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Changes in serum triglycerides, retinoic acid, and retinol associated with with drawal of retinoic acid from the diet of rats
NUTRITION RESEARCH, Vol. l , pp. 509-517, 1981 0271-5317/81/050509-09502.00/0 Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
CHANGES IN SERUMTRIGLYCERIDES, RETINOIC ACID, HiD RETINOL ASSOCIATED WITH WITHDRAWAL OF RETINOIC ACID FROM THE DIET OF RATS L. E. Gerber, L. S. Wasserman, B. H. S. Cho* and J. W. Erdman, Jr. Department of Food Science University of I l l i n o i s Urbana, I l l i n o i s 61801 and *Harlan E. Moore Heart Research Foundation Champaign, I l l i n o i s 61820
ABSTRACT Several experiments were performed to measure changes in serum triglycerides, retinoic acid, and retinol following removal of all-trans retinoic acid from the diet of rats previously fed 25 ug all-trans retinoic acid/g of diet for periods of 8-13 days. I n i t i t a l l y , rats had hypertriglyceridemia, but within 24-36 hours after withdrawal of retinoic acid, serum triglycerides returned to the level of control rats not fed retinoic acid. Serum retinoic acid also decreased after withdrawal, but did not return to the level of the control rats until 3 days afterwards. Serum retinol was depressed compared to the level of control rats until 72 hours after retinoic acid withdrawal. The fatty acid composition of the individual l i p i d fractions was shifted towards oleate and linoleate and away from palmitate and arachidonate after retinoic acid feeding. Following removal, restoration of a more normal pattern ensued. KEY WORDS: retinoic acid, vitamin A, retinol, triglycerides, hyperl i pi demia INTRODUCTION An elevation of serum triglycerides accompanies the administration of vitamin A at levels exceeding those normally ingested from dietary sources (I-3). Recently, clinical and experimental administration of vitamin A analogs (retinoids) has also been associated with hypertriglyceridemia (4-6). The mechanism by which this elevation occurs remains obscure, although various theories have been advanced (l, 5). Observations have also been made that administration of retinoic acid depresses the level of retinol in the blood of rat and man (3, 4, 7,8). The current study was designed to measure changes in serum l i p i d s , retinol and retinoic acid following removal of all-trans retinoic acid from the diet of rats administered this Compound for eight days. These changes were monitored for up to 72 hours after the last administration of all-trans retinoic acid. Specific goals were to determine the time required for removal of dietary retinoic acid from serum, the time required for restoration of normal triglyceride levels and the possible relationship between retinol and rezinoic acid serum levels in the retinoic acid-fed rat. 509
510
Gerber et al.
MATERIALS AND METHODS
Treatment of Animals Male Sprague-Dawley rats (Harlan Industries, Inc., Cumberland, IN) weighing 135-160 g were housed individually in hanging wire cages in a controlled temperature environment (22~ a light cycle from0600-1800 hours. The rats were fed an agar gel diet as described previously (3), composed of corn starch, sugar, casein, corn oii and adequate vitamins without vitamin A (Teklad, Madison, ~JI) and minerals (USP XVII), at the start of the dark cycle, with water offered ad libitum. Blood samples were taken by cardiac puncture between 0930-I030, after the rats were anesthetized with ether. The blood was allowed to clot, centrifuged at 1500 x g for 30 rain., and the serum was stored at -20 ~ until analysis. For the second experiment, blood was taken, rats were guillotined, and their livers removed, washed with d i s t i l l e d water, pattc~ dry, weighed and stored at -20 ~ until analysis. Extraction of Serum Retinol and Retinoic Acid for HPLC Analysis All handling of vitamin A compounds was done in indirect light. The serum extraction technique used was a modification of the procedure of Ito et al. (9). The solvent extract was evaporated to dryness under nitrogen and its residue dissolved in 200 ul methanol for use in HPLC. Samples of serum plus added retinol, retinoic acid and 14C-alltrans retinoic acid dissolved in methanol, in the range to be assayed in serum, were processed through this same procedure to assess the adequacy of extraction. Similar standards of retinol and retinoic acid were chromatographed and their peak heights used as a measure of the linear response of the compounds. High-Pressure Liquid Chromatography All chemicals and solvents used were analytical reagent grade. Reversed-phase HPLC separation and analysis were done on a DuPont Instruments Zorbax ODS column (4.0 mm x 25 cm). T~e mobile phase contained methanol: 1.0% ammonium acetate (80:20) and was degassed and f i l t e r e d through Whatman No 4 paper before i t was pumped through the system isocratically at l . l ml/min. The peaks were recorded at the maximum sensit i v i t y (0.005 AUFS). In this system retinol and retinoic acid eluted within 70 minutes. A Tracor Instruments Model 970A variable wave length detector set at 350 nm was used for monitoring the separated compounds. Additional Analysis of Serum and Liver Fluorimetric analyses of serum and l i v e r vitamin A were performed on an Aminco-Bowman spectrophotofluorometer according to the method of Thompson e_t_t a__ll. (lO) with two modifications: l) the l i v e r was f i r s t macerated, than a 0.5 g aliquot was taken and llomogenized with 0.5 ml 60% KOH in a Brinkman Polytron for 30 seconds before taking i t through the rest of the procedure; and 2) the saponification was at 80~ for 30 minutes. Serum triglyceride analysis was performed using a colorometric procedure with Sigma triglyceride assay reagents (St. Louis, MO). Analysis of the Fatty Acid Composition of Serum Lipids Total l i p i d materials were extracted from the pooled rat serum according to the method of Folch et al. (11). Individual l i p i d classes were separated by the solvent system of petroleum ether:diethyl ether: acetic acid (85:15:1, v/v/v) on a silica gel thin-layer plate. Areas corresponding to cholesteryl ester, triglyceride, free fatty acid, and phospholipid were scraped off into ampules and fatty acid methyl esters were prepared by the method of Morrison and Smith (12). Analyses of fatty acid methyl esters by gas-liquid chromatography were carried out as previously outlined (13).
Retinoic Acid Deprivation
511
Experimental Design Expt. I : Thirty male Sprague-Dawley rats of average weight 143 g, were placed on the previously described d i e t (4) with r e t i n y l acetate added at 1.2 R.E./g d i e t , a level suggested by tile NRC to maintain adequate vitamin A n u t r i t u r e . After I0 days, one group of six rats was randomly selected to continue on t h i s d i e t . All other rats were fed a similar diet with supplemental r e t i n o i c acid incorporated at I00 ug/g diet in addition to tile r e t i n y l a c e t a t e . These diets were continued f o r 9 days u n t i l 0900 hour on the last day. All rats were then re-fed the d i e t containing only supplemental 1.2 R.E. r e t i n y l acetate d i e t . At i n t e r v a l s during the next 54 hours, groups of rats were fasted f o r 6 hours and blood was sampled by heart puncture. Expt. 2: l~ale Sprague-Dawley rats of average weight 146 g were maintained on a gel diet containing 1.2 R.E. r e t i n y l acetate/g diet f o r six to seven days. Some rats were selected to continue on the r e t i n y l acetate d i e t , while others were fed a diet containing 25 ~g a l l - t r a n s - r e t i n o i c acid/g of diet without supplemental r e t i n y l acetate, for 12 to 13 days. At that time, rats were g a s t r i c a l l y intubated under l i g h t e t h e r anesthesia with 73 ~g r e t i n o i c acid in I . I ml of corn oil at intervals which would allow for blood sampling at times up to 72 hours p o s t - r e t i n o i c acid administration, but allowing the sampling to occur ~vithin the time period, 0930 to 1030 hours. Control rats were intubated with corn o i l , 5 hours prior to sampling. After intubation, rats were fed tile vitamin A - s u f f i c i e n t d i e t . All blood samples were taken in the nonfasted state. Expt. 3: Male Sprague-Dawley rats of average weight 140 g were fed a gel d i e t containing 1.2 R.E. r e t i n y l acetate/g of d i e t (RDA) f o r 8 to 9 days. A s i m i l a r design to that employed in Expt. 2 was followed with the exception that all diets were supplemented with r e t i n y l acetate at the basal level throughout the experiment, r e t i n o i c acid was fed f o r 8-9 days, control rats were intubated with corn o i l 30 hours p r i o r to sampling, and all rats were fasted 6 hours p r i o r to blood sampling. RESULTS Expt. I : Rats consumed an average of 16 g of control or r e t i n o i c acidsupplemented diets per day prior to the blood sampling period with an e f f i c i e n c y of 0.38 g weight gained per gram of food consumed. Serum was analyzed f o r total t r i g l y c e r i d e s and calculations were made to deter~aine I) i f the reduction of blood t r i g l y c e r i d e with the removal of r e t i n o i c acid from the diet followed a l i n e a r course; and 2) to determine how many hours were required f o r reduction to normal l e v e l s . A l i n e a r relationship between time a f t e r r e t i n o i c acid withdrawal and serum t r i g l y c e r i d e l e v e l s , (P_
512
Gerber et al.
assessment, the percentage of palmitic and arachidonic acid was generally lower in first fractions while oleic and l i n o l e i c (18:1, 18:2) were higher in r e t i n o i c acid-fed rats. The f a t t y acid pattern returned to normal over time. Expt. 3 : Rats consumed an average of 21 g of control or r e t i n o i c acidsupplemented diets per day p r i o r to the blood sampling period with an e f f i c i e n c y of 0.45. IJhen serum t r i g l y c e r i d e levels were analyzed s t a t i s t i c a l l y , i t was found that by 24 hours a f t e r the last dose of r e t i n o i c acid, they were no longer elevated (Table 4). Also, there was an inverse l i n e a r relationship between serum t r i g l y c e r i d e s and time after dosing (r=-0.683, P
Effect of Retinoic Acid Withdrawal from the Diet Upon Serum TriglyCerides in Rats (Experiment l) Hours Following l~ithdrawal of Retinoic Acid Supplemented Diets
6 18
Serum Tri gl yceri des mg/lO0 ml i
359.0 + 180.72.4 343.7 + 214.74
24
335.9 + 164.74
30
219.4 + 138.7
36
96.9 +
9.8
54
72.6 +
18.9
Control Rats 3
98.2 +
9.9
IMean + S.D. 2N ~ 5 for a l l groups. 3Average value for rats fed retinyl acetate diets and undergoing similar treatment to other rats. 4Elevated from control levels(P<_O.05).
Retinoic Acid Deprivation
513
TABLE 2 Effect of Retinoic Acid Withdrawal f r e t the Diet Upon Serum Triglycerides In Rats (Experiment 2) Hour Following Intubation of a Single Dose of Retinoic Acid in Corn Oil
0
Serum Triglycerides mg/lO0 ml I
954.3 + 243.92, 4
6
782.4 + 224.74
12
770.8 + 330.04
18
654.1 + 424.44
24
542.2 + 219.14
36
324.3 +
71.4
54
278.9 + 88.7
72
205.6 +
Control Rats 3
49.3
227.9 + 101.6
IMeaD + S.D. 2N > 5 f o r a l l groups. 3Average value f o r rats fed r e t i n y l acetate diets and undergoing s i m i l a r treatment to other r a t s . 4Elevated from control levels (P~O.05).
f i r s t experiment of the series, i t was apparent that serum t r i g l y c e r i d e s did not return to normal u n t i l at least 30 hours a f t e r the r e t i n o i c acid was withdrawn from the diet of rats. The lack of significance to the r e l a t i o n ship between serum t r i g l y c e r i d e levels and the time a f t e r withdrawal suggested the need for a sampling procedure which would eliminate the effects of circadian rhythms. In the second experiment, intubation of a small dose of r e t i n o i c acid precisely marked the time the last r e t i n o i c acid was consumed, and the staggering of these intubations insured that all blood sampling would occur between 0930 and 1030. A s i g n i f i c a n t linear relationship between serum t r i g l y c e r i d e s and time a f t e r withdrawal was observed with this design. A similar value of 36 hours for serum to return to normolipidemic levels a f t e r the last dose of r e t i n o i c acid was found for this experiment. An attempt was made in the last experiment to eliminate c y c l i c i t y , ensure precise knowledge of tile time of the last dose, and to minimize serum t r i glycerides stemming d i r e c t l y from dietary sources. This was accomplished by following a similar regimen to the previous experiment, but also fasting the rats for 6 hours prior to sampling. When experiment 3 was analyzed, a similar degree of significance to Expt. 2 was associated with the r e l a t i o n ship between t r i g l y d e r i d e levels and time, with 24 hours required for resumpti on of normol i pi demic Ievel s.
514
Gerber et a l .
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Retinoic Acid Deprivation
515
TABLE 4 Effect of Withdrawal of Retinoic Acid from the Diet Upon Serum Triglycerides, Retinoic Acid and Retinol in Rats (Experiment 3) Hours Following Intubation of Retinoic Acid in Corn Oil
0
Serum Triglycerides I mg/lO0 ml
443.4 + 199.12, 4
Serum Retinoic Acid pg/lO0 ml
9.9 + 3.2
Serum Retinol ~g/lO0 ml
67.3 +
7.25
3
622.2 + 126.24
37.2 + 5.44
53.3 +
3.55
6
843.8 + 103.74
42.2 + 1.84
65.0 +
5.15
12
557.5 + 231.54
45.8 + 4.34
67.9 + 12.25
18
429.9 + 156.94
20.0 + 4.84
62.4 + 9.35
24
254.6 +
18.1 + 4.24
64.5 +
5.75
36
280.2 + 184.7
9.3 + 5.2
79.9 +
9,85
54
157.3 +
12.5
15,5 + 2.84
72.6 +
6.25
72
163.6 +
72.9
112.2 +
69.5
Control 3
57,8
4.9 + 2.6 not detectable6
83.0 + 14.4 93.6 + 21.6
IMean + S.D. 2N _> 5 for all groups. 3Average value for rats fed retinyl acetate diet and undergoing similar treatment to other rats. 4Elevated from control levels (P
516
Gerber et al.
rats fed r e t i n o i c acid. Although the mechanism(s) have not been resolved, several hypotheses have been advanced. Erdman et al. monitoring l i p i d metabolism in post-mitochondrial l i v e r homogenates observed an increased incorporation of cholesterol precursors into t r i g l y c e r i d e s in the presence of r e t i n o i c acid (16). Retinoic acid, although present in small amounts in some tissues (17) is not normally detectable in blood; but, i f administered is bound to serum albumin (18). The depression in adipose and skeletal muscle l i p o p r o t e i n lipase a c t i v i t y observed when r e t i n o i c acid is consumed (5) may also be a consequence of the unregulated delivery of r e t i n o i c acid to these tissues. From the results of Experiment 3, i t can also be observed that as the r e t i n o i c acid levels return to normal; so, too, does the serum t r i g l y c e r i d e levels, A d d i t i o n a l l y , i t is also apparent that as r e t i n o i c acid disappers from serum, serum retinol levels return to normal. The mechanism whereby r e t i n o i c acid administration depresses serum retinol is v i r t u a l l y unexplored. Serum retinol usually remains within a very narrmv range in an animal with adequate zinc and protein n u t r i t u r e and a n~derate level of l i v e r vitamin A stores. Recently, the central role of r e t i n y l palmitate hydrolase in maintaining serum retinol levels has been suggested by Goodman et al. (19). Perhaps the hydrolysis of the r e t i n y l ester to retinol may be altered by the presence of r e t i n o i c acid in the l i v e r tissue. A l t e r n a t i v e l y , an e f f e c t may be occuring on e i t h e r the synthesis or release of serum r e t i n o l binding protein. Only future studies can show whether these theories may prove valid. HPLC techniques for measuring vitamin A and r e t i n o i d tissue levels have only recently allowed for establishing t h e i r presence or absence in various tissues and the changes which occur with r e t i n o i d feeding. REFERENCES I.
MISRA, U. K. Effect plasma l i p i d s of rats.
of intramuscular administration of retinol Agr. Biol. Chem. 32, 707-710, 1968.
2.
SINGH, M. and SINGH, V. N. Fatty l i v e r in hypervitarainosis A: synthesis and release of hepatic t r i g l y c e r i d e s . Am. J. Physiol. 234, E51 I-E514, 1978.
3.
GERBER, L. E. and ERDMAN' J. W., JR. Effect of r e t i n o i c ,i a c i d and r e t i n y l acetate feeding upon l i p i d metabolism in adrenalectomzed rats. J. Nutr. 109, 580-589, 1979.
4.
GERBER, L. E. and ERDMAN, J. W., JR. Comparative effects of a l l - t r a n s and 13-cis r e t i n o i c acid administration on serum and l i v e r l i p i d s in rats. J. Nutr. I I 0 , 343-351, 1980.
5.
GERBER, L. E. r e t i n o i c acid.
6.
KATZ, R. A., JORGENSEN, H., and NIGRA, T. P. Elevation of serum t r i glyceride levels from oral i s o t r e t i n o i n in disorders of k e r a t i n i z a t i o n . Arch. Dermatol. 116, 1369-1372, 1980.
7.
KEILSON, B., UNDERWOOD, B. A. and LOERCH, J. D. Effects of r e t i n o i c acid on the mobilization of vitamin A from the l i v e r in rats. J. Nutr. 109, 787-795, 1979.
8.
BESNER, J.-G. and LECLAIRE, R. High-performance l i q u i d chromatography of 1 3 - c i s - r e t i n o i c acid and of endogenous retinol in human plasma. ~. Chrom., Biomed. Appl. 183, 346-351, 1980.
and ERDMAN, J. W., JR. Lipids, 1981 (in press).
Hyperlipidemia
in
rats
on
fed
Retinoic Acid Deprivation
9.
ITO, Y. L., ZILE, M., AHRENS, H. and DELUCA, H. F. Liquid-gel p a r t i tion chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo. J. Lipid Res. 15, 517-524, 1974,
I0.
THOMPSON,J. N., ERDODY, P., BRIEN, R. and ~iURRAY, T. K. Fluorometric determination of vitamin A in human blood and l i v e r . Biochem. Med. 5, 67-89, 1971.
II.
FOLCH, J . , LEES, M. and SLOANE STANLEY, G. H. A simple method for the isolation and p u r i f i c a t i o n of total lipides from animal tissues. J. Biol. Chem. 226, 497-509, 1957.
12.
MORRISON, W. R. and SMITH, L. M. Preparation of f a t t y acid methyl esters and dimethylacetate from l i p i d s with boron fluoride-methanol. J. L i p i d Res. 5, 600-608, 1964.
13.
CHO, B.H.S. and KUMMEROW, F. A. Lipid composition and metabolic a c t i v i t y of the microsomal fractions from the arterial and l i v e r tissues of swine. Biochem. Med. 20, 267-278, 1978.
14.
NATIONAL RESEARCH COUNCIL. Nutrient requirements of domestic animals. Nutrient requirements of laboratory animals. Third revised edition. National Academy of Sciences, Washington, D.C., 1978, pp. 20-21.
15.
KRAUSE, R. F., PALLOTTA, M. A. and YODER, I. I. Lipid alterations in beagles produced by atherogenic stress and vitamin A. J. Atheroscler. Res. 8, 277 -289, 1968.
16.
ERDMAN, J. W., JR., ELLIOT, J. G. and LACHANCE, P. A. The effect of three forms of vitamin A upon in v i t r o lipogenesis from three cholesterol precursors. Nutr. Rep. I n t . 16, 47-57, 1977.
17.
KLEINER-BOESSALER, A. and DELUCA, H. F. Formation of retinoic acid from retinol in the kidney. Arch. Biocbem. Biophys. 142, 371-377, 1971.
18.
SMITH, J. E., MILCH, P. 0., MUTO, Y. and GOODMAN, D. S. The plasma transport and metabolism of retinoic acid in the rat. Biochem. J. 132, 821-827, 1973.
19.
HARRISON, E. H., SMITH, J. E. and GOODMAN, D. S. Unusual properties of retinyl palmitate hydrolase a c t i v i t y in rat l i v e r . J. Lipid Res. 20, 750-771, 1979.
517
CHANGES IN SERUMTRIGLYCERIDES, RETINOIC ACID, HiD RETINOL ASSOCIATED WITH WITHDRAWAL OF RETINOIC ACID FROM THE DIET OF RATS L. E. Gerber, L. S. Wasserman, B. H. S. Cho* and J. W. Erdman, Jr. Department of Food Science University of I l l i n o i s Urbana, I l l i n o i s 61801 and *Harlan E. Moore Heart Research Foundation Champaign, I l l i n o i s 61820
ABSTRACT Several experiments were performed to measure changes in serum triglycerides, retinoic acid, and retinol following removal of all-trans retinoic acid from the diet of rats previously fed 25 ug all-trans retinoic acid/g of diet for periods of 8-13 days. I n i t i t a l l y , rats had hypertriglyceridemia, but within 24-36 hours after withdrawal of retinoic acid, serum triglycerides returned to the level of control rats not fed retinoic acid. Serum retinoic acid also decreased after withdrawal, but did not return to the level of the control rats until 3 days afterwards. Serum retinol was depressed compared to the level of control rats until 72 hours after retinoic acid withdrawal. The fatty acid composition of the individual l i p i d fractions was shifted towards oleate and linoleate and away from palmitate and arachidonate after retinoic acid feeding. Following removal, restoration of a more normal pattern ensued. KEY WORDS: retinoic acid, vitamin A, retinol, triglycerides, hyperl i pi demia INTRODUCTION An elevation of serum triglycerides accompanies the administration of vitamin A at levels exceeding those normally ingested from dietary sources (I-3). Recently, clinical and experimental administration of vitamin A analogs (retinoids) has also been associated with hypertriglyceridemia (4-6). The mechanism by which this elevation occurs remains obscure, although various theories have been advanced (l, 5). Observations have also been made that administration of retinoic acid depresses the level of retinol in the blood of rat and man (3, 4, 7,8). The current study was designed to measure changes in serum l i p i d s , retinol and retinoic acid following removal of all-trans retinoic acid from the diet of rats administered this Compound for eight days. These changes were monitored for up to 72 hours after the last administration of all-trans retinoic acid. Specific goals were to determine the time required for removal of dietary retinoic acid from serum, the time required for restoration of normal triglyceride levels and the possible relationship between retinol and rezinoic acid serum levels in the retinoic acid-fed rat. 509
510
Gerber et al.
MATERIALS AND METHODS
Treatment of Animals Male Sprague-Dawley rats (Harlan Industries, Inc., Cumberland, IN) weighing 135-160 g were housed individually in hanging wire cages in a controlled temperature environment (22~ a light cycle from0600-1800 hours. The rats were fed an agar gel diet as described previously (3), composed of corn starch, sugar, casein, corn oii and adequate vitamins without vitamin A (Teklad, Madison, ~JI) and minerals (USP XVII), at the start of the dark cycle, with water offered ad libitum. Blood samples were taken by cardiac puncture between 0930-I030, after the rats were anesthetized with ether. The blood was allowed to clot, centrifuged at 1500 x g for 30 rain., and the serum was stored at -20 ~ until analysis. For the second experiment, blood was taken, rats were guillotined, and their livers removed, washed with d i s t i l l e d water, pattc~ dry, weighed and stored at -20 ~ until analysis. Extraction of Serum Retinol and Retinoic Acid for HPLC Analysis All handling of vitamin A compounds was done in indirect light. The serum extraction technique used was a modification of the procedure of Ito et al. (9). The solvent extract was evaporated to dryness under nitrogen and its residue dissolved in 200 ul methanol for use in HPLC. Samples of serum plus added retinol, retinoic acid and 14C-alltrans retinoic acid dissolved in methanol, in the range to be assayed in serum, were processed through this same procedure to assess the adequacy of extraction. Similar standards of retinol and retinoic acid were chromatographed and their peak heights used as a measure of the linear response of the compounds. High-Pressure Liquid Chromatography All chemicals and solvents used were analytical reagent grade. Reversed-phase HPLC separation and analysis were done on a DuPont Instruments Zorbax ODS column (4.0 mm x 25 cm). T~e mobile phase contained methanol: 1.0% ammonium acetate (80:20) and was degassed and f i l t e r e d through Whatman No 4 paper before i t was pumped through the system isocratically at l . l ml/min. The peaks were recorded at the maximum sensit i v i t y (0.005 AUFS). In this system retinol and retinoic acid eluted within 70 minutes. A Tracor Instruments Model 970A variable wave length detector set at 350 nm was used for monitoring the separated compounds. Additional Analysis of Serum and Liver Fluorimetric analyses of serum and l i v e r vitamin A were performed on an Aminco-Bowman spectrophotofluorometer according to the method of Thompson e_t_t a__ll. (lO) with two modifications: l) the l i v e r was f i r s t macerated, than a 0.5 g aliquot was taken and llomogenized with 0.5 ml 60% KOH in a Brinkman Polytron for 30 seconds before taking i t through the rest of the procedure; and 2) the saponification was at 80~ for 30 minutes. Serum triglyceride analysis was performed using a colorometric procedure with Sigma triglyceride assay reagents (St. Louis, MO). Analysis of the Fatty Acid Composition of Serum Lipids Total l i p i d materials were extracted from the pooled rat serum according to the method of Folch et al. (11). Individual l i p i d classes were separated by the solvent system of petroleum ether:diethyl ether: acetic acid (85:15:1, v/v/v) on a silica gel thin-layer plate. Areas corresponding to cholesteryl ester, triglyceride, free fatty acid, and phospholipid were scraped off into ampules and fatty acid methyl esters were prepared by the method of Morrison and Smith (12). Analyses of fatty acid methyl esters by gas-liquid chromatography were carried out as previously outlined (13).
Retinoic Acid Deprivation
511
Experimental Design Expt. I : Thirty male Sprague-Dawley rats of average weight 143 g, were placed on the previously described d i e t (4) with r e t i n y l acetate added at 1.2 R.E./g d i e t , a level suggested by tile NRC to maintain adequate vitamin A n u t r i t u r e . After I0 days, one group of six rats was randomly selected to continue on t h i s d i e t . All other rats were fed a similar diet with supplemental r e t i n o i c acid incorporated at I00 ug/g diet in addition to tile r e t i n y l a c e t a t e . These diets were continued f o r 9 days u n t i l 0900 hour on the last day. All rats were then re-fed the d i e t containing only supplemental 1.2 R.E. r e t i n y l acetate d i e t . At i n t e r v a l s during the next 54 hours, groups of rats were fasted f o r 6 hours and blood was sampled by heart puncture. Expt. 2: l~ale Sprague-Dawley rats of average weight 146 g were maintained on a gel diet containing 1.2 R.E. r e t i n y l acetate/g diet f o r six to seven days. Some rats were selected to continue on the r e t i n y l acetate d i e t , while others were fed a diet containing 25 ~g a l l - t r a n s - r e t i n o i c acid/g of diet without supplemental r e t i n y l acetate, for 12 to 13 days. At that time, rats were g a s t r i c a l l y intubated under l i g h t e t h e r anesthesia with 73 ~g r e t i n o i c acid in I . I ml of corn oil at intervals which would allow for blood sampling at times up to 72 hours p o s t - r e t i n o i c acid administration, but allowing the sampling to occur ~vithin the time period, 0930 to 1030 hours. Control rats were intubated with corn o i l , 5 hours prior to sampling. After intubation, rats were fed tile vitamin A - s u f f i c i e n t d i e t . All blood samples were taken in the nonfasted state. Expt. 3: Male Sprague-Dawley rats of average weight 140 g were fed a gel d i e t containing 1.2 R.E. r e t i n y l acetate/g of d i e t (RDA) f o r 8 to 9 days. A s i m i l a r design to that employed in Expt. 2 was followed with the exception that all diets were supplemented with r e t i n y l acetate at the basal level throughout the experiment, r e t i n o i c acid was fed f o r 8-9 days, control rats were intubated with corn o i l 30 hours p r i o r to sampling, and all rats were fasted 6 hours p r i o r to blood sampling. RESULTS Expt. I : Rats consumed an average of 16 g of control or r e t i n o i c acidsupplemented diets per day prior to the blood sampling period with an e f f i c i e n c y of 0.38 g weight gained per gram of food consumed. Serum was analyzed f o r total t r i g l y c e r i d e s and calculations were made to deter~aine I) i f the reduction of blood t r i g l y c e r i d e with the removal of r e t i n o i c acid from the diet followed a l i n e a r course; and 2) to determine how many hours were required f o r reduction to normal l e v e l s . A l i n e a r relationship between time a f t e r r e t i n o i c acid withdrawal and serum t r i g l y c e r i d e l e v e l s , (P_
512
Gerber et al.
assessment, the percentage of palmitic and arachidonic acid was generally lower in first fractions while oleic and l i n o l e i c (18:1, 18:2) were higher in r e t i n o i c acid-fed rats. The f a t t y acid pattern returned to normal over time. Expt. 3 : Rats consumed an average of 21 g of control or r e t i n o i c acidsupplemented diets per day p r i o r to the blood sampling period with an e f f i c i e n c y of 0.45. IJhen serum t r i g l y c e r i d e levels were analyzed s t a t i s t i c a l l y , i t was found that by 24 hours a f t e r the last dose of r e t i n o i c acid, they were no longer elevated (Table 4). Also, there was an inverse l i n e a r relationship between serum t r i g l y c e r i d e s and time after dosing (r=-0.683, P
Effect of Retinoic Acid Withdrawal from the Diet Upon Serum TriglyCerides in Rats (Experiment l) Hours Following l~ithdrawal of Retinoic Acid Supplemented Diets
6 18
Serum Tri gl yceri des mg/lO0 ml i
359.0 + 180.72.4 343.7 + 214.74
24
335.9 + 164.74
30
219.4 + 138.7
36
96.9 +
9.8
54
72.6 +
18.9
Control Rats 3
98.2 +
9.9
IMean + S.D. 2N ~ 5 for a l l groups. 3Average value for rats fed retinyl acetate diets and undergoing similar treatment to other rats. 4Elevated from control levels(P<_O.05).
Retinoic Acid Deprivation
513
TABLE 2 Effect of Retinoic Acid Withdrawal f r e t the Diet Upon Serum Triglycerides In Rats (Experiment 2) Hour Following Intubation of a Single Dose of Retinoic Acid in Corn Oil
0
Serum Triglycerides mg/lO0 ml I
954.3 + 243.92, 4
6
782.4 + 224.74
12
770.8 + 330.04
18
654.1 + 424.44
24
542.2 + 219.14
36
324.3 +
71.4
54
278.9 + 88.7
72
205.6 +
Control Rats 3
49.3
227.9 + 101.6
IMeaD + S.D. 2N > 5 f o r a l l groups. 3Average value f o r rats fed r e t i n y l acetate diets and undergoing s i m i l a r treatment to other r a t s . 4Elevated from control levels (P~O.05).
f i r s t experiment of the series, i t was apparent that serum t r i g l y c e r i d e s did not return to normal u n t i l at least 30 hours a f t e r the r e t i n o i c acid was withdrawn from the diet of rats. The lack of significance to the r e l a t i o n ship between serum t r i g l y c e r i d e levels and the time a f t e r withdrawal suggested the need for a sampling procedure which would eliminate the effects of circadian rhythms. In the second experiment, intubation of a small dose of r e t i n o i c acid precisely marked the time the last r e t i n o i c acid was consumed, and the staggering of these intubations insured that all blood sampling would occur between 0930 and 1030. A s i g n i f i c a n t linear relationship between serum t r i g l y c e r i d e s and time a f t e r withdrawal was observed with this design. A similar value of 36 hours for serum to return to normolipidemic levels a f t e r the last dose of r e t i n o i c acid was found for this experiment. An attempt was made in the last experiment to eliminate c y c l i c i t y , ensure precise knowledge of tile time of the last dose, and to minimize serum t r i glycerides stemming d i r e c t l y from dietary sources. This was accomplished by following a similar regimen to the previous experiment, but also fasting the rats for 6 hours prior to sampling. When experiment 3 was analyzed, a similar degree of significance to Expt. 2 was associated with the r e l a t i o n ship between t r i g l y d e r i d e levels and time, with 24 hours required for resumpti on of normol i pi demic Ievel s.
514
Gerber et a l .
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Retinoic Acid Deprivation
515
TABLE 4 Effect of Withdrawal of Retinoic Acid from the Diet Upon Serum Triglycerides, Retinoic Acid and Retinol in Rats (Experiment 3) Hours Following Intubation of Retinoic Acid in Corn Oil
0
Serum Triglycerides I mg/lO0 ml
443.4 + 199.12, 4
Serum Retinoic Acid pg/lO0 ml
9.9 + 3.2
Serum Retinol ~g/lO0 ml
67.3 +
7.25
3
622.2 + 126.24
37.2 + 5.44
53.3 +
3.55
6
843.8 + 103.74
42.2 + 1.84
65.0 +
5.15
12
557.5 + 231.54
45.8 + 4.34
67.9 + 12.25
18
429.9 + 156.94
20.0 + 4.84
62.4 + 9.35
24
254.6 +
18.1 + 4.24
64.5 +
5.75
36
280.2 + 184.7
9.3 + 5.2
79.9 +
9,85
54
157.3 +
12.5
15,5 + 2.84
72.6 +
6.25
72
163.6 +
72.9
112.2 +
69.5
Control 3
57,8
4.9 + 2.6 not detectable6
83.0 + 14.4 93.6 + 21.6
IMean + S.D. 2N _> 5 for all groups. 3Average value for rats fed retinyl acetate diet and undergoing similar treatment to other rats. 4Elevated from control levels (P
516
Gerber et al.
rats fed r e t i n o i c acid. Although the mechanism(s) have not been resolved, several hypotheses have been advanced. Erdman et al. monitoring l i p i d metabolism in post-mitochondrial l i v e r homogenates observed an increased incorporation of cholesterol precursors into t r i g l y c e r i d e s in the presence of r e t i n o i c acid (16). Retinoic acid, although present in small amounts in some tissues (17) is not normally detectable in blood; but, i f administered is bound to serum albumin (18). The depression in adipose and skeletal muscle l i p o p r o t e i n lipase a c t i v i t y observed when r e t i n o i c acid is consumed (5) may also be a consequence of the unregulated delivery of r e t i n o i c acid to these tissues. From the results of Experiment 3, i t can also be observed that as the r e t i n o i c acid levels return to normal; so, too, does the serum t r i g l y c e r i d e levels, A d d i t i o n a l l y , i t is also apparent that as r e t i n o i c acid disappers from serum, serum retinol levels return to normal. The mechanism whereby r e t i n o i c acid administration depresses serum retinol is v i r t u a l l y unexplored. Serum retinol usually remains within a very narrmv range in an animal with adequate zinc and protein n u t r i t u r e and a n~derate level of l i v e r vitamin A stores. Recently, the central role of r e t i n y l palmitate hydrolase in maintaining serum retinol levels has been suggested by Goodman et al. (19). Perhaps the hydrolysis of the r e t i n y l ester to retinol may be altered by the presence of r e t i n o i c acid in the l i v e r tissue. A l t e r n a t i v e l y , an e f f e c t may be occuring on e i t h e r the synthesis or release of serum r e t i n o l binding protein. Only future studies can show whether these theories may prove valid. HPLC techniques for measuring vitamin A and r e t i n o i d tissue levels have only recently allowed for establishing t h e i r presence or absence in various tissues and the changes which occur with r e t i n o i d feeding. REFERENCES I.
MISRA, U. K. Effect plasma l i p i d s of rats.
of intramuscular administration of retinol Agr. Biol. Chem. 32, 707-710, 1968.
2.
SINGH, M. and SINGH, V. N. Fatty l i v e r in hypervitarainosis A: synthesis and release of hepatic t r i g l y c e r i d e s . Am. J. Physiol. 234, E51 I-E514, 1978.
3.
GERBER, L. E. and ERDMAN' J. W., JR. Effect of r e t i n o i c ,i a c i d and r e t i n y l acetate feeding upon l i p i d metabolism in adrenalectomzed rats. J. Nutr. 109, 580-589, 1979.
4.
GERBER, L. E. and ERDMAN, J. W., JR. Comparative effects of a l l - t r a n s and 13-cis r e t i n o i c acid administration on serum and l i v e r l i p i d s in rats. J. Nutr. I I 0 , 343-351, 1980.
5.
GERBER, L. E. r e t i n o i c acid.
6.
KATZ, R. A., JORGENSEN, H., and NIGRA, T. P. Elevation of serum t r i glyceride levels from oral i s o t r e t i n o i n in disorders of k e r a t i n i z a t i o n . Arch. Dermatol. 116, 1369-1372, 1980.
7.
KEILSON, B., UNDERWOOD, B. A. and LOERCH, J. D. Effects of r e t i n o i c acid on the mobilization of vitamin A from the l i v e r in rats. J. Nutr. 109, 787-795, 1979.
8.
BESNER, J.-G. and LECLAIRE, R. High-performance l i q u i d chromatography of 1 3 - c i s - r e t i n o i c acid and of endogenous retinol in human plasma. ~. Chrom., Biomed. Appl. 183, 346-351, 1980.
and ERDMAN, J. W., JR. Lipids, 1981 (in press).
Hyperlipidemia
in
rats
on
fed
Retinoic Acid Deprivation
9.
ITO, Y. L., ZILE, M., AHRENS, H. and DELUCA, H. F. Liquid-gel p a r t i tion chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo. J. Lipid Res. 15, 517-524, 1974,
I0.
THOMPSON,J. N., ERDODY, P., BRIEN, R. and ~iURRAY, T. K. Fluorometric determination of vitamin A in human blood and l i v e r . Biochem. Med. 5, 67-89, 1971.
II.
FOLCH, J . , LEES, M. and SLOANE STANLEY, G. H. A simple method for the isolation and p u r i f i c a t i o n of total lipides from animal tissues. J. Biol. Chem. 226, 497-509, 1957.
12.
MORRISON, W. R. and SMITH, L. M. Preparation of f a t t y acid methyl esters and dimethylacetate from l i p i d s with boron fluoride-methanol. J. L i p i d Res. 5, 600-608, 1964.
13.
CHO, B.H.S. and KUMMEROW, F. A. Lipid composition and metabolic a c t i v i t y of the microsomal fractions from the arterial and l i v e r tissues of swine. Biochem. Med. 20, 267-278, 1978.
14.
NATIONAL RESEARCH COUNCIL. Nutrient requirements of domestic animals. Nutrient requirements of laboratory animals. Third revised edition. National Academy of Sciences, Washington, D.C., 1978, pp. 20-21.
15.
KRAUSE, R. F., PALLOTTA, M. A. and YODER, I. I. Lipid alterations in beagles produced by atherogenic stress and vitamin A. J. Atheroscler. Res. 8, 277 -289, 1968.
16.
ERDMAN, J. W., JR., ELLIOT, J. G. and LACHANCE, P. A. The effect of three forms of vitamin A upon in v i t r o lipogenesis from three cholesterol precursors. Nutr. Rep. I n t . 16, 47-57, 1977.
17.
KLEINER-BOESSALER, A. and DELUCA, H. F. Formation of retinoic acid from retinol in the kidney. Arch. Biocbem. Biophys. 142, 371-377, 1971.
18.
SMITH, J. E., MILCH, P. 0., MUTO, Y. and GOODMAN, D. S. The plasma transport and metabolism of retinoic acid in the rat. Biochem. J. 132, 821-827, 1973.
19.
HARRISON, E. H., SMITH, J. E. and GOODMAN, D. S. Unusual properties of retinyl palmitate hydrolase a c t i v i t y in rat l i v e r . J. Lipid Res. 20, 750-771, 1979.
517