Effect of Strain, Sex and Dietary Riboflavin on Pyridoxamine (Pyridoxine) 5 -Phosphate Oxidase Activity in Rat Tissues1 Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 KATHLEEN M. RASMUSSEN,2 PATRICE M. BARSA, DONALD B. MCCORMICK3 AND DAPHNE A. ROE Division of Nutritional Sciences, Cornell University, Savage Hall, Ithaca, NY 14853

ABSTRACT Weanling male rats of four strains—Buffalo, Long-Evans, Sprague-Dawley (SD) and Wistar—were fed a control or a riboflavin-deficient purified diet for 6 weeks. Both strain and diet had significant effects on pyri- doxamine (pyridoxine) 5'-phosphate (PMP) oxidase activity in liver and kidney. No single strain had extreme PMP oxidase values or was consistently more re sponsive than the others to riboflavin deficiency. Weanling male and female SD rats were fed purified diets containing 2.5, 1.0, 0.5 or 0 ing of riboflavin/kg diet for 9 weeks. PMP oxidase values generally were lower in the females than in the males. In both sexes, increases in dietary riboflavin level were reflected in increases in PMP oxidase activity in liver and kidney. These results confirm that PMP oxidase activity is a sensitive indicator of riboflavin status in the rat. J. Nutr. 110: 1940-1946, 1980. INDEXING KEY WORDS riboflavin •pyridoxamine-5-phosphate oxidase

Recent results from our laboratory indi ings allow the possibility of genetic dif cate that the activity of pyridoxamine ferences in PMP oxidase activity and raise (pyridoxine) 5'-phosphate oxidase (EC the practical issue of selection of the ap 1.4.3.5) has potential as an index of ribo propriate rat strain for use in future flavin status (1). This flavin mononucleo- investigations. tide-dependent enzyme catalyzes the con Other investigators have shown that version of pyridoxamine 5'-phosphate PMP oxidase activity in rat kidneys was (PMP) and pyridoxine 5'-phosphate, two responsive to castration and to testosterone of the phosphorylated vitaminic forms of administration (3, 4). Furthermore, PMP B-6, to the active coenzyme, pyridoxal oxidase activity in weanling male rats was 5'-phosphate (PLP). In rat liver, PMP consistently higher than in weanling or oxidase activity was sensitive to riboflavin adult female rats (1, 5). These findings depletion and was a more specific index suggest that sex differences in PMP oxi of riboflavin status than the erythrocyte dase activity may be important. glutathione reducíase assay commonly This report describes an experiment employed for assessment of riboflavin undertaken to establish if there are strain nutriture (1). differences in PMP oxidase activity or in Values for PMP oxidase activity in the livers of rats fed control diets have varied several-fold in literature reports (1, 2). Received tor publication 25 January 1980. 1This investigation was supported in part by USDA/SEA Agree These experiments differed in two im ment 5901-0410-8-0102-0 and XIH Research Grant AM-045S5, portant respects—the manner in which USPHS. 1 Postdoctoral lellow supported by NIH Nutrition Training Grant the control diet was fed (ad libitum or 5-T32-AM-07158, USPHS. To whom reprint requests should be sent. restricted) and the strain of rat studied s Present address: Department oí Biochemistry, Emory Uni (Sprague-Dawley or Buffalo). These find versity School of Medicine, Atlanta, GA 30322.

1940 PMP OXIDASE IN RAT TISSUES 1941

TABLE 1 control (diet 3) or riboflavin-deficient Amount of riboflatin added to the liutai clict'~ diet (diet 0, table 1). After 3 weeks and again after 6 weeks, Ribo flavin three rats from each dietary group of each Diet added strain were killed by decapitation without anesthesia. Liver, kidney and brain were diet removed rapidly, blotted, weighed, quick- Diet 3 (control) 2.5 frozen in liquid nitrogen and stored over 1.0 Diet 2 night at —20°.Tissues were homogenized Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 Diet 1 0.5 Diet 0 (deficient) 0 and cellular debris precipitated as de scribed previously (1). The protein con 1AIX-76™ formulation [J. Xutr. 107, 1340 (1977)] centration of the supernatants was de prepared without riboflavin in the vitamin mix. 2Prepared by ICN Pharmaceuticals Inc., Cleve termined by the biuret method (6) using land, OH. bovine serum albumin as the standard. PMP oxidase holoenzyme activity was measured using a modification of the the response of this enzyme to riboflavin method of Wada (7). Under subdued light, deficiency. Another experiment was de a 3.5-ml reaction mixture containing 0.17 M signed to explore further the sensitivity Tris-HCl buffer, pH 8.0,0.08 Mpotassium of PMP oxidase activity to riboflavin phosphate buffer, pH 8.0, 2.9 x 10~4M status. This investigation was carried out PMP and 0.4 ml of the tissue sample in both male and female rats to establish was incubated with shaking for 30 min if there are sex differences in PMP oxi utes at 37°.The reaction was stopped by dase activity or in the response of this the addition of 0.2 ml of 100% trichloro- enzyme to the level of dietary riboflavin acetic acid. A 4-nitrophenylhydrazine provided. color reagent was used to quantitate the PLP formed in this reaction mixture (1). MATERIALS AND METHODS Experiment 2. Effect of sex and amount Experiment 1. Effect of strain and ribo of dietary riboflavin on PMP oxidase flavin deficiency on PMP oxidase in rat activity in rat tissues. Male and female tissues. Male weanling ratsof four strains— weanling SD rats were obtained from the Buffalo (B), Long-Evans (LE), Sprague- same commercial supplier. Housing and Dawley (SD) and Wistar (W)—were ob adaptation procedures were the same as tained from commercial suppliers (B, those described for experiment 1. LE and W, Simonsen Laboratories, Gil- At the start of this experiment, the roy, CA; SD, ARS/Sprague-Dawley, Mad females were 28 days old and the males ison, WI). Animals were housed individu were 29 days old. Five rats of each sex ally in stainless steel cages with wire- were anesthetized with diethyl ether and mesh bottoms in a room maintained at bled from the heart until dead. The re constant temperature and humidity with a maining animals of each sex were as 12-hour light-dark cycle. Experimental signed randomly to one of five dietary diets and tap water were available ad treatments: diets 0, 1, 2 or 3 (table 1) libitum. Animals were fed the control diet fed ad libitum or diet 3 fed in amounts (diet 3, table 1) during a 5-day adaptation restricted to those consumed by the rats period before the beginning of the ex fed diet 0 (pair-fed controls). The fe periment. males weighed 62-64 g; the males weighed At the start of the experiment, B, LE 55-56 g. and SD rats were 29 days old and W rats After 3, 6 and 9 weeks, three rats from were 32 days old. Initial mean body each dietary group of each sex were weights were 59, 70, 67 and 69 g for the anesthetized with diethyl ether and bled B, LE, SD and W rats, respectively. from the heart until dead. The tissues Three animals from each strain were taken and their treatment were the same killed; the remaining rats in each strain as for experiment 1. were assigned randomly to either the Statistical analyses. The effects of 1942 RASMUSSEX, BARSA, McCORMICK AND ROE

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10 15 20 25 30 35 40 Days Fed Diet Days Fed Diet Fig. 1 Growth of four strains of weanling male rats fed control (open circles) or riboHavin-deficient (open triangles) diets. Mean ±SE is illustrated. Significant differences in body weight between dietary groups began at the time indicated by the asterisks (*P < 0.05, ***P < 0.001).

strain and diet in experiment 1 and sex experiment than rats of the other strains. and diet in experiment 2 were evaluated The B rats, however, reached a final mean by analysis of variance followed by com weight close to that of the LE and SD parison of specific means by Student's rats; the W rats were significantly heavier f-test (8); P < 0.05 was accepted as sig than the other three groups at the end of nificant. the experiment (fig. 1). Animals fed the deficient diet weighed significantly less RESULTS than those fed the control diet after 2 (SD rats) or 6 days. The weight gain of Experiment 1. The B rats were sig the B and SD rats fed the deficient diet nificantly lighter at the beginning of the was only about half that of the LE and W PMP OXIDASE IN RAT TISSUES 1943

rats, kidney PMP oxidase activity was sig nificantly lower by week 3 in animals fed 400300200Ô the deficient diet than in those fed the control diet. By week 6, this difference JL_I_lrH1J_l—L1JJ•*•.«1-^» was significant in all four strains. Brain PMP oxidase activity was also similar in all strains at week 0 (data not shown). Values in rats fed the control -Ì1-1,1-• diet increased only slightly during the Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 1000.n.TLIVER_ •"i experiment. Values in rats fed the deficient diet generally were lower than in those 036 036 036 036 fed the control diet, but this difference Buffolo Long-Evons Sprague- Wistor usually was not significant. Dawley Experiment 2. The female rats were Fig. 2 Effect of riboflavin deficiency on liver heavier than the males at the outset of the PMP oxidase activity in four strains of rats. iMean study but, as a result of the males' more ±SE is illustrated. Open bars represent values rapid growth, this difference disappeared from rats fed the control diet; hatched bars repre sent values from rats fed the deficient diet. Sig after 2 weeks in animals fed the control nificant differences between rats fed the two diets diet (diet 3, ad libitum, fig. 4). By the end are indicated by the asterisks (*P < 0.05, **P < 0.01, of the experiment, male controls were ***P < 0.001). Liver PMP oxidase values in the four about 100 g heavier than their female strains differed significantly (P < 0.01) at week 0. counterparts. Female rats fed the de ficient diet were heavier than the cor rats. By the end of the experiment, the responding males during the first 4 weeks difference in body weight between ani of the experiment, but after that time, their mals fed the control and deficient diets body weights were nearly identical. was much less for the LE rats than for In both sexes there were significant the other three strains. differences in body weight between the Two-way analysis of variance revealed dietary groups beginning at 6 days. The that both strain and diet had significant differences between rats fed diet 3 (ad effects on liver and kidney PMP oxidase libitum and pair-fed) and diet 0 were sig activity. In brain samples, however, there nificant in both sexes throughout the re- were no consistent effects of strain and diet on PMP oxidase activity. At the outset of the study, there were significant (P < 0.01) differences between 400o»"5E-1 the strains in liver PMP oxidase activity r\:<1-^-11week i (fig. 2). Values were 1.7-times higher in the LE rats than in the SD rats. In LE 300.?u and SD rats fed the control diet, liver PMP oxidase values increased during <200vl

Diet 200 •FEMALES Diet

Fig. 5 Effect of feeding various levels of dietary riboflavin on PMP oxidase activity in liver (top) and kidney (bottom) of weanling male SD rats. 1 1 1 O IO 20 30 40 50 60 Mean ±SE is illustrated. PMP oxidase values of Days Fed Diet rats fed the various diets are shown by the follow ing symbols: diet 3 (ad libitum), solid bars; diet 3 (pair-fed), bars with diagonal hatching; diet 2, open bars; diet 1, bars with horizontal hatching; and diet 300 . MALES 0, bars with vertical hatching. Significant differ ences between the dietary groups are indicated by the asterisks (*P < 0.05, **P < 0.01). 250

and kidney samples but only at week 9 — 200 o> in brain samples. The effect of sex was significant at week 6 for liver, at weeks 6 f '50 and 9 for kidney and at weeks 3 and 9 for brain. In the male rats at 3, 6 and 9 weeks, o 100 OD there were significant (P < 0.01) differ ences between the dietary groups in liver 50 PMP oxidase activity (fig. 5). At each of these times, there was a clear gradation in liver PMP oxidase activity by level IO 20 30 40 50 60 of dietary riboflavin fed. Values in rats Days Fed Diet fed the deficient diet decreased as the Fig. 4 Growth of female (top) and male (bottom) experiment progressed, whereas values weanling SD rats fed different levels of dietary in rats fed diet 3 ad libitum increased riboflavin. Mean ±SE is illustrated. Body weights to nearly double values obtained at week of rats fed the various diets are shown by the 0. Values in rats pair-fed diet 3 were higher following symbols: diet 3 (ad libitum), closed circles; diet 3 (pair-fed), open circles; diet 2, triangles; diet than in those fed diet 2; values in the pair- 1, squares; and diet 0, diamonds. Dietary groups fed rats increased only slightly during differ significantly by day 6 (*P < 0.05, ***P < 0.001). the study. PMP OXIDASE IN RAT TISSUES 1945

activity by level of riboflavin fed. Values in female rats fed diet 3 ad libitum were maximal at week 3 and then de creased. Values in female rats pair-fed diet 3 were maximal at week 6. Values in rats fed the deficient diet were low at week 3 and remained essentially con stant during the remainder of the study.

There were significant differences be Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 tween the dietary groups in kidney PMP oxidase activity in the female rats at all time periods (fig. 6). The gradation in kidney PMP oxidase activity by level of dietary riboflavin was evident only at weeks 3 and 6 and even then was not as I pronounced as for liver. Kidney values were maximal at week 3 and decreased thereafter in all dietary groups. As was the case for the males, dif ferences between dietary groups in brain £o,. PMP oxidase activity did not reach statis tical significance in the females (data not Fig. 6 Effect of feeding various levels of dietary shown). Values in rats fed the deficient riboflavin on PMP oxidase activity in liver (top) and kidney (bottom) of weanling female SD rats. Mean diet were consistently lower than in those ±SE is illustrated. PMP oxidase values of rats fed fed diet 3 ad libitum. the various diets are shown by the same symbols used in figure 5. Significant differences between DISCUSSION (*Pthe

The amount of riboflavin provided in enzyme is, indeed, a sensitive index the control diet (diet 3) used in experi of riboflavin status in the rat. ments 1 and 2 is the amount recom mended by the National Research Coun LITERATURE CITED cil, 2.5 mg/kg diet (9). This is less than 1. Rasmussen, K. M., Barsa, P. M. & McCormick, the amount usually found in the AIN- D. B. (1979) Pyridoxamine (pyridoxine) 5'- 76™diet, 6.0 mg/kg diet (10), and much phosphate oxidase activity in rat tissues during development of riboflavin or pyridoxine de less than the amount (22 mg/kg diet) in ficiency. Proc. Soc. Exp. Biol. Vied. ¡61,527- the commercially formulated diet used in 530. Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 our previous experiment (1). 2. Tryfiates, G. P. & Saus, F. L. (1976) Metab In experiment 1, rats fed diet 3 met or olism of pyridoxine in the liver of vitamin B-6- deficient rats. Biochim. Biophys. Acta 451, exceeded growth expectations (9, 10). In 331-341. experiment 2, however, both males and 3. Chatterjee, A. K. & Datta, S. C. (1972) Some females fed diet 3 ad libitum weighed studies on the formation in vivo of pyridoxal less than might be expected (9, 10), sug phosphate in rats receiving a high fat diet. J. gesting that for this longer study the Vitaminol. 18, 189-193. 4. Chatterjee, A. K. (1972) Studies on the in amount of riboflavin provided was only volvement of testicular hormone in the regula marginally adequate. tion of pyridoxal phosphate formation by kid In experiment 2, initial PMP oxidase ney tissue. Endocrinology 90, 880-884. values were the same in both males and 5. Nakahara, I., Morino, Y., Morisue, T. & Saka moto, Y. (1961) Enzymatic studies on pyri females. Significant differences between doxine metabolism. VI. Pyridoxine metabolism the sexes appeared at later times in the in pregnancy. J. Biochem. 49, 339-342. three tissues examined. Values for the 6. Layne, E. (1972) Spectrophotometric and tur- males generally were higher than those bidimetric methods for measuring proteins. for the females. These results are in ac Methods Enzymol. 3, 447-451. cord with Chatterjee's report (4) that the 7. Wada, H. (1970) Pyridoxine phosphate oxi dase. Methods Enzymol. ISA, 626-630. administration of testosterone to normal 8. Snedecor, G. W. & Cochran, W. G. (1967) or castrated male rats increased PMP Statistical Methods, 6th ed., Iowa State Uni oxidase activity. versity Press, Ames, IA. 9. National Research Council (1972) Nutrient In both liver and kidney samples from Requirements of Laboratory Animals No. 10, both sexes, PMP oxidase activity values 2nd rev. ed., National Academy of Sciences, responded to the amount of dietary ribo Washington, DC. flavin provided. This finding confirms our 10. Anonymous (1977) Report of the American Institute of Nutrition Ad Hoc Committee on previous results (1) and provides addi Standards for Nutritional Studies. J. Nutr. 107, tional evidence that the activity of this 1340-1348.