Effect of Chronic Ethctnol Administration Thiamin Metabolism in the Rat1 MESBAHEDDIN
on
BALAGHI ANDROBERT A. NEAL2
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 ABSTRACT The effect of chronic alcohol administration on the ab sorption, excretion and metabolism of thiamin in the rat has been examined. In ethanol-fed rats receiving a marginal daily intake of thiamin ( 10 ^g/day ) by stomach tube or by intraperitoneal injection, less of the vitamin and its metabolites were excreted in the urine as compared to controls administered the same diet except that sucrose replaced the energy represented by ethanol. More of the oral dose of thiamin was excreted in the feces of the ethanol-fed as compared to control rats. These data support previous re ports of decreased absorption of thiamin from the gut in animals exposed to ethanol. The studies in which the thiamin was administered by intra peritoneal injection also indicate an effect of ethanol on thiamin excretion in the urine which appears not to be related to absorption of the vitamin from the gut. An examination reveals no difference in the level of thiamin and its metabolites in the tissues of ethanol-fed as compared to control rats receiving thiamin by stomach tube. Thus, the decreased absorption of thiamin from the gut in the ethanol-fed rats seems to be balanced by a decreased excretion in the urine leading to a comparable accumulation of the vitamin in the tissues as controls. J. Nutr. 107: 2144-2152, 1977. INDEXING KEY WORDS thiamin •ethanol •thiamin acetic acid Thiamin deficiency is frequently ob served in alcoholics. Several factors may contribute to this deficiency. These in clude inadequate intake (1, 2) incom plete absorption (3-5) and altered metab olism of the vitamin (6). The purpose of the present study was to investigate the effects of chronic ethanol administration on the in vivo absorption, excretion and metabolism of thiamin in rats.
enzymatically synthesized using either the bacterial enzyme thiamin dehydrogenase (7) or horse liver alcohol dehydrogenase.4 4 Sigma Chemical
Co., St. Louis,
Missouri.
Using horse liver alcohol dehydrogenase 500 /ig of 35S-thiamin was incubated for 2 hours at 37°with 1 unit of the enzyme and 2 mg of NAD in 0.1 M phosphate buffer pH 7.4. The total volume of the incubation was 1.0 ml. The thiamin acetic acid was separated from unreacted thiamin using anión exchange liquid chromatography (see fig. 3). MATERIALS AND METHODS Male Sprague-Dawley rats weighing ap All chemicals used were reagent grade proximately 100 g were used throughout unless otherwise indicated. The 35S-thiathe experiment. The rats were housed in min 3 used in these experiments was puri pairs in screen bottom stainless steel metafied by liquid chromatography using a cation exchange column (see fig. 2). Received for publication January 20, 1977. The final product was greater than 99% 1 Supported by USPHS Grant No. AM10297. 3 To whom reprint requests should be addressed. pure as determined by thin layer chroma 3 Anici-slum Bearle Corporation, Arlington Heights, tography (6). Thiamin acetic acid was Illinois. 2144
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ETHANOL ADMINISTRATIONAND THIAMIN METABOLISM
2145
bolic cages which minimized coprophagy jection of the sample, the column was and allowed for separate collection of urine eluted with 50 ml of the phosphate buffer and feces. They were fed a stock diets or at a flow rate of 2.0 ml/minute. This was a commercially available thiamin-deficient followed by 25 ml of 0.02 M acetic acid diet.6 solution. Urine from rats administered 35S-thiamin The urine was prepared for chroma was collected in glass bottles, to which a tography by filtering through Whatman few crystals of thymol and enough of a No. 1 filter paper and concentrating to 2 M solution of acetic acid were added to about M of the original volume in a flash keep the pH below 4.5 (approximately 2 evaporator. The urine concentrate was fil ml). The urine, which was collected daily, tered through a 0.45 //, Millipore filter prior was centrifuged,7 the volume measured to injection onto the Chromatographie column. and aliquots taken for liquid scintillation counting. The remainder was kept at —18° In these experiments, 24 rats weighing under toluene for further study. Feces were about 100 g were divided randomly into collected daily and kept at —18°.Each two equal groups. The experimental group was fed the stock diet ad libitum and had week the daily feces collections of each ex access to a 10% solution of ethanol in perimental and control group were com bined and analyzed for radioactivity as water as the only drinking liquid. The control group was pair-fed to the experi described previously (8). In the studies of the 35S-thiamin content mental group, consuming an amount of sucrose mixed with the stock diet which of various tissues, the rats were decapi tated, the tissues removed, weighed and was isoenergetic to the amount of alcohol homogenized in five volumes of water. consumed by the experimental group. The Duplicate 1 ml aliquots of each tissue ho- control group was also given access to tap water ad libitum. After 2 weeks of this mogenate were transferred to glass scintil lation vials, 2 ml of l N NaOH added and regime, each group was divided into two the vials incubated at 50°overnight with subgroups (six rats each). Two groups gentle shaking. When all the tissue had received the thiamin-deficient diet and the dissolved, the vials were cooled to room 10% ethanol solution ad libitum. The temperature, a scintillation solution 8 added other groups, which served as the controls, and the radioactivity measured. A small were pair-fed the thiamin-deficient diets to portion of each tissue was reserved for the corresponding experimental groups and received tap water ad libitum. The control histopathological examination. High pressure liquid chromatography of groups also received an amount of sucrose urinary metabolites of thiamin was per formed using an insrtument °equipped 5Ralston Purina, St. Louis, Missouri. with a UV detector.10 For cation exchange 6ICN Nutritional Biochemical, Cleveland, Ohio. chromatography a 0.46 X 25 cm column 11 This diet contains vitamin-free casein, 18% ; sucrose, 68% ; vegetable oil, 10% ; and salt mixture, 4%. The mixture contained calcium biphosphate, 13.55% ; was used. The column was washed with salt 50 ml of a solution of pyridine 12:acetic calcium lactate-5H2O, 32.69%; Ferric citrate-5H2O, 2.97% ; magnesium sulfate, 13.69% ; potassium phos (dibasic), 23.98%; sodium biphosphate-2H.,O, acid:water (3:6:91, v/v) followed by 50 phate S.72% ; sodium chloride 4.35% ; CuSO4, 0.03 ; ZnCl2, This diet contained the following vitamins ml of water prior to injecting the urine 0.03%. (mg/kg) : retinyl acetate, 100 (200 U/mg) ; ergosample. After the sample was injected, the calciferol 5.6 (400 U/mg) ; a-tocopheryl acetate. 111, acid, 1.000 ; inositol, 111 ; choline chloride, column was eluted with 30 ml of water at ascorbic 1.667 ; menadione. 50 ; p-aminobenzoic acid. Ill ; a flow rate of 1 ml/minute. This was fol niacin, 100 ; riboflavin, 22 ; pyridoxine hydrochloride, ; calcium pantothenate, 67 ; blotln, 0.44 ; folle lowed by a linear gradient of water to 22 acid, 2.0 ; vitamin B,-, 0.03. 7Servali Omni-Mixer, Ivan Sorvall, Inc., Norwalls, pyridine :acetic acid: water (3:6:91, v/v) Connecticut. 8Aguasol, New England Nuclear, Boston Massa at the same flow rate. For anión exchange chromatography, a 0.46 X 25 cm column 13 chusetts. 9Waters Associates, Westwood, New Jersey. 10Schoeffel Inst. Corp., Westwood, New Jersey. was used. The column was washed with "Partisi! PXS 10/25 SCX. H. Reeve Angel and Co., Inc., Clifton, New Jersey. 50 ml of 0.2 M acetic acid followed by 50 12Spectrophotometric grade, Aldrich Chemical Co., Atlanta, Georgia. ml of 0.05 M phosphate buffer, pH 7.5, Inc., "Partisti PXS 10/25 SAX, H. Reeve Angel and prior to injection of the sample. After in Co., Inc., Clifton, New Jersey.
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2146
MESBAHEDDIN
BALAGHI AND ROBERT A. NEAL
400A_300;
II 1•
1,1-3000io001
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»100234WEEKSFig.
i
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12345678WEEKSethanol fed (A) and control (B) rats. All rats were fed the stock 1 Growth rats of After 2 weeks (arrow), they were fed the thiamin-deficient diet diet for the first 2 weeks.400II and each rat supplemented daily with either 10 ¿ig(0.3 juCi) (A) or 30 Mg (0.9 ¿iCi)(O) of ""S-thiamin. The data points starting at 3 weeks represent the mean ±so of body weight of six rats. The data points prior to 3 weeks are the average weight of six rats.
in their diet which was isoenergetic to the All statistical comparisons were made using Student's t test. amount of alcohol consumed by their re spective experimental groups. One of the RESULTS ethanol-fed and control groups were given a single daily dose of 10 /xg (0.3 /¿Ci)35SThe growth rates of the ethanol-fed and thiamin by stomach tube while the other control rats are shown in figures 1A and ethanol-fed and control group received a IB, respectively. There were no significant single daily dose of 30 /tg (0.9 ¡nCi)of 35S-thiamin by stomach tube. TABLE 1 The rats were fed the thiamin-deficient Ethanol consumption as percentage of diet for 6 weeks. At that time, three rats total energy intake1 from each group were chosen at random and killed for tissue analysis of radio 10 UK 30 jig Week "S-thiamin/day "S-thiamin/day activity. The experiment was continued with the remaining rats in the same man ner except for the route of thiamin admin IIIinIVVVIVIIVIII12.16il.6114.25 istration which was changed from daily iO.6313.08iO.3813.80il.7514.34i2.6814.71il.8814.98i2.5814.02i2. administration by stomach tube to daily intraperitoneal injections. This phase of Ì3.4216.43i2.3216.45il.1918.36i2.7018.78 the experiment lasted for 2 weeks. The remaining rats were killed 24 hours after the last intraperitoneal injection of 35Sthiamin and their tissues analyzed for 1Each number is the meanisD of 6 rats. radioactivity.
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ETHANOL
ADMINISTRATION
differences in weight gain between the rats receiving ethanol and the correspond ing controls on the same level of thiamin intake. However, in both the ethanol and control groups, the rats given 10 /xg thiamin/day gained significantly less weight than the rats receiving 30 /xg of thiamin/ day. Thus, under these experimental con ditions, 10 /xg/day of thiamin was below the optimum daily intake for growth. Table 1 shows the ethanol consumption of the experimental rats expressed as the percentage of the total energy intake. The rats given 10 /xg thiamin/day consumed between 12.16% and 14.98% of their total energy intake as ethanol. The rats given 30 /xg of thiamin/day consumed between 14.24% and 18.78% of the total energy as ethanol. The average weekly values for urinary excretion of thiamin and thiamin metab olites for the 6 week period of intragastric thiamin administration are presented in tables 2 and 3. In general, rats receiving ethanol excreted a smaller percentage of the administered thiamin and its metab olites in the urine as compared to the cor responding control rats. Furthermore, this difference was much more pronounced in the rats given a 10 /xg thiamin/day as com pared to those given a 30 /xg thiamin/day. Table 4 shows the urinary excretion of
AND THIAMIN
METABOLISM
2147
TABLE 3 Average weekly urinary excretion of thiamin, expressed as percent of the intake, in rats receiving 30 y.g of 36S-thiamin/day by gastric intubation1
Week
Ethanol fed%
Control
Differ ence in controls
±6.36229.58±5.13342.36±5.24444.91 IIIIIIIVVVI18.41 ±4.0937.93±2.7748.25±5.2451.87±2.2958.20±3.8761 14+ ±4.78355.14±3.1060.26±5.5225.4116+6+3 86 ±4.20+38+28+ 1The urine was collected daily. The urine from each group (six rats) was pooled and an aliquot taken for determination of radioactivity. These numbers represent the mean±so of the daily urinary radio activity values in each week of the experiment. 2Significantly different from control (P < 0.025). 3 (P < 0.0025). «(P < 0.05).
thiamin and its metabolites during the last 2 weeks of the experiment, a period during which the rats received thiamin by intraperitoneal injection instead of by stomach tube. The ethanol-fed rats receiving 10 /xg thiamin/day continued to excrete less thiamin and its metabolites in their urine than the appropriate controls. However, there was no difference in the amount of thiamin and its metabolites excreted in the urine of the ethanol-fed and control rats receiving 30 /xg thiamin/day.
TABLE 2 Average weekly urinary excretion of thiamin, expressed as percent of the intake, in the rats receiving 10 ng (0.3 fid) of 3SS-thiamin/day by gastric intubation
Week
Ethanol fed
Control
% Differ ence in controls
±5.73232.02±5.94345.3 IIIIIIIVVVI17.41 ±4.9247.59
TABLE 4 Average weekly urinary excretion of thiamin, expressed as percent of the dose, in animals receiving 3SS-thJamin by intraperitoneal injection1
Week
VII ±6.5759.20±4.4964.07±3.1270.32 ±3.53"48.23±3.73357.10±2.84360.40±5.18425.20 VIII
Ethanol fed
Control
10 jug thiamin/day 62.03±4.852 74.38±4.85 64.86±4.222 75.64±2.88
% Differ ence in controls +20 + 17
30 ¿igthiamin/day ±4.2174.16±6.85+45+49+31+33+23+23 VII 65.00±7.05 64.71 ±3.73 0 VIII 70.12±5.00 67.17±3.57 -4 1The urine was collected daily. The urine from 1The urine was collected daily. The urine from each group (six rats) was pooled and an aliquot taken each group (six rats) was pooled and an aliquot taken for determination of radioactivity. These numbers represent the mean±SD of the daily urinary radio for determination of radioactivity. These numbers activity values in each week of the experiment. represent the mean ±so of the daily urinary radio 2Significantly different from control (P < 0.025). activity values in each week of the experiment. 3 (P < 0.0005). " (P < 0.0025). 2Significantly different from control (P < 0.0005).
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BALAGHI AND ROBERT A. NEAL
/tg thiamin/day, the fecal excretion of thiamin and its metabolites was generally higher in the ethanol-fed as compared to the appropriate controls. In rats receiving 10 Mgthiamin /day 30 >igthiamin /day 30 ng thiamin/day, the fecal excretion of thiamin and its metabolites was also gen Ethanol Ethanol erally greater in the ethanol-fed rats, al fed Controls Controls Week fed though the difference was not as great as that seen in the rats receiving 10 ng thia IIIIIIIVvVI2.417.077.7518.9316.9015.951.654.367.3811.3010.749.674.875.146.3615.7314.9 min/day. Table 6 shows the mean concentration and the mean total content of radioactivity equivalent to thiamin in the tissues of ethanol-fed and control rats after a 6 week period during which they received 10 or 1The feces from each group was collected daily 30 /¿gof 35S-thiamin daily by stomach and pooled. The feces collected for a week was sub sequently pooled and an aliquot analyzed for radio tube. The tissue concentrations and total activity as described previously (8). contents were not significantly different in ethanol-fed and control rats given the The fecal excretion of thiamin and its same amount of thiamin. metabolites during the 6 weeks of intraAt the end of 6 weeks, only half of the gastric administration of thiamin is pre rats in each group were killed for tissue sented in table 5. In the rats receiving 10 analysis (table 6). The remaining rats TABLE 5
Fecal excretion of thiamin expressed as the percentage of the intake of 3iS-thiaminl
TABLE 6 Tissue distribution of 36S-thiamin and its metabolites in the tissues of ethanol fed and control rats after 6 weeks of administration of 10 or 30 ing"S-thiamin/day by stomach tube Rats receiving 10 Mgthiamin/day Ethanol fed Tissue
Concentration
Controls
Total content1
Concentration
Total content'
BrainLiverHeartKidneyTestesMuscle»9/91.74±0.231.12±0.261.16±0.090.75±0.061.79±0.150.25±0.02M3.53±0.4016.48±1.681.64± ±0.321.95±0.186.69 ±1.52— ±0.63—w/g1.86±0.011.12±0.111.32±0.410.75±0.041.80±0.180.28 ±0.03Mff3.80±0.0615.07±2.191.41 Rats receiving 30 /ig thiamin/day Ethanol fed Tissue
Concentration
Total content1
Controls Concentration
Total content1
BrainLiverHeartKidneyTestesMuscleW/92. ±0.282.99±0.233.26±0.442.00±0.295.04±0.970.60±0.02/*?6.16±1.0246.91 97 ±6.344.86±0.375.41 ±0.295.23±0.3014.91±1.25— ±0.6516.58±3.01—W/03.00±0.152.73±0.393.23±0.171.82±0.084.12±0.370.58 ±0.07W/Õ5.83±0.5241.03±6.824.07 1Each number is the mean±so of the individual analysis of the tissues of three rats. The concentration is expressed as radioactivity equivalent to 1 jig of thiamin/g of tissue (wet weight) and the total content is the total radioactivity in the tissue expressed as ¿igof thiamin.
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ETHANOL ADMINISTRATION AND THIAMIN METABOLISM
2149
TABLE 7 Tissue distribution of uS-thiamin and its metabolites in the tissues of ethanol fed and control rats after 6 weeks of administration of 10 or SO ¡¿g of i6S-thiamin/day by stomach tube followed by 2 weeks on the same intakes by intraperitoneal injection
Rats receiving 10 Mgthiamin/day Ethanol fed Tissue
Concentration1
Controls
Total content1
Concentration1
Total content1
±0.111.42±0.0621.20±0.0720.76±0.0521.97±0.2220.28 ±0.160.94±0.231.00±0.080.61 BrainLiverHeartKidneyTestesMuscleMl91.62
±0.201.92 ±0.126.83±0.142—M/g1.42 ±0.071.50±0.140.27±0.02Mff3.05±0.4715.36±2.341.26± ±0.02M3.38±0.1317.48±1.271.41 Rats receiving 30 jagthiamin/day
Tissue
Ethanol fed Total content1 Concentration1
Controls Total content1 Concentration1
BrainLiverHeartKidneyTestesMuscleml92.95±0.052.38±0.093.25±0.281.99
±6.545.12±0.725.45±0.8917.30±3.19†11
±0.094.76±0.1320.63±0.03M6.12±0.3445.23±2.314.83±0.646.31±0.5815.58±0.38—US/92.82±0
1Each number is the mean±SDof the analysis of the individual tissue of three rats. The concentration is expressed as radioactivity equivalent to 1 Mgof thiamin/g of tissue (wet weight) and the total content is the total radioactivity of the tissue expresses as /¡gof thiamin. ^Significantly different (P < 0.05) from the corresponding control tissue.
were continued on the same regimen but received thiamin by intraperitoneal ad ministration rather than by stomach tube. This phase of the experiment was con tinued for 2 weeks, at the end of which the rats were killed and the tissues ana lyzed in the same manner described in table 6. The results are shown in table 7. In all the tissues, except muscle the ethanol-fed rats given 10 /tg thiamin/day showed a higher concentration and a higher total content of thiamin and its metab olites than the corresponding controls. These differences are statistically signifi cant in case of the concentrations of thia min and its metabolites in heart, kidney, testes and liver and the total content in testes. In the rats given 30 ,ag thiamin/ day, the only tissue where there was a significant difference in concentration was the testes.
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Thiamin is metabolized to thiamin acetic acid by mammalian alcohol dehydrogenase enzymes (6). Therefore, it was of interest to examine the urine of the rats in all four groups for the amount of the radioactivity excreted in the urine as thiamin acetic acid. Figure 2 shows the results of cation exchange chromatography of the urine. The urinary metabolites are resolved into three peaks. Both thiamin and thiamin acetic acid appear in peak III. The column fractions corresponding to peak III, figure 2 were pooled and lyophilized to dryness. The residue was dissolved in 0.5 ml of water, 20 /¿gof unlabeled thiamin acetic acid added, and the mixture subjected to anión exchange chromatography (fig. 3). Thiamin and other uv absorbing material is eluted in peak I. Peak III corresponds to thiamin acetic acid. Using this proce dure of liquid chromatography, first on
2150
MESBAHEDDIN BALAGHI AND ROBERT A. NEAL
TABLE 8
20
Urinary excretion of ^S-tkiamin acetic acid in ethanol-fed and control rats at two levels of thiamin intake1 15S-thiamin/dayDay57585960616263Mean±8DEthanolfed1.631.361.461.281.191.281.271.35 10 Mg "S-thiamin/dayEthanolfed2.692.382.362.652.502.813.072.63 jig
I > 10
5
< J. ce
±0.25»Control1.201.051.071.421.971.942.421.58 ±0.14»Control1.060.960.790.850.921.050.980.94 ±0.53 ±0.0930
10
20 FRACTION
30
40
NUMBER
Fig. 2 Cation exchange chromatography of the urine of rats receiving 30 Mg Å“S-thiamin/day by stomach tube. The column was a 0.46 X 25 cm column of Partisil PXS 10/25 SCX.11 Two ml of a concentrated sample of urine was injected onto the column and the column eluted with 15 ml of water followed by a 30 minute linear gra dient of water to pyridine: acetic acid: water (3:6:91 v/v). The flow rate was 1 ml/minute. The fraction size is 1 ml.
cation exchange followed by an anión ex change column, the thiamin acetic acid could be separated from thiamin and other thiamin metabolites present in urine. The fractions corresponding to peak III, figure 3 were pooled and the 35S-thiamin acetic acid content determined by scintil lation counting. Table 8 shows the urinary excretion of thiamin acetic acid an all four groups ex pressed as the percentage of total urinary radioactivity. In both ethanol-fed and con trol rats, a higher percentage of the thia min was excreted as thiamin acetic acid in the rats receiving 30 /¿gas compared to 10 fig of thiamin/day. The ethanol-fed rats at both levels of intake converted a higher percentage of thiamin into thiamin acetic acid than the corresponding controls. DISCUSSION Several workers have studied the effect of ethanol on the intestinal absorption of
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The figures represent the/amount of thiamin acetic acid excreted expressed as the percent of the total urinary radio activity. ! The percentage of the urinary radioactivity that is represented by thiamin acetic acid was determined by liquid chromatography as described in figure 3. These numbers are the results obtained on analysis of the pooled urine of the six rats in each group on each of days 58 to 64 of the experiment. During days 49 to 63 the rats received *5S-thiamin by intraperitoneal injection. On days 1 to 48 of the experiment the rats received «S-thiamin by stomach tube. ! Significantly dif ferent (P < 0.05) than the corresponding control.
thiamin. Thus, Tomsaulo et al. (3) admin istered 14C-thiamin orally to alcoholic sub jects and found significant decreases in the
0
5
10
15
20
25
TIME (mm)
Fig. 3 Anión exchange chromatography of peak III from the cation exchange column de scribed in figure 2. The column was a 0.46 X 25 cm column of Partisil PXS 10/25 SAX.13Peak III from figure 2 was lyophilized to dryness, the residue dissolved in 0.5 ml of distilled water, 20 Hg of unlabeled thiamin acetic acid added and the mixture injected onto the column. The column was eluted first with 0.05 M Na phosphate buffer (pH 5.7) followed by (arrow) 0.2 M acetic acid. The flow rate was 2 ml/minute.
ETHANOL ADMINISTRATION AND THIAMIN METABOLISM
2151
urinary excretion of the radioactivity and a rats. These data suggest that ethanol not significant increase in fecal excretion. only depresses the intestinal absorption but Thomson et al. (4), examined the radio also decreases the urinary excretion of activity in the femoral artery and hepatic thiamin under conditions of marginal in vein after oral administration of [2-14C]- take. It is interesting to speculate that the thiazole thiamin. They also examined the decrease in urinary excretion of thiamin in excretion of radioactivity in the urine and ethanol-fed rats might be an adaptive re feces, and obtained data indicating severe sponse compensating for lower absorption depression of thiamin absorption under thus, slowing the rate of depletion of the the influence of ethanol given either orally tissue of thiamin. The data presented in table 6 supports this speculation showing or by iv injection. More recently, Hoyumpa et al. (5) used rat-everted gut sacs or that after 6 weeks of ethanol feeding, there intact isolated loops of rat intestine to was no significant difference in tissue levels of thiamin compounds between study the effect of ethanol on thiamin ab ethanol-fed and controls at either level of sorption. This group found that absorption of thiamin at concentrations below 2.0 JU.M intake in spite of a decreased degree of absorption of thiamin in the ethanol-fed is an active transport process which is in hibited by ethanol at a dose of 50 to 750 rats (table 5). However, when thiamin was administered by intraperitoneal in mg/100 g of body weight given by a stom ach tube or by intravenous injection. At jection for 2 weeks, the ethanol-fed rats on concentrations higher than 2.0 /*M,thiamin a marginal thiamin intake (10 /tg/day) had higher concentrations of thiamin in appears to be absorbed by passive diffu most tissues than the corresponding control sion. The passive diffusion of thiamin is un affected by ethanol administration. The (table 7). Thus, the decreased excretion of data from the studies reported in this thiamin and its metabolites in the urine of manuscript are in general agreement with the ethanol-fed rats may be unrelated to the decreased intestinal absorption. these findings of decreased intestinal ab Another aspect of the ethanol-thiamin sorption. Thus, from the data in table 2, the urinary excretion of radioactivity was interrelationship is the possible increase in depressed by 23% to 49% in ethanol-fed thiamin metabolism to inactive metabolites rats given a low level (10 /ug/day) of the in ethanol-fed rats. Previous work in this vitamin. In rats given a higher level (30 laboratory (6) has indicated that alcohol /ig/day), the difference, which is 38% for dehydrogenase is involved to an important the first week, tended to decrease and be degree in the in vivo metabolism of thia came nonsignificant by the end of the min to thiamin acetic acid in the rat. Thus, fourth week (table 3). The data on fecal it is reasonable that the increased metab excretion of thiamin (table 5) supports olism of thiamin to thiamin acetic acid in ethanol-fed rats which was demonstrated the view that the decreased urinary excre tion of thiamin seen in the ethanol-fed rats in these studies ( table 8 ) is, at least in part, given the low (10 ^g/day) thiamin intake the result of an increased activity of alco and, to a lesser degree, those given the hol dehydrogenase. high (30 /j.g/day) thiamin intake may have An increase in ethanol metabolism as a resulted from a decreased absorption of result of chronic exposure of rats to ethanol the vitamin. It was therefore expected has been demonstrated in vivo (9) and in when the rats were given thiamin by intra- vitro (10-13). The mechanism of this peritoneal injection that the difference in adaptive increase in ethanol metabolism excretion of thiamin and its metabolites in has been controversial. Videla et al. (IO) the urine in ethanol-fed and control rats and Thurman et al. (Il, 12) have sug would be eliminated since the impaired gested this increased rate of metabolism intestinal absorption would be bypassed. of ethanol is the result of a higher rate of reoxidation of NADH in the ethanolHowever, as shown in table 4, the urinary excretion of radioactivity in rats receiving exposed rats. These workers have further 10 ng/aay 33S-thiamin by intraperitoneal suggested that the increased rate of NADH injection remained lower in ethanol-fed oxidation is a result of ADP stimulation of
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the mitochondrial oxidation of NADH, the excess ADP arising from an ethanolstimulated increase in Na% K+ ATPase activity. On the other hand, Matsuzaki and Lieber (13) report that pyrazole, an inhibitor of alcohol dehydrogenase, has a greater inhibitory effect on ethanol metab olism in vitro using liver slices from nor mal as compared to chronic ethanoltreated rats. From these results they sug gest the adaptive increase of hepatic ethanol oxidation after chronic ethanol consumption is due to the enhanced activ ity of the microsomal ethanol oxidizing system (MEOS) (14). However, this adaptive increase attributed to MEOS was seen only at high ethanol concentrations (13). Experiments at lower and more physiological concentrations of ethanol have shown that 4-methylpyrazole equally inhibits ethanol uptake by perfused livers of ethanol-fed and control rats ( 12 ). These results were interpreted to mean that alco hol dehydrogenase and not catalase or MEOS is responsible for the increased rate of uptake and metabolism of ethanol in chronic ethanol-treated rats. Thiamin is not metabolized to the thiamin acetic acid under conditions compara ble to those used to measure microsomal ethanol metabolism.14 Therefore, it appears more likely that an increased activity of alcohol dehydrogenase is responsible for the increase in metabolism of thiamin to thiamin acetic acid seen in these experi ments (table 8). The difference in the per centage of the thiamin excreted in the urine as thiamin acetic acid in the ethanolfed and control rats is quite small. Conse quently, it is questionable whether an increased rate of metabolism of thiamin to thiamin acetic acid plays a significant role in thiamin deficiency seen in alcoholics. LITERATURE
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
CITED
1. Leevy, C. M., Cardi, L., Frank, O., Gellene, R. & Baker, H. (1965) Incidence and significance of hypovitaminemia in a randomly "Norman, vations.
2.
B. J. & Neal, R. A. Unpublished
obser
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14.
selected municipal hospital population. Am. J. Clin. Nutr. 17, 259-271. Neville, J. N., Eagles, J. A., Samson, G. & Olson, R. E. (1968) Nutritional status of alcoholics. Am. J. Clin. Nutr. 21, 1329-1340. Tomasulo, P. A., Kater, R. M. & Iber, F. L. ( 1968 ) Impairment of thiamine absorption in alcoholism. Am. J. Clin. Nutr. 21, 13411344. Thomson, A. D., Baker, H. & Leevy, C. M. (1970) Patterns of ""S-thiamine hydrochloride absorption in the malnourished alcoholic patient. J. Lab. Clin. Med. 76, 34-45. Hoyumpa, A. M., Jr., Breen, K. J., Schenker, S. & Wilson, F. A. (1975) Thiamine trans port across the rat intestine. II. Effect of ethanol. J. Lab. Clin. Med. 86, 803-816. Dalvi, R. R., Sauberlich, H. E. & Neal, R. A. (1974) An examination of the metabolism of thiamin by rat liver alcohol dehyrogenase. J. Nutr. 104, 1476-1483. Neal, R. A. (1970) Bacterial metabolism of thiamine. 3. Metabolism of thiamine to 3-(2'methyl-4'-amino-5'-pyrimidylmethyl)-4-methylthiazole-5-acetic acid (thiamine acetic acid) by a flavoprotein isolated from a soil micro organism. J. Biol. Chem. 245, 2599-2604. Balaghi, M. & Pearson, W. N. (1966) Tis sue and intracellular distribution of radioac tive thiamine in normal and thiamine-deficient rats. J. Nutr. 89, 127-132. Hawkins, R., Kalant, H. & Khanna, J. M. (1966) Effects of chronic intake of ethanol on rate of ethanol metabolism. Can. J. Physiol. Pharmacol. 44, 241-257. Videla, L., Bernstein, J. & Israel, Y. (1973) Metabolic alterations produced in the liver by chronic ethanol administration. Biochem. J. 134, 507-514. Thurman, R. G., McKenna, W. R. & McCaf frey, T. B. (1976) Pathways responsible for the adaptive increase in ethanol utilization following chronic treatment with ethanol: Inhibitor studies with the hemoglobin-free perfused rat liver. Mol. Pharmacol. 12, 156166. Thurman, R. G. & Scholz, R. (1976) Etha nol metabolism in perfused rat liver at low ethanol concentrations: Involvement of alcohol dehydrogenase in the adaptive increase in ethanolwith metabolism due to chronicZ. pretreat ment ethanol. Hoppe-Zeyler's Physiol. Chem. 357, 1443-1446. Matsuzaki, S. & Lieber, C. S. (1975) ADHindependent ethanol oxidation in the liver and its increase by chronic ethanol consumption. Gastroenterol. 69, 845. Lieber, C. S., Rubin, E. & DeCarli, L. M. (1970) Hepatic microsomal ethanol oxidiz ing system (MEOS): Differentiation from alcohol dehydrogenase and NADPH oxidase. Biochem. Biophys. Res. Commun. 40, 858865.
on
BALAGHI ANDROBERT A. NEAL2
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 ABSTRACT The effect of chronic alcohol administration on the ab sorption, excretion and metabolism of thiamin in the rat has been examined. In ethanol-fed rats receiving a marginal daily intake of thiamin ( 10 ^g/day ) by stomach tube or by intraperitoneal injection, less of the vitamin and its metabolites were excreted in the urine as compared to controls administered the same diet except that sucrose replaced the energy represented by ethanol. More of the oral dose of thiamin was excreted in the feces of the ethanol-fed as compared to control rats. These data support previous re ports of decreased absorption of thiamin from the gut in animals exposed to ethanol. The studies in which the thiamin was administered by intra peritoneal injection also indicate an effect of ethanol on thiamin excretion in the urine which appears not to be related to absorption of the vitamin from the gut. An examination reveals no difference in the level of thiamin and its metabolites in the tissues of ethanol-fed as compared to control rats receiving thiamin by stomach tube. Thus, the decreased absorption of thiamin from the gut in the ethanol-fed rats seems to be balanced by a decreased excretion in the urine leading to a comparable accumulation of the vitamin in the tissues as controls. J. Nutr. 107: 2144-2152, 1977. INDEXING KEY WORDS thiamin •ethanol •thiamin acetic acid Thiamin deficiency is frequently ob served in alcoholics. Several factors may contribute to this deficiency. These in clude inadequate intake (1, 2) incom plete absorption (3-5) and altered metab olism of the vitamin (6). The purpose of the present study was to investigate the effects of chronic ethanol administration on the in vivo absorption, excretion and metabolism of thiamin in rats.
enzymatically synthesized using either the bacterial enzyme thiamin dehydrogenase (7) or horse liver alcohol dehydrogenase.4 4 Sigma Chemical
Co., St. Louis,
Missouri.
Using horse liver alcohol dehydrogenase 500 /ig of 35S-thiamin was incubated for 2 hours at 37°with 1 unit of the enzyme and 2 mg of NAD in 0.1 M phosphate buffer pH 7.4. The total volume of the incubation was 1.0 ml. The thiamin acetic acid was separated from unreacted thiamin using anión exchange liquid chromatography (see fig. 3). MATERIALS AND METHODS Male Sprague-Dawley rats weighing ap All chemicals used were reagent grade proximately 100 g were used throughout unless otherwise indicated. The 35S-thiathe experiment. The rats were housed in min 3 used in these experiments was puri pairs in screen bottom stainless steel metafied by liquid chromatography using a cation exchange column (see fig. 2). Received for publication January 20, 1977. The final product was greater than 99% 1 Supported by USPHS Grant No. AM10297. 3 To whom reprint requests should be addressed. pure as determined by thin layer chroma 3 Anici-slum Bearle Corporation, Arlington Heights, tography (6). Thiamin acetic acid was Illinois. 2144
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ETHANOL ADMINISTRATIONAND THIAMIN METABOLISM
2145
bolic cages which minimized coprophagy jection of the sample, the column was and allowed for separate collection of urine eluted with 50 ml of the phosphate buffer and feces. They were fed a stock diets or at a flow rate of 2.0 ml/minute. This was a commercially available thiamin-deficient followed by 25 ml of 0.02 M acetic acid diet.6 solution. Urine from rats administered 35S-thiamin The urine was prepared for chroma was collected in glass bottles, to which a tography by filtering through Whatman few crystals of thymol and enough of a No. 1 filter paper and concentrating to 2 M solution of acetic acid were added to about M of the original volume in a flash keep the pH below 4.5 (approximately 2 evaporator. The urine concentrate was fil ml). The urine, which was collected daily, tered through a 0.45 //, Millipore filter prior was centrifuged,7 the volume measured to injection onto the Chromatographie column. and aliquots taken for liquid scintillation counting. The remainder was kept at —18° In these experiments, 24 rats weighing under toluene for further study. Feces were about 100 g were divided randomly into collected daily and kept at —18°.Each two equal groups. The experimental group was fed the stock diet ad libitum and had week the daily feces collections of each ex access to a 10% solution of ethanol in perimental and control group were com bined and analyzed for radioactivity as water as the only drinking liquid. The control group was pair-fed to the experi described previously (8). In the studies of the 35S-thiamin content mental group, consuming an amount of sucrose mixed with the stock diet which of various tissues, the rats were decapi tated, the tissues removed, weighed and was isoenergetic to the amount of alcohol homogenized in five volumes of water. consumed by the experimental group. The Duplicate 1 ml aliquots of each tissue ho- control group was also given access to tap water ad libitum. After 2 weeks of this mogenate were transferred to glass scintil lation vials, 2 ml of l N NaOH added and regime, each group was divided into two the vials incubated at 50°overnight with subgroups (six rats each). Two groups gentle shaking. When all the tissue had received the thiamin-deficient diet and the dissolved, the vials were cooled to room 10% ethanol solution ad libitum. The temperature, a scintillation solution 8 added other groups, which served as the controls, and the radioactivity measured. A small were pair-fed the thiamin-deficient diets to portion of each tissue was reserved for the corresponding experimental groups and received tap water ad libitum. The control histopathological examination. High pressure liquid chromatography of groups also received an amount of sucrose urinary metabolites of thiamin was per formed using an insrtument °equipped 5Ralston Purina, St. Louis, Missouri. with a UV detector.10 For cation exchange 6ICN Nutritional Biochemical, Cleveland, Ohio. chromatography a 0.46 X 25 cm column 11 This diet contains vitamin-free casein, 18% ; sucrose, 68% ; vegetable oil, 10% ; and salt mixture, 4%. The mixture contained calcium biphosphate, 13.55% ; was used. The column was washed with salt 50 ml of a solution of pyridine 12:acetic calcium lactate-5H2O, 32.69%; Ferric citrate-5H2O, 2.97% ; magnesium sulfate, 13.69% ; potassium phos (dibasic), 23.98%; sodium biphosphate-2H.,O, acid:water (3:6:91, v/v) followed by 50 phate S.72% ; sodium chloride 4.35% ; CuSO4, 0.03 ; ZnCl2, This diet contained the following vitamins ml of water prior to injecting the urine 0.03%. (mg/kg) : retinyl acetate, 100 (200 U/mg) ; ergosample. After the sample was injected, the calciferol 5.6 (400 U/mg) ; a-tocopheryl acetate. 111, acid, 1.000 ; inositol, 111 ; choline chloride, column was eluted with 30 ml of water at ascorbic 1.667 ; menadione. 50 ; p-aminobenzoic acid. Ill ; a flow rate of 1 ml/minute. This was fol niacin, 100 ; riboflavin, 22 ; pyridoxine hydrochloride, ; calcium pantothenate, 67 ; blotln, 0.44 ; folle lowed by a linear gradient of water to 22 acid, 2.0 ; vitamin B,-, 0.03. 7Servali Omni-Mixer, Ivan Sorvall, Inc., Norwalls, pyridine :acetic acid: water (3:6:91, v/v) Connecticut. 8Aguasol, New England Nuclear, Boston Massa at the same flow rate. For anión exchange chromatography, a 0.46 X 25 cm column 13 chusetts. 9Waters Associates, Westwood, New Jersey. 10Schoeffel Inst. Corp., Westwood, New Jersey. was used. The column was washed with "Partisi! PXS 10/25 SCX. H. Reeve Angel and Co., Inc., Clifton, New Jersey. 50 ml of 0.2 M acetic acid followed by 50 12Spectrophotometric grade, Aldrich Chemical Co., Atlanta, Georgia. ml of 0.05 M phosphate buffer, pH 7.5, Inc., "Partisti PXS 10/25 SAX, H. Reeve Angel and prior to injection of the sample. After in Co., Inc., Clifton, New Jersey.
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2146
MESBAHEDDIN
BALAGHI AND ROBERT A. NEAL
400A_300;
II 1•
1,1-3000io001
1Õ
'• Õ Î,
ti§ •0u2u 200
U'I
20002Ul3100i
0
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»100234WEEKSFig.
i
^^^^^^Jr
12345678WEEKSethanol fed (A) and control (B) rats. All rats were fed the stock 1 Growth rats of After 2 weeks (arrow), they were fed the thiamin-deficient diet diet for the first 2 weeks.400II and each rat supplemented daily with either 10 ¿ig(0.3 juCi) (A) or 30 Mg (0.9 ¿iCi)(O) of ""S-thiamin. The data points starting at 3 weeks represent the mean ±so of body weight of six rats. The data points prior to 3 weeks are the average weight of six rats.
in their diet which was isoenergetic to the All statistical comparisons were made using Student's t test. amount of alcohol consumed by their re spective experimental groups. One of the RESULTS ethanol-fed and control groups were given a single daily dose of 10 /xg (0.3 /¿Ci)35SThe growth rates of the ethanol-fed and thiamin by stomach tube while the other control rats are shown in figures 1A and ethanol-fed and control group received a IB, respectively. There were no significant single daily dose of 30 /tg (0.9 ¡nCi)of 35S-thiamin by stomach tube. TABLE 1 The rats were fed the thiamin-deficient Ethanol consumption as percentage of diet for 6 weeks. At that time, three rats total energy intake1 from each group were chosen at random and killed for tissue analysis of radio 10 UK 30 jig Week "S-thiamin/day "S-thiamin/day activity. The experiment was continued with the remaining rats in the same man ner except for the route of thiamin admin IIIinIVVVIVIIVIII12.16il.6114.25 istration which was changed from daily iO.6313.08iO.3813.80il.7514.34i2.6814.71il.8814.98i2.5814.02i2. administration by stomach tube to daily intraperitoneal injections. This phase of Ì3.4216.43i2.3216.45il.1918.36i2.7018.78 the experiment lasted for 2 weeks. The remaining rats were killed 24 hours after the last intraperitoneal injection of 35Sthiamin and their tissues analyzed for 1Each number is the meanisD of 6 rats. radioactivity.
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ETHANOL
ADMINISTRATION
differences in weight gain between the rats receiving ethanol and the correspond ing controls on the same level of thiamin intake. However, in both the ethanol and control groups, the rats given 10 /xg thiamin/day gained significantly less weight than the rats receiving 30 /xg of thiamin/ day. Thus, under these experimental con ditions, 10 /xg/day of thiamin was below the optimum daily intake for growth. Table 1 shows the ethanol consumption of the experimental rats expressed as the percentage of the total energy intake. The rats given 10 /xg thiamin/day consumed between 12.16% and 14.98% of their total energy intake as ethanol. The rats given 30 /xg of thiamin/day consumed between 14.24% and 18.78% of the total energy as ethanol. The average weekly values for urinary excretion of thiamin and thiamin metab olites for the 6 week period of intragastric thiamin administration are presented in tables 2 and 3. In general, rats receiving ethanol excreted a smaller percentage of the administered thiamin and its metab olites in the urine as compared to the cor responding control rats. Furthermore, this difference was much more pronounced in the rats given a 10 /xg thiamin/day as com pared to those given a 30 /xg thiamin/day. Table 4 shows the urinary excretion of
AND THIAMIN
METABOLISM
2147
TABLE 3 Average weekly urinary excretion of thiamin, expressed as percent of the intake, in rats receiving 30 y.g of 36S-thiamin/day by gastric intubation1
Week
Ethanol fed%
Control
Differ ence in controls
±6.36229.58±5.13342.36±5.24444.91 IIIIIIIVVVI18.41 ±4.0937.93±2.7748.25±5.2451.87±2.2958.20±3.8761 14+ ±4.78355.14±3.1060.26±5.5225.4116+6+3 86 ±4.20+38+28+ 1The urine was collected daily. The urine from each group (six rats) was pooled and an aliquot taken for determination of radioactivity. These numbers represent the mean±so of the daily urinary radio activity values in each week of the experiment. 2Significantly different from control (P < 0.025). 3 (P < 0.0025). «(P < 0.05).
thiamin and its metabolites during the last 2 weeks of the experiment, a period during which the rats received thiamin by intraperitoneal injection instead of by stomach tube. The ethanol-fed rats receiving 10 /xg thiamin/day continued to excrete less thiamin and its metabolites in their urine than the appropriate controls. However, there was no difference in the amount of thiamin and its metabolites excreted in the urine of the ethanol-fed and control rats receiving 30 /xg thiamin/day.
TABLE 2 Average weekly urinary excretion of thiamin, expressed as percent of the intake, in the rats receiving 10 ng (0.3 fid) of 3SS-thiamin/day by gastric intubation
Week
Ethanol fed
Control
% Differ ence in controls
±5.73232.02±5.94345.3 IIIIIIIVVVI17.41 ±4.9247.59
TABLE 4 Average weekly urinary excretion of thiamin, expressed as percent of the dose, in animals receiving 3SS-thJamin by intraperitoneal injection1
Week
VII ±6.5759.20±4.4964.07±3.1270.32 ±3.53"48.23±3.73357.10±2.84360.40±5.18425.20 VIII
Ethanol fed
Control
10 jug thiamin/day 62.03±4.852 74.38±4.85 64.86±4.222 75.64±2.88
% Differ ence in controls +20 + 17
30 ¿igthiamin/day ±4.2174.16±6.85+45+49+31+33+23+23 VII 65.00±7.05 64.71 ±3.73 0 VIII 70.12±5.00 67.17±3.57 -4 1The urine was collected daily. The urine from 1The urine was collected daily. The urine from each group (six rats) was pooled and an aliquot taken each group (six rats) was pooled and an aliquot taken for determination of radioactivity. These numbers represent the mean±SD of the daily urinary radio for determination of radioactivity. These numbers activity values in each week of the experiment. represent the mean ±so of the daily urinary radio 2Significantly different from control (P < 0.025). activity values in each week of the experiment. 3 (P < 0.0005). " (P < 0.0025). 2Significantly different from control (P < 0.0005).
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2148
MESBAHEDDIN
BALAGHI AND ROBERT A. NEAL
/tg thiamin/day, the fecal excretion of thiamin and its metabolites was generally higher in the ethanol-fed as compared to the appropriate controls. In rats receiving 10 Mgthiamin /day 30 >igthiamin /day 30 ng thiamin/day, the fecal excretion of thiamin and its metabolites was also gen Ethanol Ethanol erally greater in the ethanol-fed rats, al fed Controls Controls Week fed though the difference was not as great as that seen in the rats receiving 10 ng thia IIIIIIIVvVI2.417.077.7518.9316.9015.951.654.367.3811.3010.749.674.875.146.3615.7314.9 min/day. Table 6 shows the mean concentration and the mean total content of radioactivity equivalent to thiamin in the tissues of ethanol-fed and control rats after a 6 week period during which they received 10 or 1The feces from each group was collected daily 30 /¿gof 35S-thiamin daily by stomach and pooled. The feces collected for a week was sub sequently pooled and an aliquot analyzed for radio tube. The tissue concentrations and total activity as described previously (8). contents were not significantly different in ethanol-fed and control rats given the The fecal excretion of thiamin and its same amount of thiamin. metabolites during the 6 weeks of intraAt the end of 6 weeks, only half of the gastric administration of thiamin is pre rats in each group were killed for tissue sented in table 5. In the rats receiving 10 analysis (table 6). The remaining rats TABLE 5
Fecal excretion of thiamin expressed as the percentage of the intake of 3iS-thiaminl
TABLE 6 Tissue distribution of 36S-thiamin and its metabolites in the tissues of ethanol fed and control rats after 6 weeks of administration of 10 or 30 ing"S-thiamin/day by stomach tube Rats receiving 10 Mgthiamin/day Ethanol fed Tissue
Concentration
Controls
Total content1
Concentration
Total content'
BrainLiverHeartKidneyTestesMuscle»9/91.74±0.231.12±0.261.16±0.090.75±0.061.79±0.150.25±0.02M3.53±0.4016.48±1.681.64± ±0.321.95±0.186.69 ±1.52— ±0.63—w/g1.86±0.011.12±0.111.32±0.410.75±0.041.80±0.180.28 ±0.03Mff3.80±0.0615.07±2.191.41 Rats receiving 30 /ig thiamin/day Ethanol fed Tissue
Concentration
Total content1
Controls Concentration
Total content1
BrainLiverHeartKidneyTestesMuscleW/92. ±0.282.99±0.233.26±0.442.00±0.295.04±0.970.60±0.02/*?6.16±1.0246.91 97 ±6.344.86±0.375.41 ±0.295.23±0.3014.91±1.25— ±0.6516.58±3.01—W/03.00±0.152.73±0.393.23±0.171.82±0.084.12±0.370.58 ±0.07W/Õ5.83±0.5241.03±6.824.07 1Each number is the mean±so of the individual analysis of the tissues of three rats. The concentration is expressed as radioactivity equivalent to 1 jig of thiamin/g of tissue (wet weight) and the total content is the total radioactivity in the tissue expressed as ¿igof thiamin.
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ETHANOL ADMINISTRATION AND THIAMIN METABOLISM
2149
TABLE 7 Tissue distribution of uS-thiamin and its metabolites in the tissues of ethanol fed and control rats after 6 weeks of administration of 10 or SO ¡¿g of i6S-thiamin/day by stomach tube followed by 2 weeks on the same intakes by intraperitoneal injection
Rats receiving 10 Mgthiamin/day Ethanol fed Tissue
Concentration1
Controls
Total content1
Concentration1
Total content1
±0.111.42±0.0621.20±0.0720.76±0.0521.97±0.2220.28 ±0.160.94±0.231.00±0.080.61 BrainLiverHeartKidneyTestesMuscleMl91.62
±0.201.92 ±0.126.83±0.142—M/g1.42 ±0.071.50±0.140.27±0.02Mff3.05±0.4715.36±2.341.26± ±0.02M3.38±0.1317.48±1.271.41 Rats receiving 30 jagthiamin/day
Tissue
Ethanol fed Total content1 Concentration1
Controls Total content1 Concentration1
BrainLiverHeartKidneyTestesMuscleml92.95±0.052.38±0.093.25±0.281.99
±6.545.12±0.725.45±0.8917.30±3.19†11
±0.094.76±0.1320.63±0.03M6.12±0.3445.23±2.314.83±0.646.31±0.5815.58±0.38—US/92.82±0
1Each number is the mean±SDof the analysis of the individual tissue of three rats. The concentration is expressed as radioactivity equivalent to 1 Mgof thiamin/g of tissue (wet weight) and the total content is the total radioactivity of the tissue expresses as /¡gof thiamin. ^Significantly different (P < 0.05) from the corresponding control tissue.
were continued on the same regimen but received thiamin by intraperitoneal ad ministration rather than by stomach tube. This phase of the experiment was con tinued for 2 weeks, at the end of which the rats were killed and the tissues ana lyzed in the same manner described in table 6. The results are shown in table 7. In all the tissues, except muscle the ethanol-fed rats given 10 /tg thiamin/day showed a higher concentration and a higher total content of thiamin and its metab olites than the corresponding controls. These differences are statistically signifi cant in case of the concentrations of thia min and its metabolites in heart, kidney, testes and liver and the total content in testes. In the rats given 30 ,ag thiamin/ day, the only tissue where there was a significant difference in concentration was the testes.
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Thiamin is metabolized to thiamin acetic acid by mammalian alcohol dehydrogenase enzymes (6). Therefore, it was of interest to examine the urine of the rats in all four groups for the amount of the radioactivity excreted in the urine as thiamin acetic acid. Figure 2 shows the results of cation exchange chromatography of the urine. The urinary metabolites are resolved into three peaks. Both thiamin and thiamin acetic acid appear in peak III. The column fractions corresponding to peak III, figure 2 were pooled and lyophilized to dryness. The residue was dissolved in 0.5 ml of water, 20 /¿gof unlabeled thiamin acetic acid added, and the mixture subjected to anión exchange chromatography (fig. 3). Thiamin and other uv absorbing material is eluted in peak I. Peak III corresponds to thiamin acetic acid. Using this proce dure of liquid chromatography, first on
2150
MESBAHEDDIN BALAGHI AND ROBERT A. NEAL
TABLE 8
20
Urinary excretion of ^S-tkiamin acetic acid in ethanol-fed and control rats at two levels of thiamin intake1 15S-thiamin/dayDay57585960616263Mean±8DEthanolfed1.631.361.461.281.191.281.271.35 10 Mg "S-thiamin/dayEthanolfed2.692.382.362.652.502.813.072.63 jig
I > 10
5
< J. ce
±0.25»Control1.201.051.071.421.971.942.421.58 ±0.14»Control1.060.960.790.850.921.050.980.94 ±0.53 ±0.0930
10
20 FRACTION
30
40
NUMBER
Fig. 2 Cation exchange chromatography of the urine of rats receiving 30 Mg Å“S-thiamin/day by stomach tube. The column was a 0.46 X 25 cm column of Partisil PXS 10/25 SCX.11 Two ml of a concentrated sample of urine was injected onto the column and the column eluted with 15 ml of water followed by a 30 minute linear gra dient of water to pyridine: acetic acid: water (3:6:91 v/v). The flow rate was 1 ml/minute. The fraction size is 1 ml.
cation exchange followed by an anión ex change column, the thiamin acetic acid could be separated from thiamin and other thiamin metabolites present in urine. The fractions corresponding to peak III, figure 3 were pooled and the 35S-thiamin acetic acid content determined by scintil lation counting. Table 8 shows the urinary excretion of thiamin acetic acid an all four groups ex pressed as the percentage of total urinary radioactivity. In both ethanol-fed and con trol rats, a higher percentage of the thia min was excreted as thiamin acetic acid in the rats receiving 30 /¿gas compared to 10 fig of thiamin/day. The ethanol-fed rats at both levels of intake converted a higher percentage of thiamin into thiamin acetic acid than the corresponding controls. DISCUSSION Several workers have studied the effect of ethanol on the intestinal absorption of
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The figures represent the/amount of thiamin acetic acid excreted expressed as the percent of the total urinary radio activity. ! The percentage of the urinary radioactivity that is represented by thiamin acetic acid was determined by liquid chromatography as described in figure 3. These numbers are the results obtained on analysis of the pooled urine of the six rats in each group on each of days 58 to 64 of the experiment. During days 49 to 63 the rats received *5S-thiamin by intraperitoneal injection. On days 1 to 48 of the experiment the rats received «S-thiamin by stomach tube. ! Significantly dif ferent (P < 0.05) than the corresponding control.
thiamin. Thus, Tomsaulo et al. (3) admin istered 14C-thiamin orally to alcoholic sub jects and found significant decreases in the
0
5
10
15
20
25
TIME (mm)
Fig. 3 Anión exchange chromatography of peak III from the cation exchange column de scribed in figure 2. The column was a 0.46 X 25 cm column of Partisil PXS 10/25 SAX.13Peak III from figure 2 was lyophilized to dryness, the residue dissolved in 0.5 ml of distilled water, 20 Hg of unlabeled thiamin acetic acid added and the mixture injected onto the column. The column was eluted first with 0.05 M Na phosphate buffer (pH 5.7) followed by (arrow) 0.2 M acetic acid. The flow rate was 2 ml/minute.
ETHANOL ADMINISTRATION AND THIAMIN METABOLISM
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urinary excretion of the radioactivity and a rats. These data suggest that ethanol not significant increase in fecal excretion. only depresses the intestinal absorption but Thomson et al. (4), examined the radio also decreases the urinary excretion of activity in the femoral artery and hepatic thiamin under conditions of marginal in vein after oral administration of [2-14C]- take. It is interesting to speculate that the thiazole thiamin. They also examined the decrease in urinary excretion of thiamin in excretion of radioactivity in the urine and ethanol-fed rats might be an adaptive re feces, and obtained data indicating severe sponse compensating for lower absorption depression of thiamin absorption under thus, slowing the rate of depletion of the the influence of ethanol given either orally tissue of thiamin. The data presented in table 6 supports this speculation showing or by iv injection. More recently, Hoyumpa et al. (5) used rat-everted gut sacs or that after 6 weeks of ethanol feeding, there intact isolated loops of rat intestine to was no significant difference in tissue levels of thiamin compounds between study the effect of ethanol on thiamin ab ethanol-fed and controls at either level of sorption. This group found that absorption of thiamin at concentrations below 2.0 JU.M intake in spite of a decreased degree of absorption of thiamin in the ethanol-fed is an active transport process which is in hibited by ethanol at a dose of 50 to 750 rats (table 5). However, when thiamin was administered by intraperitoneal in mg/100 g of body weight given by a stom ach tube or by intravenous injection. At jection for 2 weeks, the ethanol-fed rats on concentrations higher than 2.0 /*M,thiamin a marginal thiamin intake (10 /tg/day) had higher concentrations of thiamin in appears to be absorbed by passive diffu most tissues than the corresponding control sion. The passive diffusion of thiamin is un affected by ethanol administration. The (table 7). Thus, the decreased excretion of data from the studies reported in this thiamin and its metabolites in the urine of manuscript are in general agreement with the ethanol-fed rats may be unrelated to the decreased intestinal absorption. these findings of decreased intestinal ab Another aspect of the ethanol-thiamin sorption. Thus, from the data in table 2, the urinary excretion of radioactivity was interrelationship is the possible increase in depressed by 23% to 49% in ethanol-fed thiamin metabolism to inactive metabolites rats given a low level (10 /ug/day) of the in ethanol-fed rats. Previous work in this vitamin. In rats given a higher level (30 laboratory (6) has indicated that alcohol /ig/day), the difference, which is 38% for dehydrogenase is involved to an important the first week, tended to decrease and be degree in the in vivo metabolism of thia came nonsignificant by the end of the min to thiamin acetic acid in the rat. Thus, fourth week (table 3). The data on fecal it is reasonable that the increased metab excretion of thiamin (table 5) supports olism of thiamin to thiamin acetic acid in ethanol-fed rats which was demonstrated the view that the decreased urinary excre tion of thiamin seen in the ethanol-fed rats in these studies ( table 8 ) is, at least in part, given the low (10 ^g/day) thiamin intake the result of an increased activity of alco and, to a lesser degree, those given the hol dehydrogenase. high (30 /j.g/day) thiamin intake may have An increase in ethanol metabolism as a resulted from a decreased absorption of result of chronic exposure of rats to ethanol the vitamin. It was therefore expected has been demonstrated in vivo (9) and in when the rats were given thiamin by intra- vitro (10-13). The mechanism of this peritoneal injection that the difference in adaptive increase in ethanol metabolism excretion of thiamin and its metabolites in has been controversial. Videla et al. (IO) the urine in ethanol-fed and control rats and Thurman et al. (Il, 12) have sug would be eliminated since the impaired gested this increased rate of metabolism intestinal absorption would be bypassed. of ethanol is the result of a higher rate of reoxidation of NADH in the ethanolHowever, as shown in table 4, the urinary excretion of radioactivity in rats receiving exposed rats. These workers have further 10 ng/aay 33S-thiamin by intraperitoneal suggested that the increased rate of NADH injection remained lower in ethanol-fed oxidation is a result of ADP stimulation of
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MESBAHEDDIN
BALAGHI AND ROBERT A. NEAL
the mitochondrial oxidation of NADH, the excess ADP arising from an ethanolstimulated increase in Na% K+ ATPase activity. On the other hand, Matsuzaki and Lieber (13) report that pyrazole, an inhibitor of alcohol dehydrogenase, has a greater inhibitory effect on ethanol metab olism in vitro using liver slices from nor mal as compared to chronic ethanoltreated rats. From these results they sug gest the adaptive increase of hepatic ethanol oxidation after chronic ethanol consumption is due to the enhanced activ ity of the microsomal ethanol oxidizing system (MEOS) (14). However, this adaptive increase attributed to MEOS was seen only at high ethanol concentrations (13). Experiments at lower and more physiological concentrations of ethanol have shown that 4-methylpyrazole equally inhibits ethanol uptake by perfused livers of ethanol-fed and control rats ( 12 ). These results were interpreted to mean that alco hol dehydrogenase and not catalase or MEOS is responsible for the increased rate of uptake and metabolism of ethanol in chronic ethanol-treated rats. Thiamin is not metabolized to the thiamin acetic acid under conditions compara ble to those used to measure microsomal ethanol metabolism.14 Therefore, it appears more likely that an increased activity of alcohol dehydrogenase is responsible for the increase in metabolism of thiamin to thiamin acetic acid seen in these experi ments (table 8). The difference in the per centage of the thiamin excreted in the urine as thiamin acetic acid in the ethanolfed and control rats is quite small. Conse quently, it is questionable whether an increased rate of metabolism of thiamin to thiamin acetic acid plays a significant role in thiamin deficiency seen in alcoholics. LITERATURE
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
CITED
1. Leevy, C. M., Cardi, L., Frank, O., Gellene, R. & Baker, H. (1965) Incidence and significance of hypovitaminemia in a randomly "Norman, vations.
2.
B. J. & Neal, R. A. Unpublished
obser
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14.
selected municipal hospital population. Am. J. Clin. Nutr. 17, 259-271. Neville, J. N., Eagles, J. A., Samson, G. & Olson, R. E. (1968) Nutritional status of alcoholics. Am. J. Clin. Nutr. 21, 1329-1340. Tomasulo, P. A., Kater, R. M. & Iber, F. L. ( 1968 ) Impairment of thiamine absorption in alcoholism. Am. J. Clin. Nutr. 21, 13411344. Thomson, A. D., Baker, H. & Leevy, C. M. (1970) Patterns of ""S-thiamine hydrochloride absorption in the malnourished alcoholic patient. J. Lab. Clin. Med. 76, 34-45. Hoyumpa, A. M., Jr., Breen, K. J., Schenker, S. & Wilson, F. A. (1975) Thiamine trans port across the rat intestine. II. Effect of ethanol. J. Lab. Clin. Med. 86, 803-816. Dalvi, R. R., Sauberlich, H. E. & Neal, R. A. (1974) An examination of the metabolism of thiamin by rat liver alcohol dehyrogenase. J. Nutr. 104, 1476-1483. Neal, R. A. (1970) Bacterial metabolism of thiamine. 3. Metabolism of thiamine to 3-(2'methyl-4'-amino-5'-pyrimidylmethyl)-4-methylthiazole-5-acetic acid (thiamine acetic acid) by a flavoprotein isolated from a soil micro organism. J. Biol. Chem. 245, 2599-2604. Balaghi, M. & Pearson, W. N. (1966) Tis sue and intracellular distribution of radioac tive thiamine in normal and thiamine-deficient rats. J. Nutr. 89, 127-132. Hawkins, R., Kalant, H. & Khanna, J. M. (1966) Effects of chronic intake of ethanol on rate of ethanol metabolism. Can. J. Physiol. Pharmacol. 44, 241-257. Videla, L., Bernstein, J. & Israel, Y. (1973) Metabolic alterations produced in the liver by chronic ethanol administration. Biochem. J. 134, 507-514. Thurman, R. G., McKenna, W. R. & McCaf frey, T. B. (1976) Pathways responsible for the adaptive increase in ethanol utilization following chronic treatment with ethanol: Inhibitor studies with the hemoglobin-free perfused rat liver. Mol. Pharmacol. 12, 156166. Thurman, R. G. & Scholz, R. (1976) Etha nol metabolism in perfused rat liver at low ethanol concentrations: Involvement of alcohol dehydrogenase in the adaptive increase in ethanolwith metabolism due to chronicZ. pretreat ment ethanol. Hoppe-Zeyler's Physiol. Chem. 357, 1443-1446. Matsuzaki, S. & Lieber, C. S. (1975) ADHindependent ethanol oxidation in the liver and its increase by chronic ethanol consumption. Gastroenterol. 69, 845. Lieber, C. S., Rubin, E. & DeCarli, L. M. (1970) Hepatic microsomal ethanol oxidiz ing system (MEOS): Differentiation from alcohol dehydrogenase and NADPH oxidase. Biochem. Biophys. Res. Commun. 40, 858865.
