An automated biotin-streptavidin procedure for progesterone evaluation.

An automated biotin-streptavidin procedure for measuring progesterone in serum is described. The method is linear up to 98.3 nmol/L and the calibration curve is stable for at least 14 days. The lower limit of progesterone detection is 0.70 nmol/L. Although serum is the preferred specimen, progesterone levels can be measured in EDTA, heparin, or citrated plasma. There is no interference from samples with monoclonal proteins or from hemoglobin and bilirubin at concentrations of 9.62 g/L and 899 mumol/L, respectively. Lipemic samples will lower the progesterone levels. Total imprecision of the method in the range of 19-80.4 nmol/L gave CVs between 4.6 and 7.3%. Progesterone values obtained with this biotin-streptavidin procedure agreed with those obtained by the DPC RIA assay (r = 0.991). The biotin-streptavidin procedure can be used as an alternative to RIA for measurement of progesterone.

Hyperamylasemia in bulimia nervosa.

To determine the value of total serum amylase levels and salivary and pancreatic isoenzyme levels as biologic indices of behavioral disturbance in bulimia nervosa, the authors monitored these levels in 40 bulimic patients participating in a placebo-controlled trial of desipramine and in 25 controls. In the patients, the total and salivary amylase levels were significantly elevated and a significant correlation existed between the frequencies of binge eating and vomiting and the level of salivary amylase. However, the ability to discriminate patients from controls on the basis of serum amylase levels was limited. In addition, a significant positive relationship between binge frequency and level of serum amylase was observed in less than one quarter of 22 patients with five or more amylase determinations. Therefore, although hyperamylasemia is associated with bulimia nervosa, we believe that serum amylase determinations have limited utility in the assessment of patients with this syndrome.

A novel CK isoenzyme migrating between CK-MB and CK-BB.

A CK isoenzyme migrating between CK-MB and CK-BB was detected in the serum of three patients with metastatic prostatic carcinoma. CK-BB was detected in the serum of all three patients and mitochondrial CK in two of the patients. Total CK activity was either normal or elevated, and the atypical CK isoenzyme, CK-BB and the mitochondrial CK isoenzyme were present in serum for up to 1.5 months. This atypical CK isoenzyme was not CK-MB, an albumin-ligand complex, or adenylate kinase, and was not bound to an immunoglobulin. This atypical CK isoenzyme did not contain immunologically normal CK-M subunits but had some CK-B subunits and could be a variant CK-BB or CK-MB isoenzyme. Its appearance in serum could be indicative of a serious illness.

Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate.

A fluorescence polarization immunoassay (FPIA) procedure for measuring methotrexate was evaluated. The dynamic range of the assay is from 0.05 to 810 microM, and the calibration curve can be stored for at least 2 weeks. The FPIA procedure is automated and rapid; one result can be obtained in 18 min and five results in 25 min. There was no interference from hemoglobin (800 mg/dl), triglycerides (500 mg/dl), bilirubin (20 mg/dl), and protein (12.1 g/dl). Cross-reactivity with 7-hydroxy methotrexate and 2,4-diamino-N-methylpteroic acid was 0.6 and 44%, respectively. The coefficient of variation for the within-run and between-run precision was less than 5.0%. For the comparison studies, the samples were divided into four groups. The methotrexate concentrations in group 1 were 0.05-2.1 microM; in group 2, 2.2-9.3 microM; in group 3, 10-80 microM; and in group 4, greater than 80 microM. Linear regression analysis of the results obtained with the FPIA procedure and the enzyme multiplied immunoassay gave a correlation coefficient of at least 0.95 for all groups.

Fluorometric measurement of serum magnesium with a centrifugal analyzer.

We developed a rapid fluorometric assay for measurement of serum magnesium using the ligand, 8-hydroxy-quinoline-5-sulfonate and adapted the procedure to the Multistat Fluorescence Light Scattering Centrifugal Analyzer. The standard curve extends from 0.26 to 4.11 mmol/l. There was no interference from calcium or inorganic phosphorus at concentrations of 4.95 and 5.0 mmol/l, lead, iron, zinc or copper at twice the normal levels found in serum, bilirubin at concentrations of 10 mg/dl, or lipemic samples with triglyceride concentrations of 2400 mg/dl. Citrate and EDTA lowered magnesium concentrations in serum. Analytical recovery of magnesium added to four serum specimens averaged 97%. Between-run and within-run precision of the assay gave CVs which ranged from 2.9 to 7.6%. Magnesium concentrations, measured by our fluorometric procedure, were compared with those obtained by atomic absorption and colorimetric procedures. Correlation coefficients of 0.91 and 0.88 were obtained.

Nephelometric measurement of ceruloplasmin with a centrifugal analyzer.

We describe a rapid nephelometric assay for measurement of ceruloplasmin in serum with the Multistat centrifugal analyzer. In establishing the optimum conditions, the concentration of polyethylene glycol was critical. With a 30 g/L solution of polyethylene glycol, the calibrators and samples react at the same rate, while with a 60/L solution the calibrators react faster than the samples. The reaction rate was not affected by temperatures of 30 or 37 degrees C or pHs between 6.0 and 8.4. There was no interference from bilirubin at a concentration of 180 mg/L, hemoglobin at a concentration of 5000 mg/L, or lipemic serum. The standard curve extends from 50 to 1300 mg/L. Analytical recovery of ceruloplasmin added to four serum specimens averaged 97%. The within-run precision (n = 17) gave CVs of 5.4, 6.1, and 4.0% for samples containing ceruloplasmin at concentrations of 240, 360, and 650 mg/L. Ceruloplasmin concentrations measured by our procedure were compared with those obtained by nephelometric, radial immunodiffusion, and enzymic procedures; the correlation coefficients were 0.93, 0.88, and 0.86.

A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes.

A new and sensitive assay for measuring galactose-1-phosphate in erythrocytes is described. Galactose-1-phosphate is determined by mixing an aliquot of deproteinized hemolysate with a reagent containing uridine diphosphoglucose, NADP+, hexose-1-phosphate uridylyltransferase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase and measuring the NADPH formed fluorometrically. Under the conditions of this assay 2 mol of NADPH are formed per mol of galactose-1-phosphate. The assay is linear from 0 to 1160 micrograms of galactose-1-phosphate per gram of hemoglobin. Recovery of galactose-1-phosphate added to four hemolysates averaged 99%. Galactose-1-phosphate concentrations were measured in erythrocytes from five heterozygous subjects not under dietary control and seven transferase-deficient galactosemic individuals who were receiving galactose restricted diets. In all samples from the heterozygous individuals, the galactose-1-phosphate concentrations were normal. Of the samples from galactosemic subjects, two showed extreme elevations of galactose-1-phosphate, four showed moderate elevations, and one was normal. Galactose-1-phosphate levels are used to monitor the degree of dietary control in the transferase-deficient galactosemic individual.

Clinical significance of plasma galactose and erythrocyte galactose-1-phosphate measurements in transferase-deficient galactosemia and in individuals with below-normal transferase activity.

We correlated the clinical symptoms of transferase-deficient galactosemia with the plasma galactose and erythrocyte galactose-1-phosphate concentrations in six galactosemic patients during dietary treatment, in a child before treatment, and in 12 individuals with below-normal erythrocyte hexose-1-phosphate uridylyltransferase activity. All the treated patients were asymptomatic. Normal galactose and either normal or above-normal galactose-1-phosphate concentrations were found. Three of these patients were clinically normal as newborns while ingesting galactose-containing foods and may resemble the asymptomatic Negro galactosemic. The clinical symptoms of galactosemia were observed in the untreated patient, who showed markedly above-normal concentrations of galactose and galactose-1-phosphate, protein and reducing substances in the urine, above-normal bilirubin and alkaline phosphatase in the plasma, with normal values for glucose, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase. Clinical improvement in this patient paralleled the decline in erythrocyte galactose-1-phosphate. The individuals with below-normal hexose-1-phosphate uridylyltransferase activity (range 7--17 U/g of hemoglobin) had normal galactose and galactose-1-phosphate concentrations and were asymptomatic.

Enzyme immunoassay of gentamicin with use of a centrifugal analyzer.

Gentamicin was determined in serum by the homogeneous enzyme immunoassay system (EMIT) adapted to the Multistat centrifugal analyzer. The standard curve can be extended to 16 mg/L; however, poor precision is obtained at concentrations greater than 10 mg/L because a small difference in change in absorbance there will produce a significant variation in gentamicin concentration. For example, the within-run precision of the method (CV) was 3.4% for a 5.0 mg/L sample (range 4.6--5.4 mg/L) and 11.1% for a 12.2 mg/L sample (range 10.4--15.4 mg/L). We recommend that all samples containing gentamicin at concentrations exceeding 10 mg/L be diluted with Tris buffer. Recovery of gentamicin added to four serum specimens averaged 102%. There is no interference from bilirubin at concentrations up to 200 mg/L or from moderately lipemic samples. Hemoglobin in serum in excess of 625 mg/L results in lower gentamicin values. Gentamicin is stable for six days in serum stored at 23, 4, or -20 degrees C. Comparison of gentamicin results by the proposed method with those by a manual EMIT procedure, and with those by a homogeneous fluorescence immunoassay method gave correlation coefficients of 0.986 and 0.947.

Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.

Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.

Problems in the diagnosis of transferase and galactokinase deficient galactosemia.

Galactose in serum and galactose-1-phosphate in erythrocytes were measured in six transferase deficient children to determine if these metabolites could be used in detecting transferase deficient galactosemia. In all six children the galactose levels were normal and the galactose-1-phosphate elevated. The galactose level depends on diet and the rate of metabolism to galactose-1-phosphate and, therefore, should not be used to predict transferase deficient galactosemia. The galactose-1-phosphate level was elevated in all the transferase deficient children because once formed it cannot be metabolized. Measurement of galactose-1-phosphate is difficult and is usually requested to determine whether or not the child is following the galactose restricted diet. In transferase deficient galactosemia, the enzyme hexose-1-phosphate uridylyltransferase is absent. The diagnosis should be determined by measurement of the activity of the enzyme hexose-1-phosphate uridylyltransferase in erythrocytes. In galactokinase deficient galactosemia, the enzyme galactokinase is absent. Galactose levels are elevated but the amount present depends on diet and how soon the blood was collected after the ingestion of galactose containing foods. The diagnosis of galactokinase deficient galactosemia is based on the measurement of the enzyme galactokinase in erythrocytes.

Enzymic measurement of cholesterol in serum with the CentrifiChem centrifugal analyzer.

Cholesterol is measured by mixing 5 mul of sample with 350 mul of a reagent consisting of phenol, 4-aminoantipyrine, and the enzymes cholesterol oxidase, cholesterol esterase, and peroxidase. After 12 min, the resulting quinoneimine is measured at 520 nm. Readings and cholesterol concentrations are linearly related up to 4.0 g/liter. Lipemic sera and samples containing uric acid (up to 200 mg/liter), hemoglobin (up to 1 g/liter), and certain drugs (clofibrate, phenobarbital, nicotinic acid, Ketochol, Ovral-28), gave no interference. Abnormally high concentrations of bilirubin and ascorbic acid in serum lowered the cholesterol values. This enzymic assay, compared with the method of Abell and with a rate method that uses the Hantzsch reaction, gave correlation coefficients of 0.987 and 989, respectively.

Interference with the enzymic measurement of cholesterol in serum by use of five reagent kits.

We describe the effects of uric acid, hemolysis, drugs, ascorbic acid, lipemia, and bilirubin on the enzymic measurement of cholesterol in serum by use of reagent kits from Abbott, Beckman, Boehringer Mannheim, Calbiochem, and Worthington. In all of these, the chromogen formed from the reaction of hydrogen peroxide with phenol and 4-aminoantipyrene is measured. The absorbance was measured at 500 nm vs. a serum blank for each kit--except Abbott's with which the recorded absorbances were the differences between readings at 500 and 600 nm. With all reagents kits, there was no interference from uric acid up to 200 mg/liter, hemoglobin up to 1.0 g/liter, or drugs (clofibrate, phenobarbital, Ketochol, Ovral-28), but negative interferences from ascorbic acid. Except for the Abbott kit, the cholesterol values obtained for lipemic samples were lower than found with the comparison method [Abell et al., Stand. Methods Clin. Chem. 2, 26 (1958)]. With Abbott's reagents, for most lipemic samples, the values were the same. Bilirubin at concentrations of 200 mg/liter significantly decreased the cholesterol values with Beckman, Calbiochem, and Worthington reagent kits. With Boehringer Mannheim reagent a small negative interference was observed and with Abbott reagent a small positive interference was observed when the bilirubin concentrations were 200 mg/liter.

Enzymatic rate method for measuring cholesterol in serum.

An enzymatic rate assay is described for measuring cholesterol in serum. Cholesterol is analyzed by mixing 5 mul of sample with a reagent consisting of cholesterol esterase, cholesterol oxidase, catalase, acetylacetone, methanol, and hydroxypolyethoxydodecane in a ammonium phosphate buffer at pH 7.0. The rate of increase in absorbance of the dihydrolutidine product is measured at 37 degrees C and 405 nm. The change in absorbance between 4 and 10 min is used to calculate the cholesterol concentrations by using simultaneously determined free cholesterol standards. The change is linearly related to cholesterol concentration up to 4 g/liter. Samples containing bilirubin up to 200 mg/liter, uric acid up to 200 mg/liter, and hemoglobin up to 1 g/liter, or certain drugs (clofibrate, phenobarbital, nicotinic acid, salicylate, Ketochol, and Ovral) gave no interference. Ascorbic acid added to serum caused a positive interference. Lipemic samples gave values that were slightly lower than did the method of Abell et al., used for comparison. Our kinetic assay, compared with the method of Abell et al., the enzymatic assay used with Abbott's Bichromatic Analyzer, and the Technicon SMA 12/60 enzymatic procedure gave correlation coefficients of 0.992, 0.985, and 0.986, respectively.

Rapid kinetic measurement of lactate in plasma with a centrifugal analyzer.

In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 mul of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.