We established a simple and rapid kinetic assay for measurement of calcium in serum by using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on inhibition of the enzyme by calcium. In the assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH; EC 1.4.1.4); then in the presence of urea amidolyase, urea, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion production was inversely proportional to calcium ion concentration in serum. The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then monitored the change of absorbance at 340 nm. The within-run CVs of this method were 1.7-3.2% (n = 10) at 1.53-3.08 mmol/L, respectively. Day-to-day (total) CVs were 2.8-4.1%. Analytical recovery was 92-112%. The presence of other ions, ascorbic acid, reduced glutathione, bilirubin, hemoglobin, citrate, lipemic material, or human serum albumin did not affect this assay system. The correlation between values obtained with our method (y) and o-cresolphthalein complexone method (CPC) (x) was: y = 1.001x + 0.077 mmol/L (r = 0.949, Sy[symbol: see text]x = 0.079, n = 100); with the other enzymatic method (x) it was: y = 0.952x + 0.021 mmol/L (r = 0.955, Sy[symbol: see text]x = 0.074, n = 100). The SEs for each method were: 0.025 mmol/L, our method; 0.023 mmol/L, CPC method; and 0.025 mmol/L, the other enzymatic method.

We measured thyroid hormones and thyroxin-binding proteins in serum from 62 normal lactating and 52 nonlactating women at three months postpartum, and compared these values with those for 42 nonpregnant control women of similar age. Mean thyroxin concentrations in the lactating and nonlactating women were significantly (P less than 0.001) lower than that of the nonpregnant controls, but there was no significant difference (P greater than 0.2) in triiodothyronine concentration among these three groups. Free T4 concentration was significantly (P less than 0.01) lower in lactating women than in controls. The reverse-T3 concentrations in both lactating and nonlactating women were significantly (P less than 0.001) lower than in controls, and were significantly (P less than 0.001) lower in lactating than in nonlactating women. The concentration of thyroxin-binding globulin was significantly higher in lactating women than in controls, and the albumin concentration was significantly lower in women postpartum than in controls. Evidently, regulation of thyroid hormone in women postpartum, especially during lactation, differs from that in nonpregnant women.

We describe a double-antibody enzyme immunoassay for determination of free thyroxin (FT4) in dried blood samples on filter paper, with use of a T4-beta-D-galactosidase complex. The measurable range of FT4 concentration in two 3-mm blood discs, each of which contained about 2.7 microL of blood, was 1.9 to 93 ng/L, as determined by comparison with concentrations of FT4 in known serum standards. FT4 in blood samples dried on filter paper was stable for at least four weeks when kept dry at -20 degrees C, room temperature, or 37 degrees C. The mean coefficients of variation were 7.6% (within assay) and 6.4% (between assays). Results for FT4 by this method correlated well with those for serum determined by radioimmunoassay (r = 0.98). The proposed method can be used to differentiate persons with hyper- and hypothyroidism from normal subjects and those with abnormal concentrations of thyroxin-binding globulin. The procedure seems suited for screening studies.

A sensitive assay procedure for immunoenzymometric assay of serum thyrotropin (TSH) was developed by making several modifications of the Enzymun-Test TSH kit (Boehringer, Mannheim GmbH). Serum samples were first incubated in plastic tubes precoated with monoclonal antibodies specific to the beta subunit of human TSH. After the tubes were washed, the TSH bound to the tubes was detected with peroxidase-conjugated polyclonal antibodies to TSH. The sensitivity of the assay was 0.2 milli-int. unit/L, and the intra-and interassay CVs were less than 10%. Analytical recovery was 96 to 106%. The normal basal range of TSH was 0.5 to 4.8 milli-int. units/L. The basal levels of TSH in all but one of 48 thyrotoxic patients with Graves' disease were less than 0.2 milli-int. unit/L, clearly different from those of normal subjects. Thyrotoxic patients in early normal pregnancy showed TSH concentrations of 1.7 to 2.9 milli-int. units/L by conventional double-antibody radioimmunoassay, possibly from cross reactivity with human choriogonadotropin, but undetectable TSH by this method. Measurement of basal TSH by this sensitive assay can be used as an initial screening test for thyroid dysfunction.

We measured creatine kinase (CK, EC 2.7.3.2) activity in serum with a new reagent system utilizing thermostable glucokinase (EC 2.7.1.2). Automated determinations were performed with Toshiba's Model TBA-80S Biochemical Analyzer. Precision studies demonstrated within-run and between-run CVs of 0.4%-2.4% and 2.8%-3.1%, respectively. The response linearity was confirmed for CK activity up to 1000 U/L at 37 degrees C. CK activities correlated well (r = 0.997) with those obtained by the manual method recommended by the German Society for Clinical Chemistry (measuring at 37 degrees C) involving hexokinase (EC 2.7.1.1). However, CK activities measured by our method were consistently higher than those of the hexokinase method at reaction temperatures of 30, 37, and 40 degrees C. These data indicate that the new method with thermostable glucokinase is better than that with thermo-unstable hexokinase for determination of CK activity in serum.