuMce, 5, 94, 96, 98 Seminal plasma, lipid!" ino itol assay, 11 Serine, in lipides, 124-3 C'olorimt'tric method , dinitrophenyl derivative" 12 -30 ninhydrin , 130--32 sodium 1,2-naphthoquinonc-4su lfonate, 132-38 ('ontrnt in animal tissues, 129 mn.nomt'tric methodE', 125-26 separation from etha.Dollimine, 127,134,137 in lipoproteins, 150 'erpllsil. See Reserpine. erwn. See also Immune sera. creatine and creatinine in, 195-96, 203-6 immuno lectrophoretic analysis, 35 erum albumin, fatty acid acceptor, 164 erum lipopr.oteins. See Lipoproteins. Sheep, heparin assay, 255 Silicon> treated glassware, 303 Silver nitrate, heparin stain, 298 Skeletal muscle, ethanolamine and serine content, 129 lipide inositol content, 123

342

SUBJECT INDEX

Skin, ethanolamine and serine content, 129 Sodium 1,2-naphthoquinone-4-sulfonate, lipide ethanolamine and serine assay, 132-138 Sodium nitroprusside, acetaldehyde indicator, 229 in alkaloid analysis, 62 Sodium tetraphenyl boron, 52 Solanidine identification, 57, 59 Solvent partition in lipide purification, 113-14 Solvent systems, alkaloid separation, 49-51,69 heparin chromatography, 263-64, 298,299 reversed phase, 50-51 Soxhlet extraction of alkaloids, 44 Sparteine identification, 57, 59 Spectra. See Absorption spectra, Derivative spectra, Difference spectra, Infrared spectra, Reflectance spectra, Transmission spectra. Spectrophotometry. See Translucent materials, Ultraviolet spectrophotometry. Spray reagents for alkaloids, 55-57 Staining techniques, 24-25, 297-9 Starch gels, 4--5 Strychnine determination, 49,57,59, 67,68 Styphnic acid in alkaloid analysis, 64 T

Tetraethyl pyropbosphate inhibition of lipoprotein lipase, 166 Theobromine assay, 68 Thionine, heparin reaction, 258 Threonine in lipoproteins, 150 Thrombin, inhibition by heparin, 261 Thromboplastin, heparin assay, 286

Tintomctcr, Lovibond, 292-93 Tissues, alkaloid extraction, 41-44 creatine phosphate in, 194, 212-13 ethanolamine and serine content, 129 ethyl alcohol in, 217-51 heparin distribution, 264-65 extraction, 266-71 lipide ino itol content, 123 lipoprotein lipase in, 163, 167, 170 assay, 175-76 spectrophotometry, I, Toluidine blue, as antiheparinoid, 157 heparin reaction , 258, 259-60, 291 Toxicological analysis, 39-75 Transamidinase, 194 Translucent materials, attenuanc , 81, 82- 83, 94, 96, 9 integral, 105-6 R-corrected, lO3- 5 rectilinear, 101- 3 optical characteristics, 80-81 spectropbotometry, 77-109 Barer method, 100 derivative spectra, 98 difference sp~ctra, 97-98 integrating sphere transmission, 95-96 Keilin-Hartree method, 98-100 opal glass transmission, 83-95 photomultiplier tube, 96 relation between transmission and reflection speotra, 103-6 Transmission spectra of translucent materials, 83-100 Trlbutyrinase, differentiation from liproprotein lipase, 167 Triglyoerides and lipoproteins, 150, 164, 165, 172 Triketohydrindene hydrate. See Ninhydrin.

343

SUBJECT INDEX

Tropine alkaloids assay, 6 Trypsin digestion for heparin extraction,271 Turbidimetry, heparin assay, 291- 92, 305 lipide analysis, 118, 121 lipoprotein lipa.se assay, 176- 77 T\y 'ens, lipoprotein lipase reaction, 164, 166

u Ultracentrifugation, lipoproteins, 148-51, 153, 166 lipoprotein lipase assay, 185- 6 preparation, 169 Ultraviolet spectrophotometry in alkaloid analysis, 5 ,62, 67,

Veratrine determination, 57, 59 Veratrum determination, 68 Volumetric methods for ethyl alcohol determination, 223- 24

w White cell. See Leucocytes. Widmark method for alcohol in blood and urine, 232-40

x X-ray diffraction in alkaloid analysis, 62 Xylocaine identification, 57, 59

y

6

Urine analysis, arginine, 20()-' creatine and creatinine, 197-206 ethyl alcohol, 238 guanidine, 21 t guanidinoacetic acid, 206-10 heparin, 265, 270-77, 305 methylguanidine, 211 Uroheparin, 265, 27()-'77 V

Vanadio acid, determination of ethyl alcohol in blood, 227- 28

Yeasts, in lipide inositol assay, 11719, 119-23 Yohimbine identification, 57, 59

z Zeisel methods for ethyl alcohol determination, 230-31 Zinc hydroxide, paration of arginine and guanidinoacetic acid, 206-8

Methods of Biochemical Analysis CUMULATIVE INDEX, VOLUM'E

I-VII

Author Index VOL.

Ackerman, C. J ., see Engel, R . W. Ames, Stanley R., see Embree, Norr1:s D. Aspen, Anita J ., and Meister, Alton, Determination of Transaminase ...... , , .. , , . , .... . , , ' . , ......... , . , . , , ... , .... ' . A ugustinsson, Klas-Bertil, As ay Methods for Choline terases . , . . Barker, S. A. , Bourne, E. J., and Whiffen, D . H. , Use of Infrared Analysis in the Determination of Carbohydrate Strur tur ..... Bauld, W. S ., and Greenway, R. M ., Chemical iJetermination of Estrogens in Human Urine. , . . , . . . ' . , . .. , , . , , . .. , , . , ... , , , Bell, Helen J ., see Jaque.s, Louis B . Bergmann, Felix, and Dikstein, Shabla,y, New Methods for Pmification and Separation of Purines , , .. ' , ' .. , . , , ...... , .... , . Bickoff, E. M ., Determination of Carotene. ' ..... , .. , ... , , ' .. ' , Bourne, E . J ., see Barker, S. A. Bray, H. G., and Thorpe, W . V., Analysis of Phenolic Compounds of Interest in Metabolism ... , . .. . . , . .. , . .. , . ' . . ... , , . , ' .. , Brodie, Bernard B ., see Udell!riffld, Sidney Chance, Britton, see Maehly, A. C. Chinard, Francis P., and Hellerman, Leslie, Determination of Sulfhydryl Groups in Certain Biological Substances .. , , ......... . Code, Charles F., and Mclntire, Floyd C., Quantitative Determination of Histamine ... . , .. , .... , .. . , ' . , , , ... . ..... , . , , .. , .. Cohn, Waldo E., see Volkin, Elliot Curry, A. S., The Analysis of Basic Nitrogenou ompounds of Toxicological Importance . .. , , , ., , , ..... , , .. , ..... ... , ,. ' , ' Davidson, Harold M., see Fishman, William H. Davis, Neil C., and Smith, Emil L., As ay of Proteolytic Enzymes Davis, R. J ., see Stokstad, E. L. R. Dikstein, Shahtay, Bee Bergmann, Felix Dische, Zacharias, New Color Reactions for the Determination of Sugars in Polysaccharides ... .. .. . ..... , . ....... . :. , . .. , , . . Dodgson, K. S., and Spencer, B., Assay of Sulfatases . ... .. .. .. . , Dyer, John R. , Use of Periodate Oxidations in Biochemical Analysis Embree, Norri8 D" Ames, Stanley R., Lehllwn, Robert W., and Harris, Philip L., Determination of Vitamin A, . . , . . ... .. ... . . . . Engel, Lewis L ., The Assay of Urinary Neutral 17-Ketosteroids .. Engel, R. W., Salmon, W . D., and Ackerman, C. J., Chemical Estimation of Choline ..... ................. . .... . ... .... .. .. . Ernster, Lars, see Lindberg, Olov 345

PAGE

VI V

131

III

213

V

337

VI IV

79

I

27

1

1

I

III

49

VII

39

II

215

II IV III

313 211 III

IV I

479

I

265

43

346

CUMULATIVE INDEX, VOLUMES I- Vll VOL.

Fink, Frederick C., see Kersey, Roger C. Fishman, WiUiam H., and Davidson, Harold M ., Detel,nination of Serum Acid Pbosphatases . ............. . . . . . ..... , , , ' ... , . Fraenlal-Conrat, H., Harris, J. leuan, and Levy, A. L., Recent Developments in Techniques for Terminal and Sequence Studies in Pep tides and Proteins . . . ...... , ... , .... . ...... , .. . ' , , .. . , , FriseU, Wilhelm R., and Mackenzie, Cosmo G., Determination or Formaldehyde and Serine in Biological Systellll' . . . , . , . .... , , , Gale, Ernest F., Determination of Amino Acids by Use of Bacterial Amino Acid Decarboxylases . . . . . . .. .. '... ' .. .... , . " .. , . Gardell, Sven, Determination of Hexosamines . . ' . " """" ' " Go/man, John W. , see Lalla, Oliver F. de Grabar, Pierre, Immunoelectrophoretic Analysis . , , , , . , . ' , . ' .... Greenway, R. M., soo Bauld, W. S. Gr088, D., see Whalley, H. C. S. de Haines, William J., and Karnemaat, John N., Chromatographic Separation of the Steroids of tbe Adrenal Gland ... . , ........ . Harris, J . leuan, see Fraenkel-Conrat, H. Harris, Phili p L., see Embree, Norris D. Hellerman, Leslie, Bee Chinard, Francis P. Hoif-J,rgensen, E., Microbiological Assay of Vitamin B12 • • . , •• , • Holman, Ralph T ., Measurement of Lipoxidase Activi ty, . ..... , , Measurement of Polyunsaturated Acids .. . . . , ... , . , ... , .... . Hough, Leslie, Analysis of Mixtures of Sugars by Paper and Cellulose Column Chromatography .. ...... . .... . . . . , ... , ....... . Hughes, Thomas R., and Klotz, Irving M., Analysis of Metal-Protein Complexes .............. , . .. . .... . . .... . , ... . .. , . .... , .. . Humphrey, J. H., Long, D. A. , and Perry, W. L . M., Biological Standards in Biochemical Analysis . . . , .. .... .......... , , . . ' , Htl/ner, S . H ., Bee S[()kstad, E. L . R . Jacobsen, C. F., Leonis, J., Linderstrpm-Lang, K. ! and Ottesen, M ., The pH-Stat and Its Use in Biochemistry ....... .... .. , .... . Jaques, IAuis B., and Bell, Helen J., Determination of Heparin .. Jukes, Thomas H ., Assay of Compounds with Folic Acid Activity. Kalckar, Herman M ., see Plesner, Paul Karnemaat, John N ., see Haines, William J. Kearney, Edna B., see Singer, Thomas P. Keenan, Robert G., see Saltzman, Bernard E. Kersey, Roger C. , and Fink, Frederick C., Microbiological Assay of Antibiotics. , ...... , . . , , ... .. , .. . ..... . . , . .... , . . . , .... , . Klotz, Irving M., see Hughe8, Thomas R . Kolin, Alexander, Rapid Electrophoresis in Density Gradients Combined with pH and/or Conductivity Gradients .. . . . . . . . . . Korn, Edward D., The Assay of Lipoprotein Lipase in Vivo and in Vitro . . , . ..... , , ... .. . .. , .. ' .. . , . .. , . . . . . . . . . . . . . . . . . . Kunkel, Henry G., Zone Electrophoresis .. . . , ... ... . . .. .. , .. , . . Lalla, Oliver F. de, and Go/man, John W., Ultracentrifugal Analysis of Serum Lipoproteins .. . ... .. .. .. . . . . ..... . , . . , . . . . .. , , . . Lazarow, Arnold, see Patterson, J. W. Lehman, Robert W., Determination of Vitamin E . , . . . . . . . . . . . . . See also Embree, Norris D.

PAGE

IV

257

II

359

VI

63

IV VI

285 289

VII

I

171

I II IV

113

I

205

III

265

V

65

IV VII II

171 253 121

I

53

VI

259

vn I

145 141

I

459

II

153

81 99

347

CUMULATIVE INDEX, VOLUMES I- VII VOL.

LAonia, J., see Jarobsen, C. F. Levy, A. L., see Fraenkel-Conrat, H . Levy, Ht'lton B ., see Webb, JuniWJ M. Lindberg, OZOV, and EmBter, Lar8, Determination of Organic Phosphorus Compounds by Phosphate Analysis . .. . .. .. ..... .... . Linderstr;m-Lang, K., see Jacobsen, C. F. Li8sitzky, Serge, see Roche, Jean Long, D. A., see Humphrey, J. H. Loveridge, B. A., and Smale8, A. A., Activation Analysis and Its Application in Biochemistry .. ..... ... .. .. . .. .......... . .. . Lundquist, Frank, The Determination of Ethyl Alcohol in Blood and Tissues ....... . ... . .... .. .. ... .. ....... .. .... .... . .. . McIntire, Floyd C., see Code, Charles F. Mackenzie, Cosmo G., see Frisell, Wilhelm R. ~AfcKibbin, John M., The Determination of Inositol, Ethanolamine, and Serine in Lipides . . ... ... . .. ... .. ..... ....... . .. ..... . Maehly, A. C., and Chance, Britton, The Assay of Catalases and Peroxidases ..... ... ... .. .... .. .. . ......... . ... . ......... . Malmstrom, Bo G., Determination of Zinc in Biological Materials. :Afargoshes, Marvin, and Vallee, Bert L ., Flame Photometry and Spectrometry: Principles and Applications ... . .. .... ... . .. . . lvfeister, Alton, see Aspen, Anita J. Michel, Raymond, see Roche, Jean Mickelsen, Olaf, and Yamamoto, Richard S ., Methods for the Determination of Thiamine ..... .... . ........... . ......... . Miller, Herbert K., Microbiological Assay of Nucleic Acids and Their Derivatives .... .. . .. ..... ... ........ .. . . . ....... .. . Montgomery, Rex, see Smith, Fred Neish, William J. P., a-Keto Acid Determina.tions . ....... .. .. . Novelli, G. David, Methods for Determination of Coenzyme A . . . . Ottesen, M., see Jacobsen, C. F. Patterson, J . W., and Lazarow, Arnold, Determination of Glutathione . .. . ... ...... ... ... ... ............ . . . ... . ... . .... . PemJ, W. L. M., see Humphrey, J. H. Persky, Harold, Chemical Determination of Adrenaline and Noradrenaline in Body Fluids and Tissues . ........ . ........ ... . Plesner, Paul, and Kalckar, Hernw,n M., Enzymic Micro Determinations of Uric Acid, Hypoxanthine, Xanthine, Adenine, and Xanthopterine by Ultraviolet Spectrophotometry ............ . Porter, Curt C., Bee Si lber, Robert H . Raaflaub, Jurg, Applications of Metal Buffers and Metal Indicators in Biochemistry .. . . ..... . . .... . ............. . ....... . ... . Radin, Norman S ., Glycolipide Determination .. .. ... ..... ..... . Roche, Jean, Lissitzky, Serge, and Michel, Raymond, Chromatographic Analysis of Radioact.ive Iodine Compounds from the Thyroid Gland and Body Fluids .... . ........... . ...... ... . Roe, Joseph H., Chemical Determinations of Ascorbic, Dehydroascorbic, and Diketoguionic Acids ........... . .. .. ... . . .. . . . Rosenkrantz, Harris, Analysis of Steroids by Infrared Spectrometry, Infrared Analysis of Vitamins, Hormones, and Coenzymes ..... Salmon, W . D., Bee Engel, R. W.

PAGE

III

1

V

225

VII

217

vn

III

I III

357 327

III

353

VI

191

VI

31

V II

107 189

II

259

II

57

III

97

III VI

301 163

I

243

I II V

115 1 407

34

CUMULATIVE INDEX, VOLUMES I- VII VOL.

Saltzm.an, B ernard E ., and Keenan, Robert G., Microdetermination of Cobalt in Biological Materials . ... . .. . .. .. . . . . . . .. . ... . . . Schubert, Jack, Measurement of Complex Ion Stability by the Use of Ion Exchange Resins . ... ... .. ... . .. ... .. . . ..... . . . . . . . . Seaman, G. R., soo Stokstad, E. L. R . Shibata, Kazuo, Spectrophotometry of Trauslucent Biological Materials; Opal Glass Transmission Method . ... .... .. . . ... . Silber, Robert H ., and Porter, Curt C., Determination of 17,21-Dihydroxy-20-ketosteroids in Urine and Plasma ... . .. .. .. . . .. . . S inger, Tharnas, P ., and Kearney, Edna B ., Determination of Succinic Dehydrogenase Activity . .. ... .. . .... . . . . . ...... . . . Smales, A . A ., soo Loveridge, B. A. Smith, Emil L ., soo Davis, Neil C. Smith, Fred, and Mantgamery, R ex, End Group Analysis of Polysaccharides . . ..... . . . .... .. . . . .. . . .... .. .. . ... .. . ..... .. . Smith, Uu.cile, Spectrophotometric ASRay of Cytochrome c Oxidase Spencer, B., see Dodgson, K . S . Sperry, Warren, M. , Lipide Analysis .. . . . . ... . .. .. .... . ... . . . . Stokstad, E . L . R ., Seaman, G. R ., Davis, R . J ., and Hunter, S . H ., Assay of Thioctic Acid . .... . . . . ... ..... . . . . ...... . ... . ... . Strehler, B. L. , and Totter, J . R ., Determination of ATP and Related Compounds: Firefly Luminescence and Other Methods . . Thiers, Ralph E ., Contamination in Trace E lement Analysis and Its Control. . . . . . ... . .. . .. . . .. . .... ... .. ... . . . ... ... . . . . . Thorpe, W . V., see Bray, H. G. TolkadorJ, Sibylle, The in Vitro Determination of Hyaluronidase . . Totter, J. R., soo Strehler, B . L. Udenfriend, Sidney, Weissbach, H erbert, and Brodie, Bernard B., Assay of Serotonin and Related Metabolites, Enzymes, and Drugs . . ....... .. . .. . . . . . . , . ... . . . . . . . . ... .. . . . . .. ... . . . . Vallee, Bert L ., soo Margoshes, Marvin Van Pilsum, John F., D etermination of Creatinine a.nd Relatf'd Guanidinium Compounds . . .. . . . . . . ..... . ...... . .. .. ... . . . Volkin, Elliot, and Cohn, Waldo E ., Estimation of Nucleic Acids .. Webb, Juniu8 M ., and Levy, Hi lton B ., New Developments in the Chemical Determination of Nucleic Acids . . .. . ... . . . ... . . . . . Wei88bach, H erbert, soo U denf riend, Sidney. Whalley, H. C. S. de, and Gr088, D ., Determination of Raffinose and Kestose in Plant Products ... . .... . . ..... . . . . .. . . . . ... . WhiiJen, D. H., soo Barker, S . A. lVinzler, Richard J ., Determination of Serum Glycoproteins . . . . . . Yamamoto, Richard S., see M ickelsen , Olaf

PAGE

V

181

III

247

VII

77

IV

139

IV

307

HI II

153 427

II

83

III

23

I

341

V

273

J

425

VI

95

VII I

193 287

VI

1

I

307

II

279

V

225

III

97

I

171

Subject Index Activation Analysis and 11.8 Application in Biochemistry (Loveridge and Smales) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenine, Enzymic !Ificro Determination, by Ultraviolet Spectrophotometry (Plesner and Kalckar) . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenal Gland, Steroid8 of, Chrorna.tographic Separatian (Haines and Karnemaat). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .

349

CUMULATIVE INDEX, VOLUMES r-Vll VOL.

Adrenaline, Chemical Determination, in Body Fluids and Ti8sues (Persky) . . .. . .... .. ...... .. ... ... .... . ........... . ..... . Amino Acids, Determination by Use oj Bacterial Amino Acid Dccarboxylasc8 (Gale) ..... . . ... . . .......... . ... ..... .... ... . A ntibiotic8, Microbiological A8say (Kersey and Fink) ........ ... . Ascorbic Acid, Chemical Determination (Roe) . ...... . ........ . . . ATP, Detennination by Firefly Luminescence (Strehler and Totter) BCU'terial A ?nino Acid Decarboxylase8 in Determination of Amino Acids (Gale) ......... ... ... .......... .. ..... ..... . ...... . Biological Materials, Determination oj Zinc in (Malmstrom) ..... . Biological Materials, Microdetermination oj CobaU in (Saltzman and Keenan) ... .. . ...... ...... ... . . ... . . ............. . .. . Biological Materials, Translucent, Spectrophotometry oj; Opal Glas8 Method ( hibata) . ... ......... ................ . .... . . Biological Standards in Biochemical Analysis (Hu mphrey, Long, and Perry) ......... ... ..... ..... .. ........ . ..... . .... .. . Biolorrical Systems, Determination oj Serine in (Fril!ell and Mackenzie) ..... . . .. .. .. ... .... ... .... ...... . ........ .. . . Blood, Determination oj Ethyl Alcohol in (Lundquist) ........... . Body Fluids, Chemical Determination of Adrenaline and Noradrenaline in (Persky) ...... .. ................. . . . .. .. ... . Body Fluids, Chromatographic AnalY8is oj Radioactive Iodine C~ pounds from (Roche, Lissitzky, and Michel) .. ....... . ... . ... . Body Tissues, Chemical Determination oj Adrenaline and Noradrenaline in (Persky) .......... . .... . ... ... .......... .. . . Carbohydrate Structure, U8e oj Infrared Analysis in Determination (Barker, Bourne, and Whiffen) . . ..... . . . ...... .... .... .. .. . Carotene, Detennination (Bickoff) .......... . ............ . .... . Catalase8, Assay (Maehly and Chance) .. .. ......... .. .... . .. . . Cellulose Column Chromatography, Jor Analysis oj Mixtures of Sugars (Hough) . .... . ........... . .... ................. .. . Choline, Chemical Estimation (Engel, Salmon, and Ackerman) . .. . Cholinesterases, Assay MetllOds for (Augustinsson) .. . ........... . Chromatographic AnalY8is of Radioactive Iodine Compounds from the Thyroid Gland and Body Fluids (Roche, Lissitzky, and Michel) . Chromatographic Separation of Steroids oj the Adrenal Gland (Haines and Karnemaat) ........................ . ........ . Chromatography, Paper and Cell-ulose Column, for Analysis of Mixtures oj Sugar8 (Bough) .. . .............. . .............. . . . Cobalt, Microdetermination of, in Biological Materials (Saltzman and Keenan) ... ...... . ......... . .. ....... ... . .. ... .. . . . . . Coenzyme A, Methods lor Determination (Novelli) . . .. ....... . .. . Coenzymes, Infrared Analysis oj (Rosenkrantz) . . . ...... , ..... . . Color Reactions, New, for Determination of Sugars in Polysaccharides (Dische) . ... . .. . .. .. ................... .. ...... .... .. .. . Complexes, Metal-Protein AnalY8is (Hughes and Klotz ) ......... . Complex Ion Stability, 111easurement Inj Use of Ion Exchange Resins (Schubert) . ............. .. ......... . ... . .. . ............. . Contamination in Trace Element Anal1/sis and Its Control (Thiers). Creatinine and Related Guanidiniulll Compounds, Determination of (Van Pileum) .... . .... . .... . ..... ......... . . .... . ....... .

PAGE

II

57

IV I I I

285 53 115 341

IV III

285 327

V

181

VII

77

V

65

VI VII

63 217

II

57

I

243

II

57

III IV

213 1 357

I

I

205 265

V

1

I

243

I

171

I

205

V II V

181

I

189

407

III

313 265

III V

247 273

VII

193

II

350

CUMULATIVE INDEX, VOLUMES I- VIl VOL.

Cytochroml! c Oxida8e, Spectrophotol1utric A8say (Smith) . .. ...... . Dehydroascorbic Acid, Chemical Determination (Roe) ......... . . . 17,fl-Dihydroxy-fO-ketosteroids, Determination in Urine and Plasma (Silber and Porter) ...... ...... .. ... .. .. .. . . ....... . . ... . . Density Gradients, Rapid Electrophoresis in (Kolin) ............. . Diketogulonic Acid, Chemical Determination (Roe) . ...... ..... .. . Electrophoresis, Rapid, in Density Gradients Combined with pH and/or Conductivity Gradients (Kolin) .......... . ...... .. ... . Electrophoresis, Zone (Kunkel) ... ... .. .......... . ..... . ..... . Enzymu, Proteolytic, Assay (Davis and Smith) .... . ..... . ..... . Enzymu, Related to Serotonin, Assay of (Udenfriend, Weissbach, and Brodie) . ... .............. . .. .. ..................... . Estrogens, Chemical Determination of, in Human Urine (Bauld and Greenway) .... .. . ...... ... ...... .. ................. . . . . . Ethanolamine, Determination of, in Lipides (McKibbin ) . ....... . Firefly Luminescence, Detemlination of ATP by (Strehler and Totter) ............. .. . ..... . ... . . . ......... . ....... . . . .... . Flame Photometry, Principles and Applications (Margoshes and Vallee) ..... . ........ . ........ .. .......... . ............. . Fluids, Body, Chemical Determination of Adrenaline and Noradrenaline in (persky) ................................... . Fluids, Body, Chromatographic Analysis of Radioactive Iodine Compounds from (Roche, Li88itzky, and Michel) ... .. .. . ........ . Folic Acid Activity, Assay of Compounds with (Jukes) . . ..... . .. . Fommldehyde, Determination of, in Biological Systems (Frisell and Mackenzie) . . ...... . ..... .. . ... .. .... .. . . ........ .. .. ... . Glutathione, Determination (Patterson and Lazarow) . .......... . Glycolipide Determination (Radin) .. ......... . ............ . .. . Glycoproteins, Serum, Determination (Winzler) . ................ . Gradients, Density, Rapid Electrophoresis in (Kolin) ....... .. .. . . Heparin, Determination of (Jaques and Bell) ....... . ..... . ... . . Hexosamines, Determination of (Gardell) .. . .. .. ..... ... .. ..... . Histamine, Quantitative Determination (Code and McIntire) .. . .. . Hormones, Infrared Analysis of (Rosenkrantz) .. ....... ........ . Hyaluronida8e, in Vitro Determination (Tolksdorf) ............ . . Hypoxanthine, Enzymic Micro Determination, by Ultraviolet Spectrophotometry (Plesner and Kalckar) . . . . .. . . ....... ....... . Immunoelectrophoretic A.nalysis (Grabar) ... . . ....... .. . .... . . . . Infrared Analysis, Use of, in the Determination of Carbohydrate Structure (Barker, Bourne, and Whiffen) . . ..... .. ... . . .. ... . Infrared A.nalysis of Vitamins, Hormones, and Coenzymes (Rosenkrantz) .... ............... . ........ . . ..... . . . .... ...... . Infrared Spectro11U!try, Analysis of Steroids by (Rosenkrantz) .... . Inositol, Determination 0/, in Lip!.du (McKibbin) .... ...... .... . Iodine Compounds, Radioactive, from Thyroid Gland and Body Fluids Chromatographic Analysis (Roche, Lissitzky, and Michel) . Ion Exchange Resins, Measure11U!n.t of Complex Ion Stability by U8e of (Schubert) ................ . ....... .... ... . .. . . . .. . ... . o-Keto Acid Determinations (N eish) . . .... . ................... . Keatose, Determination, in Plant Products (de Whalley and Gross). 17-Ketosteroids, Urinary Neutral, A8say (Engel) .. .. , ... . .... . . .

II

PAGE

I

427 115

IV VI I

139 259 115

VI

J

II

259 141 215

VI

95

V

337

VII

111

I

341

III

353

II

57

I II

243 121

VI

63

II VI II

259 163 279 259

VI VIl VI

III V I

253

289 49 407 4.25

VII

III

97 1

III

213

V II

VII

407 1 111

I

243

III

247 107 307 459

V I I

351

CUMULATIVE INDEX, VOLUMES I- VI! VOL.

Lipase, Lipoprotein, A88ay of, in Vivo and in Vitro (Korn). .. . . . . Lipide AnalY8is (Sperry). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipides, Determination of Inositol, Ethanolamine, and Serine in (McKibbin) .... .. .... . .. ..... . . ........... . .... ........ Lipoprotein Lipase, AS8ay of, in Vivo and in Vitro (Korn) . . . . . . . Lipoproteins, Serum, UUracentrifugal Analysill (de Lalla and Gofman). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipoxidase Activity, Measurement (Holman). . . . . . . . . . . . . . . . . . . . Metaboli8m, AnalY8i8 of Phenolic CompoundB of Intere8t in (Bray and Thorpe). . . . . . . ........... . . ............. ......... .. Metal Buffers, Applicatio1t8, in Biochemistry (Jiirg).... .......... Meta! Indicators, Applications, in Biochemistry (Jiirg). . . . . . . . . . . Metal-Protein Compleu8, Analysi8 (Hugbes and Klotz) . ... . ' " . . Microbiological A8say of Antibiotics (Kersey and Fink) . . . . . . . . . . Microbiological AS8ay of Vitamin Bll (Hoff.Jjilrgensen)... . . . . . . . . Nitrogenous Compounds, Basic, of Toxicological bnportance, Analysis of (Curry) . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . .. . . Noradrenaline, Chemical Determination, in Body Fluids and Tissues (Persky) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleic Acids, Chemical Determination of (Webb and Levy) . . . . . . Nucleic Acids, Estimation (Volkin and Cobn). . . . . . . . . . . . . . . . . . Nucleic Acids and Their Derivatives, Microbiological Assay of (Miller) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organic Phosphor'u.s Compounds, Determination of, by Phosphate Analysis (Lindberg and Ernster). . . . . . . . . . . . . . . . . . . . . . . . . . . OxidatiOnB, Periodate, Use of, in Biochemical Analysis (Dyer) . . . . . Paper Chromatography,Jor Analy81:s of Mixtures of Sugar8 (Hough). Peptides, Terminal and Sequence Studies in, Recent Developments in Techniques for (Fraenkel-Conrat, Harris, and Levy) . .. . . . . . . . . Periodate Oxidations, Use of, in Biochl:mical Analysi8 (Dyer) . . . . . Peroxidases, Assay (MaebIy and Cbance) . . . . . . . . . . . . . . . . . . . . . . Phenolic Compounds of Interest in Metabolism (Bray and Thorpe). Phosphate Analysis, Determination of Organic Phosphorus CmT/,poundB by (Lindberg and Ernster). . . . .. . .. . . . . . ... . . ... .... Phosphorus CompoundB, Organic, Determination of, by Phoaphate Analysi8 (Lindberg and Ernster). . . . . . . . . . . . . . . . . . . . . . . . . . . Photometry, Flame, Principles and Applications (Margoshes and Vallee) ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH-Stat and Its U8e in Biochemistry (Jacobsen, L60nis, Linderstrfijm-Lang, and Ottesen) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma, Determination of 17,21-Dihydroxy-eG-ketosteroid8 in (Silber and Porter) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polysaccharides, End Group Analysis (Smith and Montgomery). . . PolY8accharide8, Sugars in, New Color Reactio1t8 for Determination (Dische). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyunsaturated Fatty Acid8, MeCl8t"ement (Holman) ......... " . Protein, Terminal and Sequence Studies in, Recent Developments in Techniques for (Fraenkel-Conrat, Harris, and Levy) . . . . . . . . . . Proteolytic Enzymes, Assay (Davis and Smith). . . . . . . . . . . . . . . . . Purines, New M ethod8 for Purification and Separation of (Bergmann and Dikstein) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PAGE

VII II

145 83

VII VII

145

I II

459 113

I III

27 301 301

III III

111

I I

265 53 81

VII

39

II VI I

57 287

VI

31

III III I

1 III

II

HI I I

1

205

359 111 357 27

III

III III

353

IV

171

IV III

139 153

II IV

313 99

II II

359 215

VI

79

352

CUMULATIVE INDEX, VOLUMES ' I- VJI VOL.

Radiooctive Iodine Compounds,jrom Thyroid Gland and Body Fluids, Chromatographic Analysis (Roche, Lissitzky, and Michel), , ... . Raffinose, Determination in Plant Products (de Whalley and Gross) Resins, Ion Exchange, Measurement of Complex Ion Stability, by Use of (Schubert) ....................... . ............... . Serine, Determination of, in Biological Systems (Frisell and Mackenzie) . .............. ... ...... . ..... .. ............... . . . Seline, Determination of, in Lipides (McKibbin) ......... .. .... . Serotonin and Rel.ated Metabolites, Enzymes, and Drugs, Assay of (Udenfriend, Weissbach, and Brodie) . , ........... . ........ . Serum Acid Phosphalases, Determinations (Fishman and Davidson) . Serum Glycoproteins, Determination (Winzler) ...... ......... .. . Serum Lipoproteins, Ultracentrijugal Analysis (de Lalla and Gofman) ......... . ...... .. .......... . ...... . .............. . Spectrometry, Infrared, Analysis of Steroids by (Rosenkrantz) .... . Spectrometry, Principles and Applications (Margoshes and Vallee). Spectrophotometric A ssay of Cytochrome c Oxidase (Smith) ... .... . Spectrophotometry of Translucent Biological Materials; Opal Glass Method (Shibata) .. ..... . .. . . ... . . . .......... . ... . ....... . Spectrophotometry, Ultraviolet, Enzymic Micro Determinations of Uric Acid, Hypoxanthine, Xanthine, Adenine, and XantMpterine by (plesner and KaJckar) ................ ... ...... ... ..... . Standards, Biological, in Biochemical Analysis (Humphrey, Long, and Perry) .... .... . .. .......... ...... ........... ....... . Steroids, of the Adrenal Gland, Chromatographic Separat1:on (Haines and Karnemaat) . ..... .... .. .... .... .... .......... . ..... . Steroids, Analysis by I nfrared Spectrometry (Rosenkrantz) ....... . Succinic Dehydrogenase Activity, Determination ( inger and Kearney) ...... . . . . . . . ................ ... ................... . Sugars, Analysis of Mixtures, by Paper and Cellulose Column Chromatography (Hough) ........ . . . ............ . ... . ..... . Sugars, in Polysaccharides, Determination, New 'Color Reactions/or (Dische) . . . ....... .. ...... . . . . . . .... ..... . .. ... . ... ... . . Sulfatases, Assay (Dodgson and Spencer) .... . ..... . ... .... .. . . Stdfhydryl Groups, Determination in Biological Substances (Chi nard and Hellerman) ... ... .......... . .... . ... .. ... .. . .. ...... . Thiamine, Methods for the Determination of (Mickelsen and Yamamoto) ... . . .... .. .......... . .. .......... . . . . . ...... ..... . Thioctic Acid, Assay (Stokstad, Seaman, Davis, and Rutner) ... . Thyroid Gland, Chromatographic Analysis of Radioactive Iodine Compounds from (Roche, Lissitzky, and Michel) ....... . .. . .. . Tissues, Body, Chemical Determination of Adrenaline and Noradrenaline in (Persky) .... ...... .. ..................... .. . Tissues, Determination of Ethyl A lcohol in (Lundquist) .... .. .. . . . Trace Element Analysis, Contamination in, and Its Control (Thiers). 7'raMaminase, Determination of (Aspen and Meister). " . ....... . UUracentrifugal AnalY8is of Serum Lipoproteins (de Lalla and Gorman) . .. .......... . .. .. .. . ....... . .. ................... . Ultraviolet Spectrophotometry, Enzymic Micro Determinations of Uric Acid, Hypoxanthine, Xanthine, Adenine, and Xanthopterine by (plesner and Kalckar) ... ... ...... . . ...... . ...... . . ... , .

PAGE

I I

243 307

III

247

VI

63

VII

111

VI

95 257 279

IV

II I II

459

III II

353 427

VII

77

ITI

97

V

65

I II

171 1

IV

307

I

205

IV

II

313 211

I

1

VI III

191 23

I

243

VII V VI

II

57 217 273 131

I

459

III

97

1

353

CUMULATIVE INDEX, VOLUMES I-VII VOL.

Uric Acid, Enzymic Micro Determinations, by UUraviolet Spectrophotometry (plesner and Kalckar) .......................... . Urinary Neldral17-Ketosteroids, A8say (Engel) ..... .... ....... . Urine, Determination of 17,e1-Dihydroxy-:eO-keto8teroids in (Silber and Porter) ............................................. . Urine, Human, Chemical Determination of E8trogens in (Bauld and Greenway) . .... ..... .. ....... . . ........ .. ..... . ........ . Vitamin A , Determination (Embree, Ames, Lehman, and Harris). Vitamin B 12 , Microbiological AS8ay (Hotf-Jf/Jrgensen) . .. . .. .. . .. . Vitamin E, Determination (Lehman) ........ . ...... .. ........ . Vitamins, Infrared Analysi8 of (Rosenkrantz) .. . .............. . Xanthine, Enzymic Micro Determination, by Ultraviolet Spectrophotometry (Pleaner and Kalckar) . . . .... . .......... ... ..... . Xanthopterine, Enzymic Micro Determination, by UUraviolet Spectrophotometry (pleaner and Kalckar) ................. . ..... . Zinc, Determination of, in Biol()gical Mater7:a"Ls (Malmstrom) .... . Zone Electrophoresi8 (Kunkel) ............... . .. . ..... . .. .. .. .

PAGE

III

I

97 479

IV

139

V IV I V

337 43 81 153 407

III

97

III

97 327

II

III I

141

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