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Effect of Strain, Sex and Dietary Riboflavin on Pyridoxamine

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Effect of Strain, Sex and Dietary Riboflavin on Pyridoxamine 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 300 3001 SPRAGUE-DAWLEY BUFFALO 250 250 -, 2OO- _ 200- Downloaded from https://academic.oup.com/jn/article/110/10/1940/4770942 by guest on 28 September 2021 J-I—I IDO o 100 m so 50 ~0 5 ¡0 ¡520 25 30 35 4O~ 0 5 10 15 20 25 30 35 4O Days Fed Diet Days Fed Diet 35030O250„ ii/•/yi^/ -EVANS/*"I//j/ 2009Õr 20O£Î I"^"^/ r L"I— 1--^}* ^/ 'so*fco '50St «^/ IY I^jrr300250_i— •-"V/,— I^1/¿zv' 100CDSOLONG 100co50WISTAR 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).
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