Numerous studies have shown that vitamin status of alcoholic patients differs from non-drinking subjects(Reference Bonjour1–Reference Bonjour7), and the majority have shown that blood vitamin levels are lower in alcoholic patients than in controls(Reference Leevy, Baker and TenHove8–Reference Baker, Frank and Ziffer10). In addition, several reports have suggested that chronic alcohol feeding may lead to a significant inhibition of carrier-mediated thiamin(Reference Subramanian, Subramanya and Tsukamoto11, Reference Subramanya, Subramanian and Sain12) and folate(Reference Hamid and Kaur13–Reference Wani and Kaur19) uptake in the intestine and kidney. This phenomenon is observed only in alcoholic patients who drink ethanol chronically. On the contrary, a reduction in circulating levels of B-complex vitamins often occurred without clinical evidence of hypovitaminosis(Reference Leevy, George and Ziffer20). Sorrell et al. (Reference Sorrell, Baker and Barak21) reported that the in vitro perfusion of rat liver with ethanol caused the release of all B-vitamins except biotin from the liver stores. Israel & Smith(Reference Israel and Smith22) reported that acute ethanol feeding to rats inhibited the conversion of pantothenic acid to CoA. These studies in animal models suggested that acute ethanol intake results in an increased hepatic release of vitamins and an impaired utilisation, which means increased levels of free forms of vitamins in the liver which can in turn permeate the cell membranes(Reference Sorrell, Baker and Barak21, Reference Israel and Smith22). This might lead to increases in blood vitamin contents and in urinary excretion. Although there are many reports concerning the effects of ethanol on the absorption and metabolism of vitamins, the conclusion concerning the controversy remains elusive. The reason might be that there is no study regarding the simultaneous measurement of vitamin contents of liver (as a biomarker of the storage amount of vitamins), blood (as a biomarker of the circulation amount of vitamins) and urine (as a biomarker of the reabsorption ability of kidney and an extra amount of vitamins).
In the present study, we examined the effects of ethanol consumption on the contents of B-group vitamins of the liver, blood and urine in rats fed two kinds of diets containing either a sufficient- or a low-vitamin mixture.
Materials and methods
Chemicals
Vitamin-free milk casein, sucrose and l-methionine were purchased from Wako Pure Chemical Industries. Maize oil was purchased from Ajinomoto. Gelatinised maize starch, a mineral mixture (AIN-93G mineral mixture)(Reference Reeves23) and a vitamin mixture (nicotinic acid-free AIN-93 vitamin mixture containing 25 % choline bitartrate)(Reference Reeves23) were obtained from Oriental Yeast Company, Limited.
Thiamin hydrochloride (C12H17ClN4OS-HCl; molecular weight 337·27), riboflavin (C17H20N4O6; 376·37), pyridoxine hydrochloride (C8H11NO3-HCl; 205·63), cyanocobalamin (C63H88CoN14O14P; 1355·40), nicotinamide (C6H6N2O; 122·13), calcium pantothenate (C18H32N2O10-Ca; 476·54), folic acid (C19H19N7O6; 441·40) and d(+)-biotin (C10H16N2O3S; 244·31) were purchased from Wako Pure Chemical Industries. 4-Pyridoxic acid (C8H9NO4 = 183·16) was made by ICN Pharmaceuticals and obtained through Wako Pure Chemical Industries.
N 1-Methylnicotinamide chloride (C7H9N2O-HCl; 159·61) was purchased from Tokyo Kasei Kogyo. N 1-Methyl-2-pyridone-5-carboxamide (2-Py, C7H8N2O2 152·15) and N 1-methyl-4-pyridone-3-carboxamide (4-Py, C7H8N2O2 152·15) were synthesised by the methods of Pullman & Colowick(Reference Pullman and Colowick24) and Shibata et al. (Reference Shibata, Kawada and Iwai25), respectively. All other chemicals used were of highest purity available from commercial sources.
Animals and treatment
The care and treatment of the experimental animals conformed to the University of Shiga Prefecture guidelines for the ethical treatment of laboratory animals. The animals were maintained under controlled temperature (22°C), 60 % humidity and light conditions (12 h light–12 h dark cycle).
Effects of ethanol feeding on the B-group vitamin contents of liver, blood and urine in rats fed a diet containing a sufficient-vitamin mixture (Expt 1)
Male Wistar rats (3 weeks old) obtained from CLEA Japan were fed freely with a conventional purified diet, consisting of 20 % vitamin-free milk casein, 0·2 % l-methionine, 46·9 % gelatinised maize starch, 23·4 % sucrose, 5 % maize oil, 3·5 % AIN-93-G mineral mixture(Reference Hamid, Kaur and Mahmood14) and 1 % AIN-93 vitamin mixture(Reference Hamid, Kaur and Mahmood14) containing chorine bitartrate, but without nicotinic acid, to acclimatise for 7 d. Nicotinic acid had not been added to this diet because it is supplied enough from tryptophan in casein(Reference Shibata, Mushiage and Kondo26), and a dietary fibre-free diet was used because it is a tradition not to use dietary fibre in our laboratory which is not essential for normal growth(Reference Fukuwatari, Wada and Shibata27).
The rats were divided into two groups (n 5 each). Group 1 was fed with a diet containing the 1 % vitamin mixture (a sufficient-vitamin diet) and allowed to drink water for 28 d. Group 2 was fed with a diet containing the 1 % vitamin mixture (a sufficient-vitamin diet) and forced to drink a 15 % ethanol solution instead of water for 28 d. The 24 h urine samples were collected in amber bottles containing 1 ml of 1 m-HCl at 09.00–09.00 hours of the last day and were stored at − 25°C until required. The rats were killed at about 09.00 hours; blood was collected and tissues were taken to measure the weights and the contents of B-group vitamins in the liver, blood and urine. Liver samples were preserved at − 25°C until required.
Effects of ethanol feeding on the B-group vitamin contents of liver, blood and urine in rats fed a diet containing a low-vitamin mixture (Expt 2)
A preliminary experiment revealed that the body-weight gain of young rats was the same when fed a diet containing the 1 % AIN-93 vitamin mixture and the 0·3 % AIN-93 vitamin mixture, whereas the body-weight gain was lower in rats fed a diet containing the 0·2 % AIN-93 vitamin mixture than in those fed a diet containing the 1 or 0·3 % diets. Thus, we determined tentatively whether the diet containing the 0·3 % AIN-93 vitamin mixture could supply a minimum amount of vitamins for the growing rats.
Male Wistar rats (3 weeks old) obtained from CLEA Japan were fed freely with the conventional purified diet (mentioned above) to acclimatise for 7 d. The rats were then divided into two groups (n 5 each). Group 1 was fed a diet containing the 0·3 % vitamin mixture and allowed to drink water for 28 d. Group 2 was fed a diet containing the 0·3 % vitamin mixture and forced to drink a 15 % ethanol solution instead of water for 28 d. The 24 h urine samples and tissues were collected. Levels of alanine aminotransferase, aspartate aminotransferase and γ-glutamyltranspeptidase were measured at Mitsubishi Chemical Medience (Tokyo, Japan).
Measurement of B-group vitamins in urine and blood
Preparation and measurement of the extracts of the B-group vitamins from the urine and blood are described as follows(Reference Shibata, Fukuwatari and Ohta28).
Vitamin B1
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to ten volumes of 5 % ice-cold TCA and homogenised with a Digital Homogenizer Hom (Iuchi). The acidified homogenate was centrifuged at 10 000 g for 10 min at 4°C, and the supernatant was retained and used for the measurement of vitamin B1(Reference Fukuwatari, Suzuura and Sasaki29).
Vitamin B2
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to ten volumes of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) and homogenised with a Teflon/glass homogeniser (Nikko Hansen). To 0·1 ml of the homogenate, 0·44 ml of water and 0·26 ml of 0·5 m-H2SO4 were added and then kept at 80°C for 15 min. After cooling, 0·2 ml of 10 % TCA were added and centrifuged at 10 000 g for 3 min at 4°C. From the supernatant obtained, 0·2 ml was withdrawn and added to 0·2 ml of 1 m-NaOH. The alkalised mixture was irradiated with a fluorescent lamp for 30 min and then 0·02 ml of glacial acetic acid were added to the mixture. The neutralised mixture was passed though a 0·45 μm microfilter and the filtrate was directly injected into the HPLC system for measuring lumiflavin(Reference Ohkawa, Ohishi and Yagi30).
Vitamin B6
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to 90 ml of 55 mm-HCl and homogenised with a Waring blender. The homogenate was autoclaved at 121°C for 3 h. After cooling, the mixture was adjusted to pH 5·0 with 1 m-NaOH and then made up to 100 ml with water. The solution was filtered with qualitative filter no. 2 (ADVANTEC MFS, Inc.). The filtrate was used for measuring vitamin B6 as described previously(31).
Vitamin B12
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to 2·5 ml of 0·57 m-acetic acid–sodium acetate buffer (pH 4·5) plus 5 ml of water and 0·1 ml of 0·05 % potassium cyanide (KCN). The suspension was homogenised with a Teflon/glass homogeniser. The homogenate was then put into a boiling water-bath for 5 min. After cooling, 0·15 ml of 10 % metaphosphoric acid were added and made up to 10 ml with water. The solution was filtered with qualitative filter no. 2 (ADVANTEC MFS, Inc.). The filtrate was used for measuring vitamin B12 as described previously(Reference Watanabe, Abe and Katsura32).
Nicotinamide
Frozen liver samples, about 0·6 g, were thawed, minced, and then added to five volumes of 0·1 g/ml isonicotinamide. The suspension was homogenised with a Teflon/glass homogeniser. The homogenate (1 ml) was withdrawn and added to 4 ml of water, and then autoclaved at 121°C for 10 min. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained and the precipitated materials were extracted again with 5 ml of water, and the supernatant was retained. Both the retained supernatants were combined, and the extract was used for measuring nicotinamide as described previously(Reference Shibata, Kawada and Iwai25).
Pantothenic acid
Frozen liver samples, about 0·2 g, were thawed, minced, and then added to ten volumes of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0). The suspension was homogenised with a Teflon/glass homogeniser. The homogenate was incubated at 37°C overnight to convert free pantothenic acid from the bound type of pantothenate compounds. The reaction was stopped by putting it into a boiling water-bath for 5 min. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained and the precipitated materials were extracted again with 2 ml of water, and the supernatant was retained. Both the retained supernatants were combined, and the extract was used for measuring pantothenic acid as described previously(Reference Skeggs and Wright33).
Folate
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to ten volumes of 0·1 m-KH2PO4–K2HPO4 buffer (pH 6·1). The suspension was homogenised with a Teflon/glass homogeniser. The homogenate was autoclaved at 121°C for 5 min. After cooling, 2·5 ml of pronase (5 mg/ml; Pronase MS; Kaken Pharmaceutical Company, Limited) were added and then incubated at 37°C for 3 h. The reaction was stopped by putting it into a boiling water-bath for 10 min. After cooling, 0·5 ml of conjugase (extract from porcine kidney acetone powder, Type II; Sigma-Aldrich) were added and incubated at 37°C overnight. The reaction was stopped by putting it into a boiling water-bath for 10 min. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained, and the precipitated materials were extracted again with 3 ml of water, and the supernatant was retained. Both the retained supernatants were combined, and the extract was used for measuring folate as described previously(Reference Aiso and Tamura34). The conjugase solution was made as follows: 60 ml of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) were added to 20 g porcine kidney acetone powder and stirred for 30 min at 4°C. The suspension was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was dialysed against a large amount of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) to remove endogenous folate of the kidney acetone powder. The dialysed conjugase solution was used.
Biotin
Frozen liver samples, about 0·5 g, were thawed, minced, and then added to two volumes of 2·25 m-H2SO4 and then homogenised with a Waring blender. The suspension was hydrolysed by autoclaving for 1 h at 121°C. After cooling, the suspension was centrifuged at 10 000 g for 10 min at 4°C, and the supernatant was used for measuring biotin(Reference Fukui, Iinuma and Oizumi35).
Analyses
The measurements of the B-group vitamins except for vitamin B6 were described previously(Reference Wani and Kaur19). The urinary excretion of 4-pyridoxic acid, a catabolite of vitamin B6, was measured according to the method of Gregory & Kirk(Reference Gregory and Kirk36).
Statistical analysis
Mean values between the treatment groups were compared using the Mann–Whitney U two-tailed t test. P < 0·05 was considered to be statistically significant. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software).
Results
Effects of ethanol feeding on the B-group vitamin contents of liver, blood and urine in rats fed a diet containing a sufficient-vitamin mixture (Expt 1)
There were no differences in body-weight gain and liver weights between the groups. No differences in the levels of vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide, pantothenic acid, folate and biotin were observed in the liver and blood. Although the 24 h urinary excretion of some of the vitamins was slightly lower in the ethanol-treated group than in the control, the differences were not significant (data not shown). Thus, ethanol consumption did not affect the B-group vitamin contents in the liver, blood and urine when the rats were fed a diet containing sufficient amounts of the vitamins.
Effects of ethanol feeding on the B-group vitamin contents of liver, blood and urine in rats fed a diet containing a low-vitamin mixture (Expt 2)
As shown in Table 1, body-weight gain, food intake and liver weights were lower in the ethanol-fed group than in the controls. The overall food intake was lower in the ethanol-fed group than in the controls, but energy intake was almost the same because of ethanol intake.
Table 1 Effects of ethanol consumption on rat body-weight gain, food intake, ethanol intake, water intake, energy intake, food efficiency ratio and liver weight (Expt 2)
(Mean values with their standard errors for five rats per group)
* Mean values were significantly different from those of the control group (P < 0·05; Mann–Whitney U two-tailed t test).
† The value is expressed in g of pure ethanol and not as the volume of 15 % ethanol.
‡ Energy of 1 g ethanol was calculated as 29·3 kJ (7 kcal)/g.
§ ( Body-weight gain/food intake) × 100.
∥ (Body-weight gain/energy intake) × 100.
The effects of ethanol consumption on the activities of alanine aminotransferase, aspartate aminotransferase and γ-glutamyltranspeptidase in plasma are shown in Table 2. No significant effects of ethanol consumption were observed for these indices of liver function.
Table 2 Effects of ethanol consumption on the activities of alanine aminotransferase, aspartate aminotransferase and γ-glutamyltranspeptidase in plasma
(Mean values with their standard errors for five rats per group)
The effects of ethanol consumption on the B-group vitamin contents of the liver are shown in Table 3. The contents of the vitamins in liver are measured as storage amounts of the vitamins, thus are expressed as mol/liver. The contents of vitamin B1, vitamin B2 and pantothenic acid were lower in the ethanol-fed group than in the controls, whereas the contents of vitamin B6, vitamin B12, nicotinamide, folate and biotin were not significantly different.
Table 3 Effect of ethanol consumption on liver B-group vitamin contents (Expt 2)
(Mean values with their standard errors for five rats per group)
* Mean values were significantly different from those of the control group (P < 0·05; Mann–Whitney U two-tailed t test).
The effects of ethanol consumption on the B-group vitamin contents of the blood are shown in Table 4. The contents of vitamin B1, vitamin B2, vitamin B6 and pantothenic acid were lower in the ethanol-fed group than in the controls, whereas the contents of vitamin B12, nicotinamide, folate and biotin were not significantly different.
Table 4 Effect of ethanol consumption on blood B-group vitamin contents (Expt 2)
(Mean values with their standard errors for five rats per group)
* Mean values were significantly different from those of the control group (P < 0·05; Mann–Whitney U two-tailed t test).
The effects of ethanol consumption on the 24 h urinary excretion of the B-group vitamins are shown in Table 5. The excretion of vitamin B1, vitamin B2, 4-pyridoxic acid (a catabolite of vitamin B6), vitamin B12, pantothenic acid, folate and biotin was lower in the ethanol-fed group than in the controls, whereas the contents of nicotinamide (sum of the contents of nicotinamide and its catabolites such as N 1-methylnicotinamide, 2-Py and 4-Py) were not significantly different.
Table 5 Effect of ethanol consumption on urinary B-group vitamin excretion (upper row) and urinary excretion ratio (lower row) for each of the vitamins (Expt 2)†
(Mean values with their standard errors for five rats per group)
4-PIC, 4-pyridoxic acid.
* Mean values were significantly different from those of the control group (P < 0·05; Mann–Whitney U two-tailed t test).
† Percentage urinary excretion ratio was calculated using the following equation: (24 h urinary excretion (mol/d)/intake of the vitamin during urine collection (mol/d)) × 100.
‡ A catabolite of vitamin B6.
§ Niacin content was calculated as the sum of the nicotinamide content and its catabolites such as N 1-methylnicotinamide, N 1-methyl-2-pyridone-5-carboxamide and N 1-methyl-4-pyridone-3-carboxamide.
∥ Urinary excretion ratio was not calculated as niacin was derived from tryptophan.
Food intake was different in the two groups, so that urinary excretion ratios of the vitamins were calculated. As shown in Table 5, the excretion ratios of all vitamins except for vitamin B12 were lower in the ethanol-fed group.
Discussion
An ordinary diet for rats generally contains sufficient amounts of nutrients including vitamins(Reference Reeves23). Under well-nourished conditions, rats are generally little affected by factors such as ethanol consumption. In fact, the present study proves that ethanol consumption did not affect the body-weight gain or the vitamin contents in the liver and blood when rats were fed a diet containing sufficient amounts of vitamins. On the other hand, when rats were fed a diet low in vitamins, body-weight gain was lower in the ethanol-fed group than in the control group and some vitamin contents of the liver and blood, and urinary excretion were decreased. These results show that chronic ethanol consumption affects absorption, distribution and excretion of vitamins, as reported previously(Reference Bonjour1–Reference Wani and Kaur19). The present findings are not consistent with the in vitro perfusion of rat liver with ethanol, which caused the release of all B-vitamins except biotin from the liver stores(Reference Reeves23). This phenomenon was not observed in the present whole-body experiment, because the vitamin contents of the blood were not increased by ethanol consumption. In the present in vivo experiment, any vitamins released from the liver were quickly absorbed by non-hepatic tissues. In humans, the typical dietary vitamin intakes are generally around the minimum requirements. Thus, the nutritional status of rats fed a diet low in vitamins was similar to that of humans. Ethanol consumption was 45 g over 28 d, so that daily average ethanol consumption was about 1·6 g/d, which corresponds to an energy intake of 46·9 kJ (11·2 kcal)/d. The energy intake in the ethanol-fed group, including ethanol energy, was 5845 kJ (1396 kcal) over 28 d (about 209 kJ (50 kcal)/d). Thus, ethanol accounted for 20 % of dietary energy. Under these conditions, liver functions in rats were not injured. If humans were to consume 10 467 kJ (2500 kcal)/d, the equivalent ethanol consumption would be about 70 g/d, which corresponds to 1 litre of typical beer.
Vitamin depletion, common in malnourished alcoholic patients(Reference Baker, Frank and Ziffer10), can occur despite vitamin supplementation. Vitamin malabsorption(Reference Thomson, Baker and Leevy37), exacerbated by malnutrition, contributes to this depletion(Reference Leevy and Baker38). Also, in alcoholic patients, the impaired ability of the liver to bind and store vitamins might contribute to this depletion. This may probably be due to the hepatotoxicity of ethanol, which impairs not only the vitamin-binding capacity but also the vitamin storage of the liver. In the present study, a diet containing 20 % casein supplemented with methionine was used, which is an excellent protein source from a nutritional standpoint. This suggest the reasons why ethanol consumption did not cause any severe damage, such as an extremely low food intake and body-weight gain and roughness of fur for the rats, even when they were fed a low-vitamin diet.
Sorrell et al. (Reference Sorrell, Baker and Barak21) reported that the in vitro perfusion of rat liver with ethanol caused the release of all vitamins from the liver stores, especially thiamin. It is generally considered that this phenomenon causes increased urinary excretion of vitamins, but in the present in vivo experiments, ethanol consumption did not cause increased urinary excretion, but rather decreased it. This discrepancy between the expected and the actual findings may be attributed to the difference between the in vitro and in vivo experiments. Moreover, there are differences in short-term and long-term adjustment mechanisms for ethanol toxicity. The protein nutritional status was high in the present study because the diet used 20 % casein supplemented with methionine. Protein plays a pivotal role in vitamin absorption and storage in hepatocytes. Protein malnutrition causes malabsorption, reduced storage and impaired utilisation of vitamins. Thus, an adequate intake of vitamins, and also protein, is essential for preventing ethanol toxicity.
In the present study on the low-vitamin diet, vitamin B1, vitamin B2 and pantothenic acid contents in the liver and blood were lower in the ethanol-fed group than in the controls, even when rats were fed a high-protein diet. Furthermore, the total urinary excretion and excretion ratios of all three vitamins were also lower in the ethanol-fed group. Thus, ethanol consumption reduced the intestinal absorption of these vitamins, as reported by Subramanya et al. (Reference Subramanya, Subramanian and Sain12), Hamid et al. (Reference Hamid and Kaur13, Reference Hamid, Kaur and Mahmood14, Reference Hamid, Wani and Rana16, Reference Hamid, Kiran and Rana17) and Wani & Kaur(Reference Wani and Kaur19). Vitamins such as vitamin B1, vitamin B2 and pantothenic acid might be directly and/or indirectly involved in the metabolism of ethanol, indicating that the vitamin catabolites increased and were excreted into the urine. Of these three vitamins, only the catabolic fate of vitamin B1 is relatively well known. It has been reported that the excretion of vitamin B1 metabolites usually exceeds by far the excretion of intact vitamin B1 using radioactive tracer experiments(Reference Pearson39). The major metabolites of vitamin B1 in rat urine are 2-methyl-4-amino-5-pyridinecarboxylic acid(Reference Neal and Pearson40), 4-methylthiazole-5-acetic acid(Reference Suzuoki, Tominaga and Matsuo41) and thiamine acetic acid(Reference Amos and Neal42). Pearson(Reference Pearson39) reported that the sum of the metabolites accounted for about 50 % of the total urinary excretion of vitamin B1 and its catabolites from radioactive tracer experiments. Although we cannot measure the catabolites of vitamin B1, these metabolites might increase in the urine of the ethanol-fed rats. It is likely that a similar phenomenon would apply for the fates of vitamin B2 and pantothenic acid.
The content of vitamin B6 in the blood was lower in the ethanol-fed group, but the content of vitamin B6 in the liver was slightly higher in the ethanol-fed group than in the control. The urinary excretion of vitamin B6, determined from its catabolite 4-pyridoxic acid, was much lower in the ethanol-fed group than in the control. Probably ethanol consumption resulted in an increased storage of vitamin B6 in the liver.
Other B-group vitamin contents in the liver and blood, such as vitamin B12, nicotinamide, folate and biotin, were not affected by ethanol consumption. The lack of any effect of ethanol consumption on the niacin content in this experiment was probably because nicotinamide was synthesised from tryptophan, which was present in the diet as casein and was supplied adequately(Reference Shibata and Onodera43). For rats, NAD precursors such as nicotinic acid and nicotinamide are not essential. In fact, the urinary excretion of nicotinamide did not differ between the two groups. Concerning the effect of ethanol consumption on biotin, Sorrell et al. (Reference Sorrell, Baker and Barak21) reported that the in vitro perfusion of rat liver with ethanol did not cause the release of biotin, but caused the release of vitamin B12 first. In the present experiment, a similar phenomenon was observed for biotin, but not for vitamin B12. Frank et al. (Reference Frank, Baker and Leevy44) reported that the first vitamin released into the circulation during hepatic insult by ethanol is vitamin B12. This disparity between the reported and the present findings might also arise from the difference in protein nutritional status.
There are many reports concerning how ethanol consumption affects folate absorption and metabolism(Reference Hamid and Kaur13–Reference Hamid and Kaur18, Reference Collins, Eisenga and Bhandari45–Reference Romanoff, Ross and McMartin53). Some studies have reported that ethanol consumption increased the urinary excretion of folates(Reference Tamura and Halsted46, Reference McMartin47, Reference McMartin and Collins50–Reference Romanoff, Ross and McMartin53) and caused decreased serum folate levels. Romanoff et al. (Reference Romanoff, Ross and McMartin53) reported that acute ethanol exposure inhibits the apical transport of 5-methyltetrahydrofolate in cultured human proximal tubule cells, and in subchronic ethanol studies, increasing concentrations of ethanol resulted in an up-regulation of folate transporters. Furthermore, Romanoff et al. (Reference Romanoff, Ross and McMartin53) reported that both the folate receptor and reduced folate carrier transporter proteins were up-regulated in rats receiving an ethanol diet. On the contrary, Hamid et al. (Reference Hamid and Kaur13, Reference Hamid, Kaur and Mahmood14, Reference Hamid, Wani and Rana16, Reference Hamid, Kiran and Rana17) and Wani & Kaur(Reference Wani and Kaur19) reported that ethanol reduced the intestinal uptake of folate by altering the binding and transport kinetics of the folate transport system and also the expression of folate transporters in the intestine. In addition, Hamid & Kaur(Reference Hamid and Kaur15) reported that ethanol consumption reduces folate re-uptake in the renal absorption system by the decreased expression of transporters. The present data for folate are not consistent with previous reports(Reference Hamid and Kaur13–Reference Hamid and Kaur18, Reference Collins, Eisenga and Bhandari45–Reference Romanoff, Ross and McMartin53); the contents of folate in the liver and blood were not affected by ethanol consumption, and the urinary excretion of folate and the excretion ratio were decreased markedly. A study(Reference McMartin, Collins and Eisenga52) reported that urinary folate excretion increased in ethanol-fed rats consuming folate-containing diets, but not in rats fed folate-deficient diets. In the present study, the urinary excretion of folate did not increase, but decreased. This was because the diet was low in folate. In the present study, the urinary excretion of folate was lower in the ethanol-fed group than in the non-ethanol group, suggesting that ethanol consumption and the feeding of a low-folate diet up-regulated the folate receptor and reduced folate carrier transporter proteins. This up-regulation was probably a compensatory response to counteract the effects of ethanol in inhibiting the reabsorption of folate. Therefore, the effects of ethanol would depend on the dose and duration of treatment.
In summary, these results show that ethanol consumption affects the absorption, distribution and excretion of each of the vitamins in rats fed a diet containing a low-vitamin mixture. On the other hand, when rats were fed a 20 % casein diet containing a sufficient amount of vitamins, ethanol consumption did not affect any factors that we measured.
Acknowledgements
This study was part of the project ‘Studies on the Dietary Reference Intakes for Japanese’ (principal investigator, K. S.), which was supported by a Research Grant for Comprehensive Research on Cardiovascular and Life-Style Related Diseases from the Ministry of Health, Labour and Welfare of Japan. The authors' responsibilities are as follows: A. M. designed the study, performed the experiments and prepared the manuscript; M. S. and T. F. helped in the study design, performed the experiments and assisted with the data analysis; K. S. contributed to the study design and supervised the study. The authors declare that they have no conflict of interest.
