Enzymatic Conversion of Pantothenylalcohol to Pantothenic Acid1

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1 THE JOURNAL OF VITAMINOLOGY 15, (1969) Enzymatic Conversion of Pantothenylalcohol to Pantothenic Acid1 YASUSHI ABIKO, MUNEHIRO TOMIKAWA, AND MASAO SHIMIZU2 Central Research Laboratory, Daiichi Seiyaku Co., Ltd., Edogawa-ku, Tokyo (Post No. 132) (Received May 31, 1968) Enzymatic oxidation of pantothenylalcohol to Pantothenic acid by rat liver extract was demonstrated. The enzyme responsible to the first step of the oxidative reactions was highly purified from rat liver, and identity of the enzyme with liver alcohol dehydro genase (alcohol: NAD oxidoreductase, EC ) was evidenced. It has been reported that pantothenylalcohol, an alcohol analog corresponding to pantothenic acid, had a pantothenic acid-like activity in mammalians (1-8), although it was a growth inhibitor of pantothenic acid-requiring bacteria (9-11). Pantothenic acid-like activity of pantothenylalcohol was explained to be the result of oxidation of the alcohol to pantothenic acid in vivo, and this presumption was confirmed by many investigators (12-21) who showed that pantothenylalcohol given orally or parenterally to rats was largely excreted as pantothenic acid in the urine. There has been no direct evidence, however, for the enzymatic conversion of the alcohol to pantothenic acid in vitro. In the present report, the oxidative con version of pantothenylalcohol to pantothenic acid by rat-liver extract is demon streted, and the purification of the enzyme which catalyzes the oxidation of panto thenylalcohol is reported with some evidence for identity of the enzyme with liver alcochol dehydrogenase (alcohol: NAD oxidoreductase, EC ). MATERIALS AND METHODS 1. Urinary Excretion of Pantothenic Acid Six male rats (Wister-Imamichi strain) were devided into 3 groups. Each rat was kept in an individual metabolic cage on CLEA-CA 1 diet (Central Laboratories for Experimental Animals, Tokyo) and water. The fist group was injected intra peritoneally with 20 ƒêmoles (4.1mg) of pantothenylalcohol per rat in saline solution. The second group was injected with 10ƒÊmoles of calcium pantothenate (4.76mg, corresponding to 20 ƒêmoles of pantothenic acid) per rat. The third group was 1 Investigations on Pantothenic Acid and Its Rel ated Compounds. XVI. 59

2 60 ABIKO, TOMIKAWA AND SHIMIZU 1969 injected with saline alone to serve as the control. Each 24 hour-urine was col lected, filtered through filter paper and assayed for pantothenic a cid. 2. Rat-Liver Extract Fresh livers from male rats were homogenized with 3 volumes of cold 0.02M phosphate buffer (ph 7.2). The homogenate was centrifuged at 16,000rpm for 30 minutes and the resultant supernatant was dialyzed against 200 vol umes of 0.02M phosphate buffer (ph 7.2) overnight. 3. Rat-Liver Acetone Powder Acetone-dried powder of rat liver was prepared according to the method of Hill and Smith (22) involving ethanol-chloroform treatment of the li ver homogenate. 4. Assay of Pantothenic Acid Pantothenic acid was determined microbiologically with Lactobacillus ar -5 as described previously (23) abinosus 17. Microbioassay of pantothenic acid was not affected by pantothenylalcohol when the molar ratio of the alcohol to the acid i n the assay samples was less than 100, although the growth of L. arabinosus 17-5 was inhibited in the presence of large excess amounts of pantothenylalcohol as r eported by Snell and Shive (9) (Fig. 1). FIG. 1 Effect of Pantothenylalcohof on the hue r obioassav of P antothenic A cid by Lactobaciltus arabinosus Determination of O2-Uptake 02-Uptake was measured by the Warburg manometric technique. Warburg fl asks contained 0.5ml of the rat-liver extract, 0.1ml of 1 ~10-3M NAD, 0.4ml of 0.01% methylene blue and 0.5ml of 0.1M glycine buffer (ph 9.6) in the main chamber, 0.3ml of 0.01M pantothenylalcohol in the side chamber and 0.2ml of 20% KO H in the center well. The reaction was initiated by introducing the substrate into the main chamber. Gas phase was air 110 cycles per minute at 37.. The flasks were shaken at 6. Assay of Dehydrogenase Activity "P antothenylalcohol dehydrogenase" activity was assayed at 25, according to

3 Vol. 15 CONVERSION OF PANTOTHENYLALCOHOL 61 Bonnichsen and Brink (24), by the increase in the absorbancy at 340mƒÊ caused by the reduction of NAD which was accompanied by the oxidation of the substrate. To a spectrophotometric cell (1cm light path) were added 3.0ml of 0.1M glycine buffer (ph 9.6) containing 0.075% semicarbazide hydrochloride, 0.1ml of 1.5x10-2M NAD, 0.2ml of 2M pantothenylalcohol and 0.2ml of the enzyme solution. One unit of the enzyme was defined as the amount of the enzyme which catalyzed reduction of 1 ƒêmole of NAD for initial 1 minute at Determination of Protein Protein was determined by the method of Lowry et al. (25).8. Substrates and Reagents D-Pantothenylalcohol was prepared in our laboratory (26). NAD was obtained from Daiichi Pure Chemicals Co., Ltd., Tokyo. DEAF-Cellulose (Serva, 0.6meq/g) and CM-cellulose (Serva, 0.75meq/g) were obtained from Seikagaku Kogyo Co., Ltd., Tokyo. Crystalline horse-liver alcohol dehydrogenase was a preparation of Boehringer and Soehne GmbH, Germany. RESULTS 1. In Vivo Conversion of Pantothenylalcohol to Pantothenic Acid Twenty micromoles of pantothenylalcohol was administered to rats by intra peritoneal injection and each 24 hour-urine was collected for assay of pantothenic acid. As shown in Table 1, about 40% of pantothenylalcohol given to rat was excreted as pantothenic acid in the 24 hour-urine; this was comparable to the urinary excretion of pantothenic acid from the rats which were injected with 20 ƒêmoles of pantothenic acid, indicating rapid oxidation of pantothenylalcohol to pantothenic acid in vivo. TABLE 1 Urinary Excretion of Pantothenic Acid after Intraperitoneal Injection of Pantothenylalcohol and Pantothenic Acid to Rats Twenty micromoles of pantothenylalcohol or 10 ƒêmoles of calcium pantothenate (= 20 ƒêmoles of pantothenic acid) in saline solution was injected intraperitoneally to rats. Each 24-hour urine was collected and assayed for pantothenic acid. Basal ex cretion indicates the pantothenic acid excretion of rat which was injected with saline alone. Figures represent average values from two rats. 2. In Vitro Conversion of Pantothenylalcohol to Pantothenic Acid Pantothenylalcohol was incubated with the rat-liver extract in the presence of NAD and methylene blue at ph 9.6 in the Warburg flasks. At the time intervals of 20 minutes, immediately after recording of O2-uptake, the content of each flask was heated in a boiling water bath for 1 minute to stop the reaction. An aliquot of the reaction mixture was assayed for pantothenic acid after proper dilution. Fig. 2 shows the time course of pantothenic acid formation from pantothenylalcohol

4 62 ABIKO, TOMIKAWA AND SHIMIZU 1969 FIG. 2 Formation of Pantothenic Acid from Pantothenylalcochol and O2-Uptake by Rat-Liver Extract The reaction mixture contained 3 ƒêmoles of pan tothenylalcohol, 0.1 ƒêmole of NAD, 40ƒÊg of methyl ene blue, 50 ƒêmoles of glycine and 0.5ml of the rat liver extract in a total volume of 1.8ml at ph 9.6. The reaction was performed at 37 in the Warburg flasks, which contained 0.2ml of 20% KOH in center wells., œ: complete system ;, : without NAD; œ, : pantothenic acid formation;, : O2- uptake. and NAD-requirement of this re action. No pantothenic acid was detected when methylene blue was omitted from the reaction system. After 1 hour incubation, about 20% (0.64 ƒêmole) of added panto thenylalcohol was converted to pan tothenic acid in this system. Optimal ph of this reaction was 9.5 to 10.0 in glycine buffer. alcohol 3. Purification of "Pantothenyl Dehydrogenase" The enzyme which participates in the first step of the oxidative conversion of pantothenylalcohol to pantothenic acid was purified from the rat-liver acetone powder. This enzyme was tentatively named "pantothenylalcohol d ehydrogen ase". The following operations were performed in a cold room ex cept for the heat-treatment. Step 1. Extraction and am monium sulfate fractionation - Twenty-seven grams of the rat-liver acetone powder was homogenized with 10 volumes of 0.02M phos phate buffer (ph 7.2), stirred for about 1 hour and centrifuged at 14,000rpm for 30 minutes. The crude extract of the acetone pow der was subjected to ammonium sulfate fractionation at ph 7 to 7.2 being adjusted with ammonium hydroxide. The crude extract was brought to 30% saturation with solid ammonium sulfate and the precipitate was centrifuged off. The resultant supernatant was brought to 55% saturation by adding more solid ammonium sulfate and the precipi tate was collected by centrifugation, dissolved in 25ml of 0.02M phosphate buffer (ph 7.2) and dialyzed against the same buffer. The dialyzed solution was centri fuged at 14,000rpm for 30 minutes to remove precipitate which occurred during dialysis. Step 2. Heat-treatment The supernatant solution of the step 1 w as b rought to ph 6.2 with 1N HCl, heated at 58 for 15 minutes under stirring and chilled quickly in an ice-water bath. Heat-denatured protein was centrifuged off and the supernatant solution was brought back to ph 7 with 1N NaOH. Step 3. Acetone fractionation Dry-ice cold acetone was slowly added to

5 Vol. 15 CONVERSION OF PANTOTHENYLALCOHOL 63 the heat-treated enzyme solution of the step 2 to make the concentration 32%. Inactive precipitate was re moved by centrifugation at -15. The resultant supernatant was brought to 55% acetone and the precipitate was collected by centrifugation, dissolved in 6ml of 0.02M phosphate buffer (ph 7.0) and dialyzed against the same buffer. Step 4. DEAR-cellulose and CM-cellulose chromatographies- The dialyzed solution of the step 3 was diluted 4 times with water and chromatographed on a DEAE-cellu lose column of 1.7cm ~23cm which had been equilibrated with 0.005M phosphate buffer (ph 7.0). Protein was eluted with the same buffer at a flow rate of about 50ml/hr. The elution pattern is shown in Fig. 3. The active fractions were combined and placed on a CM-cellulose column of 1cm ~7cm which had been con ditioned with 0.005M phosphate buffer (ph 7.0). The column was first washed with 50ml of the same buffer FIG. 3 DEAR-Cellulose Column Chromato graphy of Rat-Liver Pantothenylalcohol De hydrogenase Column: DEAE-cellulose (0.6meq/g), 1.7 ~23 cm; load: 209mg protein obtained from the acetone-fractionation; buffer for condition and elution: 0.005M phosphate buffer, ph 7.0; flow rate: 50-60m1/hr; fraction size: 2.2ml. Protein in the eluate was measured by Lowry's method (22)., protein, œ, activity. FIG. 4 CM-Cellulose Column Chromatography of Rat-Liver Pantothenylalcohol Dehydrogenase Column: CM-cellulose 10.75meq/g), 1 ~7cm; load: 23mg protein obtained from the DEAE-cellulose chromatography; buffer for condition: 0.005M phosphate buffer, ph 7.0; buffer for elution: 0.005M phosphate buffer containing gradient NaCl (0-0.3 M), ph 7.0; flow rate: 50-60ml/hr; fraction size: 2.3ml., protein, œ, activity.

6 64 ABIKO, TOMIKAWA AND SHIMIZU 1969 and the enzyme was eluted with the phosphate buffer employing NaCl gradient The elution pattern is shown in Fig. 4. The summary of the purification of pantothenylalcohol dehydrogenase is shown in Table 2. This procedure gave an about 330-fold purification with 27.8% recovery of the activity of the original extract. The recovery of the activity exceeded 100 % at the steps of ammonium sulfate fractionation and heat-treatment. This was probably due to removal of some members of NAD-regenerating systems contained in the crude extract. TABLE 2 Summary of Purification of Pantothenylalcohol Dehydrogenase from Rat Liver. 4. Identity of "Pantothenylalcohol Dehydrogenase" with Alcohol Dehydro genase (EC ) Since liver alcohol dehydrogenase has a wide range of substrate specificity, it seems reasonable to think that the enzyme may also catalyze oxidation of panto thenylalcohol. In the course of the purification of "pantothenylalcohol dehydrogenase ", the activity was assayed for ethanol as well as pantothenylalcohol at each step of puri fication. As shown in Table 3, the ratio of the specific activity for pantothenylalcohol to that for ethanol was constant in all steps of purification after heat-treatment Moreover, the ratio remained constant also in each fraction eluted from the DEAE. - cellulose and the CM-cellulose columns (Table 4). The mixed substrate method (27) was applied to the enzyme preparation to examine whether both activities were associated with the same enzyme. The result indicated that the enzyme could catalyze the two reactions (Fig. 5). These activities were not affected by monoiodoacetic acid (1 ~10-2M), and were inactivated almost TABLE 3 Ratio of Specific Activity for Pantothenylalcohol to That for Ethanol in the Purification Steps PaOH, pantothenylalcohol; EtOH, ethanol.

7 Vol. 15 CONVERSION OF PANTOTHENYLALCOHOL 65 TABLE 4 Ratio of Specific Activity for Pantothenylalcohol to That far Ethanol in Each Eluate FIG. 5 Mixed Substrate Analysis of Rat - Liver Pantothenylalcohol Dehydrogenase 0.2ml (0.024 unit) of the purified enzyme was added to the mixture of 3ml of 0.1M glycine buffer (ph 9.6) containing 0.75% semicarbazide hydrochloride, 0.1ml of 1.5 ~10-2M NAD, and 0.2ml of 1M pantothenylalcohol ( œ) or 0.2 ml of 1M ethanol ( ) or each 0.1ml of 2M pantothenylalcohol and 2M ethanol ( ). In crease in extinction at 340mƒÊ was recorded at 25. FIG. 6 Oxidation of Pantothenylalcohol and Ethanol by Crystalline Horse-Liver Alcohol Dehydrogenase Experimental conditions were similar to those of Fig. 5, with exception that crystalline horse liver alcohol dehydrogenase (0.028 unit) was used in place of the rat-liver enzyme. œ, pan tothenylalcohol,, ethanol;, the mixture of pantothenylalcohol and ethanol.

8 66 ABIKO, TOMIKAWA AND SHIMIZU 1969 TABLE 5 Distribution of Alcohol Dehydrogenase Activity in Ammonium Sulfate Fractionation of Rat-Liver Extract TABLE 6 Effects of Monoiodoacetic Acid and Heat on Rat -Liver Alcohol Dehydro genase Activities a Final concentration of CH2ICOOH was 1 ~10-2M (ph 9.6).b 58 f or 15 minutes at ph 6.2. to the same extent by heat treatment at 60 for 5 minutes (13.1% inactiva tion in the activity for ethanol and 13.9% for pantothenylalcohol). These results strongly suggested the identity of "pantothenylalcohol dehydrogen ase" with liver alcohol dehydrogen ase (EC ). This was also sup ported by the fact that crystalline horse-liver alcohol dehydrogenase could catalyze oxidation of pantothenylal cohol as well as ethanol, although the rate of reduction of NAD with panto thenylalcohol as a substrate was lower than with ethanol (Fig. 6). However, the ratios of the specific activities for the two substrates at the steps of extraction and ammonium sulfate fractionation were different from those at other steps (Table 3) the finding inconsistent with the identity of the two activities. This contradiction could be explained by the fact that there were two different alcohol dehydrogenase activities in rat liver. FIG. 7 Mixed Substrate Analysis of the Rat-Liver Fraction Precipitating at 30% Saturation with Ammonium Sulfate Experimental conditions were similar to those of Fig, 5, with exception that the enzyme fraction which precipitated at 30% saturation with am monium sulfate (approx. 5mg protein) was used (see text). œ, pantothenylalcohol,, ethanol,, the mixture of pantothenylalcohol and ethanol.

$fraction which precipitated at 30% saturation with ammonium sulfate. This fraction contained a very small amount of "pantothenylalcohol dehydrogenase" activity (Table 5).$

9 Vol. 15 CONVERSION OF PANTOTHENYLALCOHOL 67 In the course of the purification of "pantothenylalcohol dehydrogenase ", "ethyl alcohol dehydrogenase" activity was found to be present also in the fraction which precipitated at 30% saturation with ammonium sulfate. This fraction contained a very small amount of "pantothenylalcohol dehydrogenase" activity (Table 5). The dehydrogenase activities in this fraction was completely lost by heating at 58 for 15 minutes, whereas the specific activities for both substrates in the second fraction which precipitated at between 30 and 55% saturation with ammonium sulfate in creased on the heat-treatment. About 70% of the activities in the first fraction was inhibited by monoiodoacetate (1 ~10-2M), while in the second fraction 67% of the activity for ethanol and 80% for pantothenylalcohol were resistant to the reagent (Table 6), and after the heat-treatment the activities in the second fraction were not affected by the reagent. The mixed substrate analysis of the first fraction indicated the existence of different dehydrogenase activities (Fig. 7). These results indicated the existence of another alcohol dehydrogenase in rat liver, which was heat-labile, sen sitive to monoiodoacetate and had no or very low susceptibility to pantothenylalcohol. DISCUSSION It has been reported that pantothenylalcohol had the same effects as pantothenic acid on mammalia (1-8), although it acted as a growth inhibitor of pantothenic acid-requiring bacteria (9-11). Many investigators (12-21) suggested the oxidative conversion of the alcohol to pantothenic acid in vivo, based on the finding that pantothenylalcohol given to animals was excreted as pantothenic acid in the urine, which was confirmed by us in this report. The results of our in vitro experiments reported here gave direct evidence for the enzymatic conversion of pantothenylalcohol to pantothenic acid. Enzymatic oxidation of the alcohol by the dialyzed rat-liver extract was NAD-dependent and accompanied by O2-consumption in the presence of methylene blue (Fig. 2). Liver alcohol dehdrogenase has been demonstrated to possess a very broad specificity for its substrate: aliphatic primary and secondary alcohols (28, 29), vitamin A (30), ethyleneglycol (31), benzylalcohol (29), p-nitrobenzylalcohol (32) and cyclic secondary alcohols (29, 33). Therefore, the purification of "pantothenylalcohol de hydrogenase" was first attempted according to the method for the purification of horse-liver alcohol dehydrogenase reported by Bonnichsen and Brink (24). But the rat enzyme lost its activity during processing, indicating that the method was not suitable to rat liver. Merrit and Tomkins (33) reported the purification of alcohol dehydrogenase from acetone powder of rat liver. They described that their pro cedure resulted in a 10-fold purification over the acetone powder extract, that further purification could not been achieved in spite of various attempts, and that the enzyme was not stable against dialysis. In the present report, acetone powder was prepared from rat liver as the starting material for the purification of the enzyme according to the method of Hill and Smith (22), which had been employed for the purification of ƒ -aminoacylpeptide hydrolase (EC ) from hog kidney. The enzyme extracted from this acetone powder was stable against dialysis and even against heat-treatment, and thus permitted its higher purification as described in the text.

10 68 ABIKO, TOMIKAWA AND SHIMIZU 1969 Identity of "pantothenylalcohol dehydrogenase" with liver alcohol dehydrogenase was evidenced by the results from the mixed substrate analysis, the experiments of monoiodoacetate-inhibition and heat-inactivation and by the findings that the ratio of the specific activities for the two substrates, pantothenylalcohol and ethanol, remained constant throughout the purification procedures after heat-treatment (Tables 3 and 4). Discrepancy in the ratios of the specific activities before and after heat treatment seems to be explained by the existence of "another alcohol dehydrogen ase" in rat liver, which was heat-labile, sensitive to monoiodoacetate and had no or very low susceptibility to pantothenylalcohol and which was eliminated partially by ammonium sulfate fractionation and completely by heat-treatment. This enzyme may correspond to the "second alcohol dehydrogenase" reported by Treble (34), which was partially purified from horse liver. Further investigations on this new alcohol dehydrogenase will be reported elsewhere in respects of alcoholdehydro genase isozymes in rat liver. In contrast with the liver enzyme, yeast alcohol dehydrogenase could not catalyze oxidation of pantothenylalcohol3. The crude extract from Lactobacillus arabinosus 17-5 was also inactive for oxidation of pantothenylalcohol although it was active for ethanol3. This may be related to the inhibitory action of the alcohol on pantothenic acid-requiring bacteria, such as L, arabinosus ACKNOWLEDGEMENT The authors wish to express their deep gratitude to Dr. T. Ukita, Professor of the University of Tokyo, for his kind and helpful discussion, and to Dr. T. Ishiguro, President of this Com pany, for his encouragement. REFERENCES 1. Pfaltz, H., Z. Vitanninforsch., 13, 236 (1943). 2. Jurgens, R., and Pfaltz, H., Z. Vitaminforsch,, 14, 243 (1944). 3. Weiss, M., De Ritter, E., Rubin, S. H., and Randall, L. D., Proc. Soc. Exptl, Biol. Med., 73, 292 (1950). 4. Ludovici, P. D., and Axelrod, A. F., Proc. Soc. Exptl. Biol. Med., 77, 530 (1951). 5. Lih, H., King, T. E., Higgins, H., Baumann, C. A., and Strong, F. M., J. Nutrition, 44, 361 (1951). 6. Hegsted, D.M., Proc. Soc. Expel. Biol. Med., 69, 571 (1948). 7. Hirabayashi, M., Vitamins, 30, 114 (1964). 8. Inagawa, Y., Vitamins, 30, 245 (1964). 9. Snell, E. F., and Shive, W., J. Biol. Chem., 158, 551 (1945). 10. Drell, W., and Dunn, M. S., Arch, Biochem. Biophys., 51, 391 (1954). 11. Riekels, K., Munch, rued. Wochschr., 96, 1510 (1954). 12. Burlet, E., Z. Vitaminforsch., 14, 318 (1944). 13. Schmidt, V., Acta Pharmacol. Toxicol., 1, 120 (1945). 14. Schmidt, V., Acta Pharmacol. Toxicol., 3, 13 (1947). 15. Drekter, L., Drekter, R., Pankopf, R., Schneider, J., and Rubin, S. H., J. Am. Pharm. Asoc., 37, 498 (1948). 16. Rubin, S. H., Coopermann, J. M., Moore, M. F., and Schneider, J., J. Nutrition, 35, 499 (1948). 3 Data are not presented in this report.

11 Vol. 15 CONVERSION OF PANTOTHENYLALCOHOL Rubin, S. H., Drekter, L., Moore, M. E., and Pankopf, R., J. Nutrition, 40, 265 (1950). 18. Braekkan, O. R., Acta Pharmacol. Toxicol., 11, 215 (1955). 19. Rubin, S. H., Proc. Amer. Drug Manufrs. Assoc., (1948) Crokaert, R., Ann. soc, roy, sci. med. et nat., 6, 157 (1953); Chem. Abstr., 48, 7145 (1953). 21. Yamawaki, Y., and Ishida, T., Vitamins, 23, 169 (1961). 22. Hill, R.L., and Smith, E. L., J. Biol. Chem., 228, 577 (1957). 23. Abiko, Y., J. Biochem., 61, 290 (1967). 24. Bonnichsen, R. K., and Brink, N. G., Methods in Enzymology, 1, 495 (1955). 25. Lowry, O. H., Resebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., 193, 265 (1951). 26. Hosokawa, Y., Tomikawa, M., Nagase, O., and Shimizu, M., Chem. Pharm. Bull. (Tokyo), 17, 202 (1969). 27. Dixon, M., and Webb, E. C., Enzymes, 2nd. edition, Longmans, Green and Co., Ltd., London, p. 84 (1964). 28. Theorell, H., and Bonnichsen, R., Acta Chem. Scand., 5, 1105 (1951). 29. Winer, A. D., Acta Chem. Scand., 12, 1695 (1958). 30. Bliss, A. F., Arch. Biochem. Biophys., 31, 197 (1951). 31. Holzer, H., Goedde, H. W., and Schneider, S., Biochcmn, Z., 327, 245 (1955). 32. Gillette, J. R., J. Biol. Chem., 234, 139 (1959). 33. Merrit, A. D., and Tomkins, G. M., J. Biol., Chemn., 234, 2778 (1959). 34. Treble, D. H., Biochem. J., 82, 129 (1962).

Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro

[Agr. Biol. Chem., Vol. 34, No. 5, p. 710-714, 1970] Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro By Katuhiko NODA Department of Nutrition, School of Medicine, Tokushima