Measurement of Erythrocyte Glucose-6-Phosphate Dehydrogenase Activity with a Centrifugal Analyzer

1975 ◽  
Vol 21 (1) ◽  
pp. 134-138 ◽  
Author(s):  
Edward W Catalano ◽  
George F Johnson ◽  
Harvey M Solomon
Keyword(s):  
Dehydrogenase Activity ◽  
Regression Line ◽  
Reaction Volume ◽  
Phosphogluconate Dehydrogenase ◽  
Standard Assay ◽  
Variable Contribution ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Improved Methods ◽  
Final Reaction Volume

Abstract We describe two improved methods for determination of erythrocyte glucose-6-phosphate dehydrogenase activity. One method is an"enzyme-linked" procedure in which an excess of 6-phosphogluconate dehydrogenase is used to produce two moles of NADPH for each mole of glucose-6-phosphate oxidized. In the other method 2,3-diphosphoglycerate is used to inhibit the variable contribution of endogenous 6-phosphogluconate dehydrogenase to glucose-6-phosphate dehydrogenase activity in erythrocyte lysates. These assays require 100 µl of blood and are performed on a centrifugal analyzer in a final reaction volume of 410 µl at 37 °C. NADPH formation is monitored at 340 nm. Hemoglobin is measured as oxyhemoglobin in the same reaction mixture used to determine enzyme activity by changing the wavelength to 550 nm. Results are expressed as international units of glucose-6-phosphate dehydrogenase activity per gram of hemoglobin. The coefficient of correlation between "enzyme-linked" assay and "standard" assay was .992, the slope of the regression line was 1.07, and the intercept was at -0.76. When results of the "enzyme-linked" assay were compared to those of the "nonlinked" assay with added 2,3-diphosphoglycerate, the slope of the regression line was .994, the intercept 0.109, and the correlation coefficient .994.

scholarly journals Field Trial of the CareStart Biosensor Analyzer for the Determination of Glucose-6-Phosphate Dehydrogenase Activity in Haiti

2017 ◽  
Vol 97 (4) ◽  
pp. 1262-1270 ◽  
Author(s):  
Thomas A. Weppelmann ◽  
Elisa Aguenza ◽  
Tara D. Wilfong ◽  
Bernard A. Okech ◽  
Michael E. von Fricken ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Field Trial ◽  
Glucose 6 Phosphate Dehydrogenase

Maleimide as an inhibitor in measurement of erythrocyte glucose-6-phosphate dehydrogenase activity.

1978 ◽  
Vol 24 (6) ◽  
pp. 885-889 ◽  
Author(s):  
J Deutsch
Keyword(s):  
Dehydrogenase Activity ◽  
Coefficient Of Variation ◽  
Present Method ◽  
Reaction Rate ◽  
Hemoglobin Concentration ◽  
Statistical Correlation ◽  
Secondary Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Reagent Cost ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Erythrocyte glucose-6-phosphate dehydrogenase activity is measured with a centrifugal analyzer by use of a commercial reagent kit and of the reaction glucose-6-phosphate + NADP+ leads to 6-phosphogluconolactone + NADPH. Rate of production of NADPH is measured and related to hemoglobin concentration. Maleimide is added to inhibit further production of NADPH in a secondary reaction by endogenous 6-phosphogluconate dehydrogenase. The method is compared with others that are designed to circumvent the secondary reaction by either (a) addition of excess phosphogluconate dehydrogenase to drive the secondary reaction to completion or (b) inhibition of endogenous phosphogluconate dehydrogenase by 2,3-diphosphoglycerate. The present method has the advantages that reaction rate more quickly becomes linear and reagent cost is less as compared with other methods. The within-run coefficient of variation was 3%. The various methods investigated showed good statistical correlation.

Measurement of Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase Activites in Erythrocytes by Use of a Centrifugal Analyzer

1974 ◽  
Vol 20 (10) ◽  
pp. 1349-1352 ◽  
Author(s):  
John F Nicholson ◽  
Selma H Bodourian ◽  
Michael A Pesce
Keyword(s):  
Dehydrogenase Activity ◽  
Dehydrogenase Deficiency ◽  
Simple Modification ◽  
Phosphogluconate Dehydrogenase ◽  
Automated Method ◽  
Large Populations ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract We describe an accurate automated method for measuring activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in erythrocytes with a centrifugal analyzer. Blood is collected in microhematocrit tubes, centrifuged, and the erythrocytes are lysed with digitonin. Glucose-6-phosphate dehydrogenase activity is determined by mixing the hemolysate with glucose-6-phosphate, NADP+, and 6-phosphogluconate dehydrogenase #{ 233} (EC 1.1.1.44) in triethanolamine—EDTA buffer at pH 7.6, and measuring the rate of NADPH production for 3 min. Under these conditions 2 mol of NADPH are produced per mole of glucose-6-phosphate oxidized, ensuring the accuracy of the method and increasing its sensitivity. Activity is referred to hemoglobin, measured as cyanmethemoglobin. Activity of glucose-6-phosphate dehydrogenase added to hemolysates was well accounted for. Results obtained by our method and by the method of Bishop [J. Lab. Clin. Med. 68, 149 (1966)] are virtually identical. Our method requires a small amount of blood and is accurate and rapid; thus it is well suited for use in surveying large populations for glucose-6-phosphate dehydrogenase deficiency. A simple modification of the method may be used to determine the activity of 6-phosphogluconate dehydrogenase.

Automated Fluorometric Determination of Glucose-6-Phosphate Dehydrogenase (G6PD) and 6-Phosphogluconate Dehydrogenase (6PGD) Activities in Red Blood Cells

1969 ◽  
Vol 15 (6) ◽  
pp. 467-478 ◽  
Author(s):  
I K Tan ◽  
T P Whitehead
Keyword(s):  
Quantitative Determination ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Blood Capillary ◽  
Enzymatic Reactions ◽  
Fluorometric Determination ◽  
Phosphogluconate Dehydrogenase ◽  
Natural Fluorescence ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract An automated fluorometric microtechnic, suitable for screening purposes, has been developed for the quantitative determination of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) levels in red blood cells. The method is rapid, simple, and precise. It uses 50 µl whole-blood (capillary) samples and is based on the measurement of natural fluorescence emitted by the NADPH generated during the enzymatic reactions.

scholarly journals Stimulation of pentose phosphate pathway dehydrogenase enzyme activities in ethionine-treated mice

1968 ◽  
Vol 106 (4) ◽  
pp. 769-776 ◽  
Author(s):  
Hsien-Gieh Sie ◽  
William H. Fishman
Keyword(s):  
Dehydrogenase Activity ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Hepatic Glycogen ◽  
Synthetase Activity ◽  
Phosphogluconate Dehydrogenase ◽  
Glycogen Synthetase ◽  
Period 2 ◽  
Marked Preference ◽  
Glucose 6 Phosphate Dehydrogenase

1. Mice treated with ethionine (intraperitoneally, 5mg./day for 4 days or 10mg./day for 3 days) showed a profound loss of hepatic glycogen, a decrease of glycogen synthetase activity, a development of hypoglycaemia, a two- to five-fold increase in the activity of glucose 6-phosphate dehydrogenase but no change in 6-phosphogluconate dehydrogenase and an earlier manifestation of the solubilization of phosphorylase as compared with glycogen synthetase. The administration of ATP did not prevent these effects. 2. During the early post-injection period (2–3 days) there was a further enhancement of the activity of glucose 6-phosphate dehydrogenase (tenfold) in the liver and a clear elevation of 6-phosphogluconate dehydrogenase activity (twofold). Subsequently, the glycogen concentration was restored, followed by an earlier reassociation of glycogen particle with phosphorylase than with glycogen synthetase, along with a disappearance of ethionine effect at about the eighteenth day. 3. Glucose 6-phosphate dehydrogenase from both control and ethionine-treated animals showed a marked preference for glucose 6-phosphate as substrate rather than for galactose 6-phosphate, whose rate of oxidation was only 10% of that of the glucose 6-phosphate. 4. Since actinomycin D, puromycin, 5-fluorouracil and dl-p-fluorophenylalanine failed to block the ethionine-enhanced glucose 6-phosphate dehydrogenase activity, the possibility that new enzyme protein synthesis is responsible for the effect is doubtful.

6-Phosphogluconate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, Glucose-6-Phosphate Isomerase, and Hexokinase Activity Ratios in Some Human Tumor Cytosols

1978 ◽  
Vol 64 (6) ◽  
pp. 579-586
Author(s):  
Michele Miranda ◽  
Terenzio Ventura ◽  
Luciano Iorio ◽  
Anna M. Ragnelli ◽  
Claudio Martano ◽  
...  
Keyword(s):  
Cell Growth ◽  
Dehydrogenase Activity ◽  
Human Tumor ◽  
Hexokinase Activity ◽  
Phosphogluconate Dehydrogenase ◽  
Growth Requirements ◽  
High K ◽  
Activity Ratios ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Low K

The ratios of some key enzymatic activities of carbohydrate metabolism have been measured in human tumor cytosols. The activities of whole hexokinase (low Km, EC 2.7.1.1 and high Km, EC 2.7.1.2), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glucose-6-phosphate isomerase (EC 5.3.1.9) change according to a biochemical pattern coherent with cell growth requirements. 6-phosphogluconate dehydrogenase activity was in each sample tested higher than glucose-6-phosphate dehydrogenase activity; this indicates that 6-phosphogluconate, a powerful inhibitor of glucose-6-phosphate isomerase, is unlikely to accumulate and inhibit this enzyme and glucose-6-phosphate channelling into glycolysis.

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