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Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.

We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.

We present the results of measurements of alkaline phosphatase activity from interlaboratory transferability studies conducted by 12 laboratories in five countries. The variability, as demonstrated by within-day precision (CV less than or equal to 2.6%), between-day precision (CV less than or equal to 3.6%), and between-lab precision (CV less than or equal to 6.3%) establishes the transferability of this method for alkaline phosphatase. Some common errors encountered in enzyme measurements that affect the accuracy and precision of the measurements are listed.

We studied the kinetic properties of purified "heavy" form of human liver gamma-glutamyltransferase (EC 2.3.2.2) in the presence and absence of the acceptor substrate glycylglycine under Vmax conditions and as a function of pH. gamma-Glutamyl carboxynitroanilide was used as the donor substrate. Our data suggest that hydrolysis of donor substrate is the major pathway in the absence of acceptor. Autotransfer, if it occurs, is not important. Hydrolysis and transfer reactions have different pH profiles both for Vmax and Km. The pH dependency of Vmax and Km for both the hydrolase and the transferase reactions most probably reflects a change in the rate-limiting step: deacylation of the enzyme at acidic pH and acylation at alkaline pH. Negative cooperativity, observed for both donor and acceptor substrates near neutral pH, is interpreted in terms of more than one active site per dimer for each substrate.

We measured serum protein and calcium concentrations in 2340 individuals between 10 and 96 years of age from 900 families chosen by probability methods to give a representative population. These values were used to calculate an index, based on a regression analysis of serum protein on calcium, which was then treated as a new variable. Age-sex specific reference values and frequency distributions are presented for this index as well as for protein and calcium calculated by both parametric and nonparametric methods.

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.

A new technic using the principle of enzyme-linked immunoassay (ELISA) has been developed for the detection of complement components on red blood cells sensitized in vivo or in vitro. Using a double-antibody technic, anticomplement antisera (anti-C3c or anti-C3c/C3d) produced in rabbits was incubated with the red blood cells, followed by incubation with antirabbit alkaline phosphatase conjugated antiglobulin. The amount of the enzyme fixed was measured spectrophotometrically by the enzymatic hydrolysis of the substrate PNPP. A calibration curve was made from red blood cells on which complement was deposited by the method of Fruitstone . The technic showed a greater sensitivity than the standard antiglobulin tests and allowed simultaneous qualitative and semiquantitative estimates. The technic can be performed in any laboratory equipped with the standard equipment found in a blood bank, including a spectrophotometer. The authors made a modification of Alsever 's solution, which allowed the safe and stable preservation of complement coated red blood cells for 15 days. Significant positive results were obtained clinically using this technic, while negative or weakly positive reactions were obtained by the conventional antiglobulin tests.

A natural population has been studied for its serum urate levels and for host factors associated with these levels. Householdhood, ethnicity, drug and alcohol use were found to be negligible behavioral attributes with respect to population serum urate concentrations. Of interest among significant correlates of serum urate are smoking behavior, serum protein, creatinine, calcium and monocyte count. Predominant statistically significant host factors, however, are sex, body weight and age, body weight being linearly related in both sexes and age in females only.

Sex- and age-specific serum lactate dehydrogenase values provided by a natural population of 2,378 individuals selected by a multistage area cluster probability sampling scheme are given. The activity of this enzyme has definite sex and age associations over the age range studied (larger than or equal to 10 years). In both sexes, it is characterized by elevations in preadolescents, by a sharp decrease in adolescent groups, and by a gradual increase in adult and senescent groups, which, however, does not bring it back to earlier levels. Males have higher mean serum lactate dehydrogenase activities than females in most age groups until about 50 years of age. Unusually high activities have been found in this study, compared with those found in two other widely separated areas in North America using the same assay procedures.