Automated Biochemical Analysis of Plasma

Commercial colorimetric diagnostic kits were used to measure plasma glucose (Ciba Corning Diagnostics Corp., OH), triglycerides, cholesterol and creatine kinase (Chiron Diagnostics, Cergy Pontoise, France), lactate dehydrogenase (LDH, Bayer Healthcare, Dublin, Ireland), and uric acid levels (Pointe Scientific Inc, Canton, MI) with an automated spectrophotometer according to manufacturer’s recommendations. Briefly, glucose concentration was estimated by the formation of NADH at 340 nm absorbance. When phosphorylated with ATP and hexokinase, glucose yields glucose-6- phosphate and ADP which, in turn, is catalyzed by G-6-PD to form 6-phosphogluconate and NADH. NADH formation is directly proportional to the amount of glucose in the sample. Triglyceride levels were enzymatically determined based on the action of lipase, glycerol kinase and glycerol-phosphate oxidase at 540 nm. Cholesterol levels were enzymatically estimated in the presence of cholesterol esterase, cholesterol oxidase and peroxidase at 500 nm. Creatine kinase levels were determined based on the rate of NADPH formation measured at 340 nm in the presence of HK and G-6-PD. LDH activity was estimated following the oxidation of lactate to pyruvate at 340 nm. Uric acid levels were measured using the coupling of 4-aminoantipyrine with 2-hydroxy-2,4,6-tribromobenzoic acid and hydrogen peroxide in the presence of peroxidase at 520 nm.