scholarly journals A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status

1981 ◽  
Vol 45 (1) ◽  
pp. 53-65 ◽  
Author(s):  
A. M. Prentice ◽  
C. J. Bates
Keyword(s):  
Glutathione Reductase ◽  
Liver Weight ◽  
Strongly Correlated ◽  
Riboflavin Deficiency ◽  
Deficient Diet ◽  
Serial Measurement ◽  
Weanling Rats ◽  
Biochemical Evaluation ◽  
Activation Coefficient ◽  
Riboflavin Status

1. Chronic marginal riboflavin deficiency was induced in groups of weanling rats by feeding a deficient diet supplemented with 0, 0·5, 1·0 and 1·5 mg riboflavin/kg diet. Ad lib.- and pair-fed controls received 3·0 and 15 mg riboflavin/kg diet respectively.2. Serial measurement of erythrocyte NAD(P)H2 glutathione oxidoreductase (glutathione reductase; EC 1.6.4.2) and its activation coefficient revealed that after 12 weeks a steady-state of deficiency had been reached following initial fluctuations in status; the animals were then killed, and their tissues analysed.3. Food intake, growth rate and the appearance of pathological signs were directly proportional to riboflavin content; however relative liver weight was increased above control levels only in the most-severelydeficient group, and anaemia was not detected in any group.4. The activation coefficient of glutathione reductase in erythrocytes and liver was closely related to dietary riboflavin content; that of skin responded maximally even in the least-severelydepleted animals.5. Hepatic and renal flavin contents were directly proportional to dietary riboflavin, FAD being conserved at the expense of ribotlavin and FMN. ATP: riboflavin 5-phosphotransferase (flavokinase; EC 2.7.1.26) activity was reduced, even in the least-severely deficient animals; ATP: FMN adenylyltransferase (FAD pyrophosphory1ase; EC 2.7.7.2) was increased in liver, but only in the most-severely-deficient animals.6. Hepatic succinate: (acceptor) oxidoreductase (succinate dehydrogenase; EC 1. 3.99.1) activity fell sharply between 1·5 and 0·5 mg riboflavin/kg diet, producing an S-shaped dose-response curve; it showed smaller or less specific changes in other tissues such as brain, skin and intestine. NADH: (acceptor) oxidoreductase (NADH dehydrogenase; EC 1.6.99.3) activity declined in liver and intestine, but not in skin or brain.7. Theactivation coefficient of glutathione reductase was correlated strongly with nearly all the riboflavin-sensitive variables measured, once equilibrium had been reached in this chronic deficiency model, and it was particularly strongly correlated with hepatic and renal FAD levels. Under equilibrium conditions, therefore, it appears to represent a good index of the extent of riboflavin deficiency, and significant changes in flavin levels and enzymes in the internal organs were detected even under conditions of marginal deficiency, associated with relatively small increases in the activation coefficient.

scholarly journals A biochemical evaluation of the erythrocyte glutathione reductase (EC1.6.4.2) test for riboflavin status

1981 ◽  
Vol 45 (1) ◽  
pp. 37-52 ◽  
Author(s):  
A. M. Prentice ◽  
C. J. Bates
Keyword(s):  
Glutathione Reductase ◽  
Dehydrogenase Activity ◽  
Early Stage ◽  
Mean Value ◽  
Riboflavin Deficiency ◽  
Deficient Diet ◽  
Biochemical Evaluation ◽  
Final Extent ◽  
Riboflavin Status ◽  
Deficient Group

1. Acute riboflavin deficiency was produced in weanling rats by feeding a deficient diet and using tailcups to prevent refection. Animals were killed at weekly interval for 7 weeks, by the end of which they had become severely deficient, and mortality was high.2. Growth of the deficient animals virtually ceased in the early stages of deficiency; food intake was severely and progressively depressed. Liver: body-weight increased markedly; packed cell volume fell at a late stage only. Pathological signs accumulated throughout the deficiency but were not closely related to the biochemical changes within the deficient group.3. The activation coefficient (stimulated: basal activity; AC) of glutathione oxidoreductase (EC 1.6.4.2; glutathione reductase; GR) in erythrocytes rose to a mean value of 3–8 after 3 weeks, and subsequently remained almost constant: this change was not seen in pair-fed or ud lib.-fed controls. Both deficient and pair-fed animals exhibited a twofold reduction in FAD-stimulated erythrocyteGR activity at an early stage. In liver, both deficient and pair-fed groups showed a major progressive fall in FAD-stimulated GR activity, but only the deficient group showed an increase in AC, which occurred towards the later stages of the experiment. In skin, too, the deficient group showed an increase in AC during the terminal stages.4. Hepatic, intestinal and brain succinate: (acceptor) oxidoreductase (EC 1.3.99,1; succinate dehydrogenase) activity fell relatively early during deficiency; in liver and intestine this was at least partly shared by the pair-fed group, and therefore attributable to inanition. Changes in hepatic NADH:(acceptor) oxidoreductase (EC I.6.99.3; NADH dehydrogenase) activity appeared to be entidy attributable to inanition.5. An early reduction was observed in hepatic ATP: riboflavin 5-phosphotransferase (EC2.7.1.26; flavo- kinase) activity in the deficient group, values falling by nearly half within 1 week, and then remaining almost constant. Similar but smaller changes were seen in renal flavokinase activity. Hepatic ATP: FMN adenylyltrans- ferase (EC2.7.7.2; FAD pyrophosphorylase) was unchanged until the third week, at which point it rose sharply to a new plateau in the deficient group; in kidney it did not respond. These changes were not observed in pair-fed or ad-lib.-fed controls.6. Hepatic flavin levels fell dramatically during the first 2 weeks of deficiency, FAD being conserved at the expense of FMN. Smaller changes were observed in kidney.7. Of the processes which are affected by riboflavin deficiency, AC of erythrocyte GR (EGRAC) responds earlier, more dramatically and more specifically than most others, with the possible exception of hepatic flavin levels and flavokinase. Potentially, it is therefore a good index of over-all body riboflavin status, but in acute deficiency the rate of response of many variables is not related to the final extent of response; consequently the correlation between EGRAC and other riboflavin-sensitive processes is less satisfactory than it would be in an equilibrium situation.

scholarly journals Effects of methodological variation on assessment of riboflavin status using the erythrocyte glutathione reductase activation coefficient assay

2008 ◽  
Vol 102 (2) ◽  
pp. 273-278 ◽  
Author(s):  
Marilyn H. E. Hill ◽  
Angela Bradley ◽  
Sohail Mushtaq ◽  
Elizabeth A. Williams ◽  
Hilary J. Powers
Keyword(s):  
Glutathione Reductase ◽  
Intervention Study ◽  
Flavin Adenine Dinucleotide ◽  
Riboflavin Deficiency ◽  
Study Results ◽  
Erythrocyte Enzyme ◽  
Activation Coefficient ◽  
The Uk ◽  
Riboflavin Status

Riboflavin status is usually measured as thein vitrostimulation with flavin adenine dinucleotide of the erythrocyte enzyme glutathione reductase, and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). This method is used for the National Diet and Nutrition Surveys (NDNS) of the UK. In the period between the 1990 and 2003 surveys of UK adults, the estimated prevalence of riboflavin deficiency, expressed as an EGRAC value ≥ 1·30, increased from 2 to 46 % in males and from 1 to 34 % in females. We hypothesised that subtle but important differences in the detail of the methodology between the two NDNS accounted for this difference. We carried out an evaluation of the performance of the methods used in the two NDNS and compared against an ‘in-house’ method, using blood samples collected from a riboflavin intervention study. Results indicated that the method used for the 1990 NDNS gave a significantly lower mean EGRAC value than both the 2003 NDNS method and the ‘in-house’ method (P < 0·0001). The key differences between the methods relate to the concentration of FAD used in the assay and the duration of the period of incubation of FAD with enzyme. The details of the EGRAC method should be standardised for use in different laboratories and over time. Additionally, it is proposed that consideration be given to re-evaluating the basis of the EGRAC threshold for riboflavin deficiency.

scholarly journals Refection in rats fed on a sucrose-based, riboflavin-deficient diet

1980 ◽  
Vol 43 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A. M. Prentice ◽  
C. J. Bates
Keyword(s):  
Biochemical Tests ◽  
Riboflavin Deficiency ◽  
Deficient Diet ◽  
Biochemical Effects ◽  
Severe Deficiency ◽  
Riboflavin Deficient ◽  
Riboflavin Status

1. Refection, resulting in an increased supply of riboflavin to riboflavin-deficient rats through coprophagy, was demonstrated on a sucrose-based diet when sensitive biochemical tests of riboflavin status were employed: these included measurements of NAD(P)H2:glutathione oxidoreductase (EC 1.6.4.2); succinate:(acceptor) oxidoreductase (EC 1.3.99.1) and NADH:(acceptor) oxidoreductase (EC 1.6.99.3).2. The use of tail-cups to eliminate coprophagy, and hence refection, resulted in a more rapid and reproducible progress into severe deficiency.3. The occurrence of refection on a sucrose-based diet may account for hitherto unexplained differences between previous publications on the biochemical effects of riboflavin deficiency.

scholarly journals Incubation of NAD(P)H2: glutathione oxidoreductase (EC1.6.4.2) with flavin adenine dinucleotide for maximal stimulation in the measurement of riboflavin status

1982 ◽  
Vol 48 (3) ◽  
pp. 459-466 ◽  
Author(s):  
D. I. Thurnham ◽  
Prapimporn Rathakette
Keyword(s):  
Quality Control ◽  
Glutathione Reductase ◽  
Flavin Adenine Dinucleotide ◽  
Maximal Stimulation ◽  
Maximal Coupling ◽  
Analysis Of Results ◽  
Cusum Analysis ◽  
Activation Coefficient ◽  
Riboflavin Status ◽  
Initial Results

1. Some modifications to the erythrocyte glutathione reductase assay for riboflavin status are described.2. Cusum analysis of results collected on a quality-control (QC) haemolysate, analysedseparately at the beginning and end of each batch of samples over a period of 20 weeks, suggested that the activation coefficient (AC) was higher at the end of a batch than at the beginning.3. The higher AC was due to higher FAD-stimulated enzyme activities of the QC samples measured at the end of the day, by comparison with the beginning, and this suggested that the conditions of assay were not optimal.4. The conditions required to achieve maximal coupling of FAD to glutathione reductase(NAD(P)H2: glutathione oxidoreductase;EC1.6.4.2) were therefore examined and found to be 15 min at 35° by comparison with the 5–7 min incubation used by most workers.5. Alternatively, where samples are prepared in batches, the enzyme and FAD should be pre-incubated in the reaction mixture for 2 h at 4° or 1 h at 25° before the standard incubation of 5 min at 35°.6. Additionally, the use of cummulative sum (cusum) analysis on the QC results suggested that there was a slight deterioration of QC sample after 4-weeks storage. However, theQC results obtained, remained within 2 standard deviations of initial results over a 20-week period, suggesting that the deterioration was very slight.

scholarly journals Riboflavin deficiency: early effects on post-weaning development of the duodenum in rats

2001 ◽  
Vol 86 (5) ◽  
pp. 593-599 ◽  
Author(s):  
Catherine A Yates ◽  
Gareth S. Evans ◽  
Hilary J. Powers
Keyword(s):  
Wistar Rats ◽  
Developmental Changes ◽  
Riboflavin Deficiency ◽  
Show Evidence ◽  
Activation Coefficient ◽  
Early Effects ◽  
Riboflavin Deficient ◽  
Rates Of Growth ◽  
Riboflavin Status

The aim of this present study was to identify the earliest point at which riboflavin deficiency affects post-weaning bowel development in rats. After weaning, eighty Wistar rats were weight-matched as pairs, one animal being fed a normal synthetic diet and the other being fed the same diet but deficient in riboflavin. Body weight, feeding and rates of growth were monitored and eight pairs of animals were taken for analysis at 45, 69, 93, 117 and 141 h. Riboflavin status was monitored by determining the erythrocyte glutathione reductase activation coefficient (EGRAC), and hepatic flavins were measured by a fluorescence assay. Changes to the number and dimensions of villi and crypts in the duodenum were determined, as well as crypt division (bifurcation) and the DNA synthesis index of the crypt epithelium by bromodeoxyuridine (BrdU) labelling. Riboflavin deficiency was established in the experimental rats, as demonstrated by a significant increase in EGRAC after 45 h (P<0·001) and decreased liver flavins after 96 h (P<0·001). After 96 h a significant increase in the size and cellularity of the crypts (P<0·001 in both cases) was seen in these riboflavin-deficient animals, with a decreased incidence of bifurcating crypts and of BrdU-labelled cells. No changes to villus number or size were observed. The present study has demonstrated that developmental changes to the duodenal crypt arise shortly after circulating riboflavin measurements show evidence of deficiency. These changes primarily affect cell proliferation and crypt bifurcation, and precede long-term changes such as the reduction of villus number.

scholarly journals Effect of riboflavin deficiency on reproductive performance and on biochemical indices of riboflavin status in the rat

1985 ◽  
Vol 53 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Julia M. Duerden ◽  
C. J. Bates
Keyword(s):  
Post Partum ◽  
Basal Diet ◽  
Young Female ◽  
Female Rats ◽  
Riboflavin Deficiency ◽  
Diet Control ◽  
Activation Coefficient ◽  
Riboflavin Deficient ◽  
Biochemical Indices ◽  
Riboflavin Status

1. Young female rats were made riboflavin-deficient by feeding a purified diet containing casein (210 g/kg). This basal diet provided 0.40 mg riboflavin/kg diet, to which was added additional riboflavin at 0, 0.12 or 0.25 mg/kg diet. Control animals received the same diet with 15 mg added riboflavin/kg. The diets were given for 4 weeks before mating, then throughout pregnancy and for 15 d of lactation.2. With no added riboflavin in the diet, reproduction was severely impaired and fetal resorption was usually observed. With 0.12 mg added riboflavin/kg diet, however, reproduction was usually successful, and the growth of dams and pups was only marginally depressed in comparison with pair-fed controls optimally supplied with riboflavin.3. The activation coefficient (stimulated: basal activity) of erythrocyte glutathione reductase (NAD(P)H) (EC 1.6.4.2)was high, and the concentration of riboflavin in the liver was correspondingly low in the dams receiving diets containing 0.12 or 0.25 mg added riboflavin/kg and in their sucking pups at 15 d post partum. Riboflavin levels in the milk from both groups of dams were about eightfold lower than in controls. There was little evidence that the sucking pups could maintain their riboflavin level at the expense of that in the maternal tissues.

scholarly journals The effect of riboflavin deficiency on methylenetetrahydrofolate reductase (NADPH)(EC 1.5.1.20) and folate metabolism in the rat

1986 ◽  
Vol 55 (2) ◽  
pp. 455-464 ◽  
Author(s):  
C. J. Bates ◽  
N. J. Fuller
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Folate Metabolism ◽  
Hepatic Tissue ◽  
Riboflavin Deficiency ◽  
Weanling Rats ◽  
Metabolizing Enzyme ◽  
Riboflavin Deficient ◽  
Riboflavin Status

1. Riboflavin deficiency at two levels of severity was produced in weanling rats by feeding deficient diets for 6 weeks and using neck collars to prevent coprophagy. The severity of deficiency was monitored by growth, liver flavin levels and the activation coefficient of erythrocyte glutathione oxidoreductase (NAD(P)H) (EC 1. 6. 4. 2 ) Control groups, receiving the same diet with ample added riboflavin, were fed either ad lib., or were pair-fed with the deficient animals.2. The hepatic flavoenzyme, methylenetetrahydrofolate reductase (NADPH) (EC 1.5.1.20), was very markedly affected by severe riboflavin deficiency and was significantly, but less markedly, affected by the intermediate level of deficiency. This reduction in activity was due primarily to the direct effect of the diminished supply of riboflavin, and occurred to only a small extent as a result of inanition, demonstrated by a moderate reduction in activity in the more severely food-restricted of the two pair-fed groups. Since the enzyme is assayed in the presence of its flavin cofactor, FAD, it clearly cannot be reactivated in vitro, as some other depleted flavoenzymes can. The discriminatory ability in distinguishing between severe and moderate riboflavin deficiency in vivo confers some potential advantages on this oxidoreductase as a possible index of riboflavin status.3. The hepatic activity of another key folate-metabolizing enzyme, dihydrofolate reductase (EC 1.5.1.3), was not diminished by riboflavin deficiency in the present study.4. The ratio, labelled 5-methyltetrahydrofolic acid: other labelled compounds derived from intraperitoneally injected pteroylglutamic acid in extracts of hepatic tissue was significantly reduced in the riboflavin-deficient groups, indicating the possibility of an effect of riboflavin deficiency on folate metabolism in vivo.

Effect of Riboflavin Deficiency on the Metabolism of Oxypurines in Chicks

1971 ◽  
Vol 49 (12) ◽  
pp. 1059-1062 ◽  
Author(s):  
S. T. Chou
Keyword(s):  
Uric Acid ◽  
Acid Synthesis ◽  
Xanthine Dehydrogenase ◽  
Broiler Chicks ◽  
Uric Acid Concentration ◽  
Riboflavin Deficiency ◽  
Deficient Diet ◽  
High Incidence ◽  
Liver And Kidney ◽  
Riboflavin Deficient

Day-old broiler chicks of both sexes were used in three experiments to determine the effect of riboflavin deficiency on oxypurine metabolism catalyzed by xanthine dehydrogenase, a riboflavin-containing enzyme. Chicks fed a riboflavin-deficient diet (1.38 mg/kg) for 3 weeks exhibited depressed growth and a high incidence of curled-toe paralysis (higher than 80%) as compared to control chicks (15.1 mg riboflavin per kilogram diet; no incidence of curled-toe paralysis). In addition, the precursors of uric acid, hypoxanthine and/or xanthine, accumulated in the liver and kidney of deficient chicks showing curled-toe paralysis. These observations show that dietary riboflavin being incorporated into xanthine dehydrogenase is essential for oxypurine metabolism. Moreover in the chick, the liver and the kidney may be important sites of uric acid synthesis. The low uric acid concentration in the plasma of the deficient chicks appeared to be indicative of a disturbance in uric acid synthesis in the liver and kidney.

scholarly journals Identification of Plasmatic Biomarkers of Foie Gras Qualities in Duck by Metabolomics

2021 ◽  
Vol 12 ◽  
Author(s):  
Zohre Mozduri ◽  
Nathalie Marty-Gasset ◽  
Bara Lo ◽  
Ali Akbar Masoudi ◽  
Mireille Morisson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Linear Models ◽  
Glucuronic Acid ◽  
Liver Weight ◽  
Quality Parameters ◽  
Strongly Correlated ◽  
H Nmr ◽  
Non Invasive ◽  
Technological Quality ◽  
Foie Gras

The foie gras is an emblematic product of French gastronomy composed of waterfowl fatty liver. The organoleptic qualities of this product depend on the liver characteristics such as liver weight (LW) and technological yield (TY) at cooking. One of the main issues for producers is to classify the foie gras with high or low technological quality before cooking them. Thus the study aims at identifying biomarkers of these characteristics with non-invasive biomarkers in duck. 1H-NMR (nuclear magnetic resonance of the proton) analyses were performed on plasma of male mule ducks at different time points during the overfeeding period to obtain a large range of liver characteristics so as to identify plasmatic biomarkers of foie gras. We used two methods, one based on bucket data from the 1H-NMR spectra and another one based on the fingerprints of several metabolites. PLS analyses and Linear models were performed to identify biomarkers. We identified 18 biomarkers of liver weight and 15 biomarkers of technological yield. As these two quality parameters were strongly correlated (−0.82), 13 biomarkers were common. The lactate was the most important biomarker, the other were mainly amino acids. Contrary to the amino acids, the lactate increased with the liver weight and decreased with the technological yield. We also identified 5 biomarkers specific to LW (3 carbohydrates: glucuronic acid, mannose, sorbitol and 2 amino acids: glutamic acid and methionine) that were negatively correlated to liver weight. It was of main interest to identify 2 biomarkers specific to the technological yield. Contrary to the isovaleric acid, the valine was negatively correlated to the technological yield.

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