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A continuous spectrophotometric method for measuring serum alkaline phosphatase activity is described. The effects of temperature, pH, substrate concentration, type and molarity of the buffer, sample size, cofactors, and inhibitors on the enzymatic hydrolysis of p-nitrophenyl phosphate were studied. The optimal conditions for assay of serum alkaline phosphatase at 30° were found to be 0.75 M 2-amino-2-methyl-1-propanol buffer, pH30° 10.15, 4 mmole substrate, and 100 µl. or less sample size. Studies of the factors affecting analytical precision-i.e., control of reaction temperature, of reagent manufacture, and of standardization-are discussed. The precision of this method was 2.3% (relative standard deviation) on 10 within day replicates and 5.0% on day-to-day replicates spread over a 5-week period. The range of activity for 258 apparently healthy adult blood donors was 6-110 mU./ml. (International milliunits per milliliter), with a mean of 49 and a standard deviation of 14.

This candidate Reference Method for measuring total bilirubin in serum is based on the Jendrassik-Gróf principle (Clin Chem 29: 297-301, 1983). Standard Reference Material no. 916 bilirubin (National Bureau of Standards) is used as the standard. Bilirubin standard solutions may be prepared either in human serum or in 40 g/L albumin solution (human or bovine), because we found the molar absorptivity of the azopigment at 598 nm to be identical in these media. The absorptivities of the unconjugated and conjugated azopigments appear to be identical, but the conjugated azopigment is completely hydrolyzed in the final reaction mixture. Bilirubin added to serum from adults or neonates was quantitatively accounted for. Interference by hemoglobin (up to 2 g/L), ascorbic acid (up to 20 mg/L), or zinc (at physiological concentrations) is negligible. Of the therapeutic drugs we tested, only L-dopa and alpha-methyldopa interfere. We established normal adult reference values for total bilirubin and examined the intraindividual variation in 19 subjects.

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The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high-purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose-6-phosphate by NAD+ in the presence of glucose-6-phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 degrees C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.

Optimum reaction conditions at 30° ± 0.5° for two continuous spectrophotometric assay procedures, lactate to pyruvate (L → P) and pyruvate to lactate (P → L), were determined with respect to pH at 30° (pH30) substrate concentration, and coenzyme concentration for the human LDH isoenzymes. For the P → L procedure, broad pH30 optima were within the range of 7.20-7.40 for all the LDH isoenzymes. The coenzyme optima were identical for all of the isoenzymes tested at a reduced NAD concentration of 1.5 x 10-4 M. Pyruvate substrate optima ranged from 7.5 x 10-4 M for LDH1 to 1.7 x 10-3 M for LDH5 at pH30 7.30. For the L → P procedure, the pH30 optima were within the range of 8.30-8.88 for the LDH5 through LDH1 isoenzymes, respectively. Optimum activity was obtained at a NAD concentration of 6.0 x 10-3 M and remained constant at least to 1.8 x 10-2 M for each of the isoenzymes tested. L-Lactate substrate optima ranged from 4.0 x 10-2 M for LDH1 to at least 7.2 x 10-2 M for LDH5. From the isoenzyme studies, the degree of variation possibly involved in either method due to variations in isoenzyme distribution was calculated for total LDH samples. These calculations showed that both methods, P → L and L → P, were essentially equivalent. This equivalency was verified by a comparative study of the two methods on human serum samples.

We have compared 23 compounds, with and without transphosphorylating properties, as buffer systems for human serum alkaline phosphatase activity, with p-nitrophenylphosphate as substrate. Relative enzyme activity in four representative buffers at near-optimal conditions was ethylaminoethanol > diethanolamine > 2-amino-2-methyl-1-propanol > carbonate. Transphosphorylation was demonstrated in the two buffers in which the enzyme was most active, ethylaminoethanol and diethanolamine. The optima for pH, buffer concentration, and substrate for these four systems were studied in detail. 2-Ethylaminoethanol supports the highest enzyme activity of any of the compounds tested. Diethanolamine was shown to have many of the favorable characteristics required for a reference enzyme procedure.

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