We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.

We describe our optimization and evaluation of a candidate Reference Method for uric acid in serum. Reaction parameters were optimized for a manual, enzymic method for uric acid in which highly purified microbial uricase is used to quantitate uric acid by a differential ultraviolet procedure. We evaluated the method in terms of freedom from interferences, analytical recovery, precision, and comparison with five other uric acid methods. We conclude that (a) the candidate uric acid Reference Method exhibits the least interference; (b) all six methods exhibit satisfactory analytical recoveries and precision; and (c) results by all six methods agree well. As a result of this evaluation study, the manual ultraviolet uricase method for uric acid, with Tris as buffer, was chosen as the candidate Reference Method for uric acid.

We describe the interlaboratory testing of a candidate Reference Method (Part I) for uric acid in serum. The method is based on the ultrasound spectrophotometric quantitation of uric acid before and after incubation with uricase. A comprehensive investigation involving 12 laboratories was organized to document the transferability, intra- and interlaboratory precision, and general reliability of the candidate Reference Method. The interlaboratory CV with this method was about 2 to 6% for uric acid concentrations ranging from 0.12 to 0.60 mmol/L. The results detailed here demonstrate that the method can be successfully duplicated among different laboratories.

A recently developed gel-filtration technique allows protein-bound calcium fractions to be separated and quantitated; the protein is separated under physiological conditions of pH, temperature, and concentrations of Na, Mg, and Ca to assure that the calcium-proteinate equilibrium is not disturbed. We used this gel-filtration technique to study the protein-bound calcium fractions in 18 patients with hyperparathyroidism, multiple myeloma, diabetes, osteoporosis, or liver cirrhosis. We calculated the amount of calcium bound per gram of protein for each of the three protein peaks and the intrinsic association constant (Ka) for calcium/albumin. Results with the multiple myeloma patients (three IgG, one IgA) indicated that IgG did not bind calcium appreciably, that IgA had about the same affinity as albumin for Ca, and that Ka was slightly low for one patient of the IgG type (79 L/mol) and normal for the other three myeloma patients (106, 90, and 91 L/mol). Results for patients with the other diseases were also essentially normal, except for the osteoporesis patients (two men, one woman), whose Ka values (69, 75, and 73 L/mol) were lower than normal.

This study evaluates whether hemodialysis alters the affinity of plasma proteins for calcium. We have used a new automated methodology for the determination of the dialyzable calcium fraction. Protein-bound calcium was estimated as the difference between the total and dialyzable calcium fractions. All measurements were made after adjustment of the sample pH to 7.40 +/- 0.02. The calcium fractions were determined in 12 patients before and after hemodialysis. In addition, we added calcium to the predialysis specimens to evaluate the effect of increased ionic calcium on protein binding and corrected the protein-bound calcium in these specimens for the increase in total protein observed in the postdialysis specimens. Hemodialysis significantly increased total, dialysable, and protein-bound calcium. The mean ratio of protein-bound calcium in the postdialysis specimens to the corrected protein-bound calcium in the predialysis specimens with calcium added was 0.97 +/- 0.02. We conclude that (1) the increase in protein-bound calcium to the predialysis specimens, and that (2) the binding affinity of serum proteins for calcium is not altered by hemodialysis.