Influence Dietary Riboflavin Deficiency on Lenticular Glutathione Redox Cycle, Lipid Peroxidation, and Free Radical Scavengers in the Rat

Abstract

(first set of animals) or 0.05 M sodium phosphate buffer, pH 7.2 (second set of animals) using a Potter/Elvehj am glass homogenizer. A 7.5% homogenate was prepared from two lenses (third set of animals) in 5% ice-cold TCA. Aliquots of J. Clin. Biochem. Nutr. INFLUENCE OF RIBOFLAVINDEFICIENCY ON RAT LENS 197 the lens homogenate (Tris-EFTA buffer) were analysed for GSH and GSSG measured fluorimetrically [11] in a Hitachi 650-lOS spectrofluorimeter and for total and non-protein sulfhydryl groups with Ellman's reagent [12]. Lipid peroxide level was estimated (in terms of malondialdehyde (MICA)) in the phosphate buffer by the thiobarbituric acid (TBA) reaction [13] and ascorbic acid in 5,000>< g supernatant of 5% TCA homogenate, using orthophosphoric acid, ferric chloride and aa '-dipyridyl [14]. The remaining homogenate (Tris-EFTA buffer) was centrifuged at 20.000>< g in a Sorvall RCSB super-speed centrifuge at 4°C for 30 min. The 20,000>< g supernatant was used for the assay of glutathione redox cycle enzymes. Glucose 6-phosphate dehydrogenase (G6PDH) was assayed spectrophotometrically as described by Selvaraj, and Bhat [15]. The reaction mixture contained in 1.2 ml: 50,u mol glycylglycine buffer (pH 8.7), 12 ,u mol KCl, 12 ,u mol MgC12, 3 ,u mol nicotinamide, 61u mol glucose 6-phosphate, and 75 1a l lens tissue supernatant. The reaction was started by adding 0.12 ,u mol NADP+. GSH-R was assayed by the method of Bayoumi, and osalki [16] as described elsewhere [9]. Glutathioneperoxidase (GSH-PX) was assayed as described by Hochstein and Utley [17] with the following modification: The reaction mixture contained in 1 ml: 50 ,u mol phosphate buffer (pH 7.2), 2.5 ,u mol GSH, 0.5 ,u mol NaN3, 0.3 ,u mol EFTA, 0.1 ,u mol NADPH, 0.5 IU GSH-R, and 75 ,u 1 supernatant (enzyme preparation, 1 : 5 dilution). The reaction mixture was incubated at 37°C for 10 min and the reaction started by the addition of 0.251a mol tert-butyl hydroperoxide. Catalase in the 750 X g supernatant fraction of the homogenate prepared in phosphate buffer was assayed polarographically as described by Goldstein [18] with a polarograph (YSI, Model 5301) equipped with an oxygen monitor (model 53) and a recorder (LKB 2210). The activity of superoxide dismutase (SOIL) in supernatant fractions of the lens homogenate (phosphate buffer) obtained by centrifugation at 35,000>< g and 0-4°C for 25 min was determined spectrophotometrically by measuring the inhibition of autooxidation of pyrogallol [19]. Protein concentration was measured by the method of Lowry et al. [20]. Statistical analysis. The data was analysed by one way analysis of variance. The mean differences were also further tested by Duncan's multiple range test. RESULTSThis study spans three different animal experiments in every one of which riboflavin-deficient rats showed a severe growth retardation, hair loss, and skin lesions, all of which are characteristic features of riboflavin deficiency.Ocular examination revealed severe corneal vascularization in riboflavindeficient rats (30%) as comp...

Riboflavin (vitamin B2) and oxidative stress: a review

“…Bate et al (27) also reported elevated lipid peroxidation in riboflavin deficiency with no change in glutathione levels and GPx activity. Moreover, in some studies, an increased GPx activity was observed simultaneously with increased lipid peroxidation in riboflavin deficiency (22,29,40) . As has been mentioned previously, this increased GPx activity, most probably, is a compensatory response to increased lipid peroxidation occurring in riboflavin deficiency.…”

“…The only human study published has reported no significant difference in erythrocyte glutathione concentrations between normal and riboflavin-deficient subjects ( 26 ) . Furthermore, some animal studies also did not report any change in glutathione content in different tissues (such as the liver, lens and erythrocytes) in riboflavin-deficient animals ( 27 – 29 ) . A mechanism for the unchanged tissue glutathione content in riboflavin deficiency could be the increased biosynthesis of glutathione from its precursor amino acids as a compensatory action ( 30 ) .…”

“…Erythrocyte GPx activity and lipid peroxidation product levels were higher in riboflavin-deficient rats than in the control rats, while erythrocyte SOD and catalase activities were not affected Liang et al (23) Experimental Blood MDA and GSH levels and SOD activity were measured in riboflavin-deficient rats and riboflavin-sufficient control rats Riboflavin-deficient rats had lower SOD activity and GSH levels and higher MDA levels than the control rats Powers & Thurnham (26) Human study Erythrocyte glutathione levels were compared between riboflavin-deficient and normal subjects No significant differences in erythrocyte glutathione levels were observed between riboflavin-deficient and normal subjects Rao & Bhat (29) Experimental Lens MDA levels, ascorbate levels, GPx, catalase and SOD activities, and GSH levels were assessed in riboflavin-deficient rats and riboflavin-sufficient control rats…”

<i>In Vitro</i> Effects of Paclitaxel and Cremophor EL on Human Riboflavin Transporter SLC52A2

“…Alteration in energy metabolism due to mitochondria dysfunction is one of the major causes of paclitaxel-induced peripheral neuropathy. 18) Neurons require large amounts of energy to transport neuronal transmitters in axons. Flavin mononucleotide (FMN) and FAD, which are derived from riboflavin, are used for the electron transport chain to produce ATP, the primary energy carrier in cells.…”

The Importance of Nutrition in the Prevention of Ocular Disease with Special Reference to Cataract

“…The studies on Indians revealed that inadequacies of certain micronutrients, such as riboflavin, zinc and copper, were associated with cataract [13,14] . In undernourished patients, the proportion of insoluble proteins in the lens was found to be significantly higher as compared to well-nourished cataract patients, indicating a decreased solubility of soluble proteins, a common finding in nuclear cataracts [15] .…”