Method Description

Glucose-6-Phosphate Dehydrogenase:

Glucose-6-phosphate dehydrogenase (G6PD) in a hemolysate catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate. Concomitantly, nicotinamide adenine dinucleotide phosphate (NADP[+]) is changed to its reduced form (NADPH), and the reaction is measured spectrophotometrically on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:68-71; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Pyruvate Kinase:

Pyruvate kinase catalyzes the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by converting phosphoenolpyruvate to pyruvate. The amount of pyruvate formed is quantitated by adding lactate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) and measuring the rate of decrease in absorbance spectrophotometrically at 340 nm as the NADH is oxidized to NAD(+) on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:68-71; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Glucose Phosphate Isomerase:

Glucose phosphate isomerase interconverts glucose-6-P (G6P) and fructose-6-P (F6P). In this assay, the F6P is then further converted to 6-phosphogluconate (6-PG) through the G6P dehydrogenase (G6PD) reaction resulting in the reduction of NADP(+) to NADPH. The reduction of NADP(+) is measured spectrophotometrically by the increase in absorbance at 340 nm on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:40-42; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Hexokinase:

Hexokinase catalyzes the reaction of ATP and glucose to G6P and ADP. In this assay, the formation of G6P is measured by linking its further oxidation to 6-PG to the reduction of NADP(+) through the G6PD reaction. The increase in absorbance, which occurs as NADP(+) is reduced to NADPH, is measured spectrophotometrically at 340 nm on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton;1984:38-40; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Adenylate Kinase:

Adenylate kinase (myokinase) catalyzes the dismutation of ADP into adenosine-5'-monophosphate and ATP. In this assay, the reverse reaction is measured by following the formation of ADP with pyruvate kinase (PK) and lactate dehydrogenase reactions resulting in NADH being oxidized to NAD(+). The decrease in absorbance that occurs as NADH is oxidized is measured spectrophotometrically at 340 nm by an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:93-95; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Phosphofructokinase:

Phosphofructokinase catalyzes the phosphorylation of F6P by ATP to fructose-1,6-diphosphate (FDP). FDP is then converted to dihydroxyacetone phosphate (DHAP) through subsequent aldolase, and triosephosphate isomerase (TPI) catalyzed reactions. The rate of formation of DHAP is measured by linking its reduction to alpha-glycerophosphate by alpha-glycerophosphate dehydrogenase, which results in the oxidation of NADH to NAD(+). The decrease in absorbance at 340 nm is measured spectrophotometrically as the NADH is oxidized on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:pp 68-71; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Phosphoglycerate Kinase:

Phosphoglycerate kinase catalyzes the phosphorylation of ADP to ATP by conversion of 1,3-diphosphoglycerate (1,3-DPG) to 3-phosphoglyceric acid. In this assay, the reaction is driven in the reverse direction. The formation of 1,3-DPG is then measured through the glyceraldehyde phosphate dehydrogenase reaction as 1,3-DPG is converted to glyceraldehyde-3-phosphate (GAP), resulting in the oxidation of NADH to NAD(+). The decrease in absorbance that occurs as NADH is oxidized is measured spectrophotometrically at 340 nm on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984, pp 53-55; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Triosephosphate Isomerase:

Triosephosphate isomerase (TPI) interconverts GAP and DHAP. The rate of DHAP formation is measured by further converting it to alpha-glycerophosphate by alpha-glycerophosphate dehydrogenase, which results in the oxidation of NADH to NAD(+). The decrease in absorbance that occurs as NADH is oxidized is measured spectrophotometrically at 340 nm on an automated chemistry analyzer.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton 1984; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Glutathione:

Virtually all the nonprotein sulfhydryl of red blood cells is in the form of reduced glutathione (GSH). 5,5'-dithiobis (2-nitrobenzoic acid) is a disulfide compound that is readily reduced by sulfhydryl compounds, forming a highly colored yellow anion. The absorbance of this resultant yellow substance is measured by 412 nm and compared to that of a known standard.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984; Alisik M, Neselioglu S, Erel O. A colorimetric method to measure oxidized, reduced and total glutathione levels in erythrocytes. J Lab Med. 2019:43(5), 269-277. doi:10.1515/labmed-2019-0098)

Pyrimidine 5' Nucleotidase:

Pyrimidine nucleotides have a spectral absorption curve that is markedly different from that exhibited by (normally present) adenine nucleotides, eg, ATP. The former have a peak at about 270 nm; the latter at about 257 nm. Thus, pyrimidine 5' nucleotidase deficiency may be ascertained by demonstrating a very high spectral absorption maximum of 270 nm in erythrocyte extracts.(Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:100-102; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)