Idea Transcript
MEASUREMENT BY
HERBERT (From
OF THE
RATE
C. TIDWELL, CAROLYN WOOD,t AND WILLIAM the Department Southwestern (Received
of Biochemistry, Medical School, for publication,
OF FAT
ABSORPTION*
DUNKELBERG, W. BURR, JR. The University Dallas, Texas) August
WILLIAM
of
A.
Texas,
1, 1955)
* This investigation was supported Powder Company and the Lipotropic t Chilton Foundation Fellow.
in part Research 733
by a research Fund.
grant
from
the
Atlas
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The rate of fat absorption has generally been measured either directly by determining the amount of lipide remaining in the alimentary tract after feeding fat, or indirectly by following the appearance of fat in the blood stream. The latter measurement is known to be influenced by factors other than the actual absorption of fat, such as the rate of removal of the lipides from the blood. This rate, of course, is determined by the speed of lipide utilization and deposition, as well as any mobilization from the tissues. Hence, the direct recovery by an adaptation of the Cori (1) method, introduced by Irwin et al. (2) and later modified by Deuel and coworkers (3), seemsto have been preferred in studies of fat absorption with animals. The findings of the present study indicate that the results obtained with the direct method may also be influenced by factors other than the absorption of fat. Lipide recovered from the alimentary tract shortly after feeding C14-labeledfat contained considerably less activity per mg. of lipide than did the fed fat. A similar finding has been reported by Borgstrom (4). These observations suggested that it might be worth while to compare the results obtained with a number of different methods which have been used for determining, either directly or indirectly, the rate of fat absorption. Then, too, it would be advantageous to have additional information regarding the limitations of some of these indirect methods since the direct procedure is necessarily limited to studies with animals. The various determinations studied have involved the appearance of the ingested labeled fat in the blood and tissues, its disappearance from the alimentary tract, and its utilization. The rate of appearance of the fat in the blood was measured by both the chylomicrograph and the activity of the blood, whereas its disappearance from the alimentary tract was determined by the amount as well as the activity of the fat recovered. The excretion of the labeled carbon dioxide was employed as a measure of utilization.
734
MEASUREMENT
OF
FAT
ABSORPTION
EXPERIMENTAL
Methods The method of direct mounting of blood samples for Cl4 determinations and the technique for chylomicron counting have been described in detail elsewhere (6). Areas under the curves were calculated from the 1 to 4 hour values of chylomicron counts as well as those of the activities of the blood and expired CO2 of each animal. The alimentary tract and the liver of each rat were homogenized, and the lipides were extracted with ethanol and ether. The remaining carcass was ground, and similarly the fat was extracted from an aliquot of the tissue. The radioactivities of the extracted lipides and residual tissues were determined after wet oxidation of aliquots 1 Cerelose York. 2 Palmitic Massachusetts.
generously acid-l-Cl4
supplied
by
(1 me. per mmole)
the
Corn
Products
was obtained
Refining from
Tracerlab,
Company, Inc.,
New Boston,
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Procedure-Young adult male rats of the Sprague-Dawley strain received only water and 5 per cent glucose1 ad libitum during the 48 hours preceding the study. The animals were maintained at about 26” and initially conditioned to the metabolism cage by being placed in it for 3 or 4 hours a day for several days. Each rat was weighed, and a blood sample taken from the clipped tail for chylomicron counts. The rat was then given, by tube, 0.3 ml. of lipide (1.3 per cent palmitic acid-l-Cl4 in olive oi12) per sq. dm. of body surface and placed in an all-glass metabolism cage (5). Air freed of COZ was drawn through the metabolism cage at the rate of about 500 ml. per minute. The expired CO2 was trapped in N NaOH in 4 hour intervals for 4 hours. Blood samples were collected from the tail of the rat at the end of each hour for direct mounting on planchets, for carbonate formation after wet oxidation, and for use in chylomicron counting. The portion of the alimentary tract from the esophagus to the cecum was removed from the anesthetized rat at the end of the experimental period, and the unabsorbed fat was recovered by flushing the tract with 100 ml. of ether. The recovered lipides were freed of solvent and weighed in order that the amount of absorbed fat could be calculated. No corrections were made for the lipide in the intestine after the 48 hour preparation period, since the 25 mg. correction factor was approximately canceled by the 5 per cent loss in immediate recovery. The lipide residues were dissolved in petroleum ether, aliquots of the recovered as well as the ingested lipides were wet ashed, and the specific activity of each was determined. This permitted another calculation of lipide absorption on the basis of the loss of labeled lipide from the intestinal tract. The total activities of extracted lipides and the residual tissues of the organs and carcass were determined as an additional check on the total absorbed fat.
TIDWELL,
DUNKELBERG,
WOOD,
AND
735
BURR
to CO2 with a modified Van Slyke-Folch (7) oxidizing mixture and subsequent conversion of the CO2 to BaC03. Specific and total activities were obtained as previously described (6) on mounts of infinite thickness. RESULTS
AND
DISCUSSION
The amounts of lipide absorbed, as measured by the recovery method and by activity determinations on the fed and recovered lipide (Table I), are expressed in mg. per sq. dm. of body surface, since evidence has been I
TABLE
Bate
of Fat
Absorption
As Measured
by Direct
Methods
I
Total
-
Rat No. Fed
Average. * Relative lated material t Calculated
recovery
i
From activity
/
-
i4mount I ‘erpersq.hr.dm.
Lipide Amount
determinations
absorbed
Recovered
Per sq. Relative dm. per h r. activity* i
cent
lipide
9mount xretedt __~ mg.
Dilution of fed fat
cent
m&T.
mg.
nzg.
mg.
m.
804 978 822 786 786
324 413 234 154 140
27.7 28.9 19.5 13.5 12.2
490 726 573 524 501
41.9 50.8 47.6 45.8 43.5
65.5 44.6 42.3 41.5 44.1
166 313 339 370 361 -~
20.7 32.0 41.2 47.1 45.9
835
253
20.4
563
45.9
47.6
310
37.4
-
fier
activity is defined here as the ratio of the specific activity to the specific activity of the fed lipide X 100. from the amount and relative activity of the recovered
per
of the
iso-
lipide.
presented to suggest that this measurement more truly represents the rate of fat absorption (3, 8). The values for absorption based on activity are calculated from the difference in the total activities of the fed lipide and that recovered from the alimentary tract. The assumptions are made that the administered fat is absorbed as a homogeneous entity and that the disappearance of C14-labeledlipide reflects absorption of the test meal. The rates of lipide absorption, as calculated by these two methods, are in poor agreement. The absorption values based on activit.y present only such variations as are to be expected from animal to animal, and are more uniform than those based on total recovered lipide. The specific activity of the recovered lipide is considerably lessthan that
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Rats were given olive oil containing labeled fatty acid. Lipide absorption was determined after a 4 hour absorptive period by both an adapted Cori technique and by calculat.ion from the total activity of the fed and recovered lipide. The secreted lipide was calculated to be that in the tract in excess of the ingested labeled fat recovered.
736
MEASUREMENT
OF
FAT
ABSORPTION
of the fed fat (Table I). Borgstrom (4) has reported an analogous mean value of 48.8 per cent which agrees closely with the mean of 47.6 per cent obtained in this study. If the lowered specific activity of the lipide recovered from the intestine is explained by a secret,ion of body lipides into the tract, then the amount of dilut.ing lipide can be calculated from the amount and activity of the lipides recovered. Based on this assumption, TABLE Results
jrom
Methods
II
Rate of Fat Absorption
Determining
was determined by two direct methods which inof the fat ingested and recovered. Three relative counts, blood fat activity, and CO2 activity) were
-
Direct methods
Indirect
Fed lipide absorbed Rat No.
I
Alimentary Cori per cent
Average.
tract Activity per cent
methods
Area under curves, l-4 brs.
in COn -‘;‘at,abeltissues*
Chylomicron countst
Blood fat activityi
.abeled CO* exhaleda
ger cent
40.3 42.2 28.5 19.6 17.8
61.0 74.2 69.7 66.7 63.8
51.1 46.2 37.7 51.2
365 222 375
336 245 378
331 231 401
29.7
67.1
46.6
321
320
321
* In per cent of total CP fed. This did not include which was not calculated since total blood volume carcass activity was corrected for mechanical loss. t Average of counts of chylomicrons in 15 squares $ Average of relative blood activities X 36.5. $ Average of relative CO, activities X 59.
the total activity of the blood, could only be estimated. The of micrometer.
the calculated amounts secreted varied from 21 to 47 per cent of the amount fed. In Table II the results of the direct methods are expressedin percentages of the lipide and activity ingested, and are listed along with those of some indirect methods for determining the rate of fat absorption. For greater ease in comparison, t’he latter values, which are only relative ones, have been multiplied by constants which make their averages approximately equal to the average of the chylomicron counts for 15 squares of the micrometer used. Also included in Table II are the percentages of the total
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The per cent of fat absorbed volved the amount and activity or indirect methods (chylomicron also employed.
Used for
TIDWELL,
DUNKELBERG,
WOOD,
AND
BURR
737
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activity fed which were recovered from the respiratory COZ, extracted lipides, and tissues of each rat. Two facts are evident from these data (Table II). First, although a complete recovery of activity from the animal was not attempted, the summations of these activities are incompatible with the absorption data based on recovery of the unabsorbed lipide from the intestinal tract. Secondly, the amount of absorption of t.he labeled fat is 1.5 to 3.5 times that. indicated by the recovery of total lipides from the intestinal tract. The results in percentages are also considerably more variable by the latter method, even though the variations are within the range of those reported by Irwin and coworkers. Hence, it appears that these two methods may not be measuring the same phenomena or, if so, at least one of them is inaccurate in its measurement. The markedly slower rate of absorption, as determined by the generally accepted recovery method, might be accounted for in either of two days: (1) A selective or more rapid absorpt’ion of the labeled free palmitic acid would give a false measure of t’he rate of absorption of the lipide mixture ingested and explain the lack of agreement of the two methods used. (2) Secretion of lipide into the alimentary tract during the absorptive period could account for the dilution of the lipide recovered and the results oFt: incd. Bergstriim and coworkers (9) have reported that an examination of the chyle indicates a more rapid absorption of the labeled free fatty acid when fed dissolved in corn oil. However, the absorption rate was found to be the same for the labeled fatty acids, both free and incorporated into glycerides, when measured by recovery from the alimentary tract or by the CO2 expired. Also, t.he finding of a considerable amount of the labeled acid fed in an esterified form in the lipides recovered from the intestine (4, 10) indicates that not all of the labeled free acid was rapidly absorbed as fatty acid. Thus, part of this evidence does not appear to support the contention that the free fatty acid is preferentially absorbed. In the present study, the mean activit’ies of t]he blood and the expired CO2 (Fig. 1) are in good agreement with t’he chylomicron counts. These curves, as well as the previously report.ed (6) individual curves depicting the rate of change of the level of particulate blood fat and its activity, almost parallel each other and give no indication that one part of the ingested lipide is preferentially absorbed. If there had been a more rapid absorption of the labeled fatty acids, it seems unlikely that the chylomicron and activity curves would have paralleled each ot,her during the absorptive period. Furthermore, if a select,ive absorption were taking place, it would be expected that in the lat)er periods the slope of the CO2 activity curve I\-ould decrease. This change should occur prior to a decrease in chylomicron count,. It did not> occur, at least within t)he first 4 hours after fat
738
MEASUREMENT
OF
FAT
ABSORPTION
ingestion (Fig. 1). These data would seem to agree better with the interpretation that an appreciable amount of lipide is secreted into the alimentary tract during the period of fat absorption. The hypothesis of secretion into the tract need not entail the concept of total loss of this fat from the body. It may represent a process in which a major part of the lipide secreted is reabsorbed and only the remaining portion excreted. Sperry and Angevine (11) in much earlier work were able to report that there was a greater secretion of lipide into the ileum than could be recovered
1
2 HO”Rs3
4
FIG. 1. Changes in the mean values of chylomicron counts the blood and expired COz after ingestion of labeled lipide. counts per 15 squares; Curve 2, relative CO2 activity in c.p.m. blood activity in c.p.m. X 28.
and radioactivities of Curve 1, chylomicron X 56; Curve 3, relative
from the colon. These authors concluded that their results “indicate that relatively large amounts of lipids are secreted into the small intestine,” and that “A considerable portion of this secretion is reabsorbed.” However, neither the possibility of selective fatty acid absorption nor the secretion of endogenous lipide can be ruled out on the basis of the present data. Further studies on this point arc in progress. Earlier studies from this laboratory (6, 8) have demonstrated that chylomicron counts and areas under the curves from them may serve as satisfactory relative measures of the absorbed fat in the blood. If this is true, these curves should be useful as a measure of the rate of fat absorption and utilization in spite of factors other than absorption affecting the con-
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0
TIDWELL,
DUNKELBERG,
WOOD,
AND
BURR
739
SUMMARY
Carboxyl-labeled palmitic acid dissolved in olive oil was fed to rats, and the absorption of the lipide was followed by a number of direct and indirect methods. The lipide recovered from the alimentary tract had considerably less activity per mg. than the fed fat. The amounts of absorption based on the disappearance of activity from the tract were not in agreement with amounts determined by a modified Cori technique. It is suggested that the basis of this discrepancy may be either (1) a selective absorption of the labeled fatty acid or (2) a secretion of endogenous lipide into the tract during the absorptive period. Indirect methods for measuring fat absorption based on the CO2 expired, blood activity, chylomicrograph, and a recovery from the tissues mere studied. In the three animals in which comparisons were made, these relative measures were found to be in agreement. Thus, it would appear that all of these methods reflect the rate of absorption of the fat fed. BIBLIOGRAPHY
1. 2. 3. 4. 5.
.Cori, C. F., J. Bid. Chem., 66, 691 (1925). Irwin, M. H., Steinbock, H., and Templin, V. M., J. N&r., 12, 85 (1936). Deuel, H. J., Jr., Hallman, L., and Leonard, A., J. Nutr., 20, 215 (1940). Borgstrom, B., Acta physiol. Stand., 26,328 (1952). Roth, L. J., Leifer, E., Hogness, J. R., and Langham, W. H., J. Biol. Chem.,
176,
249 (1948). 6. Burr, W. W., Jr., Dunkelberg, C., McPherson, J. C., and Tidwell, H. C., J. Biol. Chem., 210, 531 (1954). 7. Van Slyke, D. D., and Folch, J., J. Biol. Chem., 136, 509 (1940). Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, R. M., and Yankwich, P. E., Isotopic carbon, New York (1949). S. Tidwell, H. C., J. Biol. Chem., 182, 405 (1950).
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centration of fat in the blood. The results from the three indirect methods (Table II) suggest that a direct relationship exists; all gave the same relative values. The same relationship is also seen in Fig. 1. This would indicate that all three measurements reflect lipide absorption in spite of the fact that different phenomena are being measured. The findings on expired CO, are in accord with those of other investigators (9, 12), who have reported that the amount of isotope expired is directly related to the amount of the fatty acid absorbed. A direct relationship also seems to exist between these three relative values and the amount of labeled fat which was recovered from the tissues, organs, and expired COZ. The results of the indirect methods do not indicate close agreement with those given by either of the direct procedures, but appear to be more in accord with the absorption values based on activity.
740
MEASUREMENT
OF
FAT
ABSORPTION
9. Bergstrijm, S., Borgstrijm, B., and Rottenberg, M., Acta physiol. Stand., 26, 120 (1952). and Biophys., 49, 268 (1954). 10. Borgstriim, B., Arch Biochem. 11. Sperry, W. M., and Angevine, R. W., J. Biol. Chem., 96, 769 (1932). 12. Reiser, R., Bryson, M. J., Carr, M. J., and Kuiken, K. A., J. Biol. Chem., 194, 131 (1952).
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MEASUREMENT OF THE RATE OF FAT ABSORPTION Herbert C. Tidwell, Carolyn Dunkelberg, William A. Wood and William W. Burr, Jr. J. Biol. Chem. 1956, 220:733-740.
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