A step forward in enzymatic measurement of creatinine.

We describe an improved enzymatic ultraviolet absorbance method for assaying creatinine in serum, plasma, and urine. Creatinine is hydrolyzed by creatinine iminohydrolase (EC 3.5.4.21) to ammonia and N-methylhydantoin. The ammonia produced combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase to yield glutamate and NADP+. The consumption of NADPH, measured by a two-point fixed-time assay, is proportional to the amount of creatinine in the sample. The assay is carried out in two steps: The first step eliminates background absorbance in hyperlipemic samples and endogenous ammonia through a "clearing system" and an isocitrate dehydrogenase-based "ammonia scavenger system"; the second step starts creatinine measurement. The method affords a simple, rapid, and sensitive assay with good precision and extended linearity; it employs working solutions stable at least 4 months. Test results compare closely with those of the isotope dilution-mass spectrometry Definitive Method, the HPLC procedure, and the fuller's earth method. The proposed method is not subject to interference from several metabolites or from the 72 drugs tested. Because it is easily automated, the method is suitable for routine work in clinical laboratories.

Clinical validity of a continuous colorimetric method for serum lipase.

The clinical validity of a continuous colorimetric method for measuring pancreatic lipase was assessed. 1,2-Diacylglycerol containing long-chain fatty acid residues was used as substrate, and the method was adapted to a discrete analyser. The dynamic range was ascertained up to at least 30-fold the upper reference limit. Precision tests on three control sera yielded overall CVs of 4.6% (mean value 21 U/l), 2.4% (115 U/l), and 1.0% (386 U/l), respectively. Using serum samples from normal subjects and patients with pancreatic and non-pancreatic disorders, the present method was compared with a turbidimetric method (r = 0.997; n = 281) and a homogeneous enzyme immunoassay (r = 0.987; n = 93). The reference interval established on 121 healthy subjects was 8-57 U/l (central 95th percentile, median 22 U/l). The sensitivity of this lipase assay in the diagnosis of acute pancreatitis (100%, median 5.6-fold the upper reference limit) was equal to that of the pancreatic isoamylase assay, and higher than that of the total alpha-amylase assay (88.2%); the specificity for acute pancreatitis with respect to a group of patients with acute and chronic non-pancreatic abdominal diseases (91%) was higher than that of both pancreatic isoamylase (76%) and total alpha-amylase (71%).

An enzyme-labeled immunometric assay for quantitation of digoxin in serum or plasma.

A heterogeneous enzyme-labeled immunometric assay for quantifying digoxin in serum or plasma was developed. The sample's drug reacts with an excess of an antidigoxin Fab'-beta-galactosidase monoconjugate and then the free monoconjugate is removed by polyacrylamide digitoxigenin-coupled beads; the beta-galactosidase activity of the supernatant measured photometrically at 634 nm is directly proportional to the digoxin concentration in the sample. The assay shares the basic reagents for the immunological reaction with the Seralyzer dry-strip immunometric assay; the reagents for the indicator enzyme reaction were instead formulated in liquid form for use with common clinical analyzers. The test requires a two-point calibration (a 3.0 micrograms/L digoxin calibrator and a reagent blank). One sample can be assayed in approximately 20 min; the assay range is from 0.3 to 5.0 micrograms/L. Dilution tests showed an average found/expected ratio of 100.6%. Mean analytical recovery was 99.2%. Overall coefficients of variation (CVs) (three replicates for 12 runs over 15 days; daily calibration) ranged from 4.9 to 10.0% (Technicon RA-50) and from 2.5 to 10.0% (Cobas Fara) for samples with digoxin concentrations of 4.7 to 0.5 micrograms/L. No interference was found by high levels of common analytes (bilirubin, triglycerides, hemoglobin, total protein, uric acid) or anticoagulants. Cross-reactivity by digoxin metabolites and structurally-related compounds was investigated. Good correlations were found with radiommunoassay (RIA) (r = 0.986) and enzyme immunoassay (EIA) (r = 0.994). Thus, the assay is a specific, reliable, and convenient method for measuring digoxin in both small and large laboratories.

Kinetic colorimetric assay of lipase in serum.

We describe a kinetic colorimetric method for assaying lipase (EC 3.1.1.3) activity in serum by using a natural long-chain fatty acid 1,2-diglyceride. In the presence of colipase, deoxycholate, and calcium ions, pancreatic lipase hydrolyzes the clear substrate solution to produce a 2-monoglyceride, which in turn releases glycerol by the action of a 2-monoglyceride lipase. Glycerol is then assayed by a sequence of enzymatic actions (glycerol kinase, glycerol phosphate oxidase, and peroxidase) that produce a violet quinone monoimine dye with peak absorption at 550 nm. The method features zero-order reaction kinetics, provides a simple and rapid assay with an extended dynamic range, is specific and precise, gives results that correlate well (r greater than or equal to 0.99) with those of methods in which emulsified triolein is the substrate, and lends itself readily to automation. For all these reasons, the method seems highly suitable for routine use in clinical laboratories.

Immunoturbidimetric method for routine determinations of apolipoproteins A-I and B.

A simple immunoturbidimetric method for quantifying apolipoproteins (apo) A-I and B in serum or plasma is described. A special reagent formulation, including large amounts of suitable detergents, obviates the need for a sample blank even with grossly lipemic specimens. The assay is rapid, easily automated, and thus convenient for routine work. For both apo A-I and apo B, the assay range is about 0.2-3.5 g/L. The performance characteristics were assessed with discrete (Optimate and Olli CD) and centrifugal analyzers (Cobas Fara and IL Monarch 2000). Average analytical recovery was 101.5% for apo A-I and 99.4% for apo B. Dilution tests showed found/expected ratios of 101.2% (apo A-I) and 101.0% (apo B). Overall precision (CV) ranged from 1.4% to 3.3% for apo A-I and from 1.1% to 8.3% for apo B. Comparisons with commercially available rate nephelometry, radial immunodiffusion, and immunoturbidimetric methods gave good correlations (r greater than or equal to 0.938). Using the immunoturbidimetric method, we also established the relationships between apolipoproteins and lipids and determined the reference intervals. We conclude that the proposed method is suitable for routine use in clinical laboratories.

A simple immunoturbidimetric method for IgG and albumin quantitation in cerebrospinal fluid and serum.

We describe a simple immunoturbidimetric method for measuring both IgG and albumin in CSF and serum, which enables the calculation of CSF indices. For each protein, only one calibration curve is used for both CSF and serum samples. The assay protocol is simple and similar for both tests. Sensitivity and versatility of the method afford measurements over a very wide range of concentrations (approx. 0.007 to 94 g/l for IgG and 0.06 to 92.40 g/l for albumin). Precision studies (triplicates for 6 runs over 15 days) gave overall CVs: less than or equal to 2.9 and 4.9% for IgG in CSF (11.5 mg/l) and serum (10.28 g/l); less than or equal to 1.3 and 1.1% for albumin in CSF (115 mg/l) and serum (76.89 g/l). Comparison studies showed good correlation with radial immuno-diffusion (r greater than or equal to 0.995 and 0.976 for IgG and albumin) and rate nephelometry (r greater than or equal to 0.967 and 0.982 for IgG and albumin). Thus, the method under investigation proved to be reliable and appears to be particularly suitable for the routine work.

Enzymatic assay of magnesium through glucokinase activation.

We describe an improved enzymatic method for assaying magnesium in serum, plasma, or urine. Magnesium participates as an Mg.ATP complex in a reaction catalyzed by glucokinase (EC 2.7.1.2) coupled to an NADP+-dependent glucose-6-phosphate dehydrogenase (EC 1.1.1.49) reaction. The increase of absorbance at 340 nm, due to the NADPH produced, is proportional to the amount of the activated glucokinase, which in turn is related to the concentration of magnesium in the sample. The method is characterized by a zero-order reaction kinetics, affording a simple and rapid assay with good sensitivity and linearity (up to 2.06 mmol/L) and by working solutions that are stable (refrigerated) for one month. The method is reliable, produces test results that compare closely with those of the atomic absorption spectrophotometry (r greater than or equal to 0.99), is suitable for routine work, and lends itself to automation.

One-step protocol for assays of total and direct bilirubin with stable combined reagents.

We describe an improved colorimetric method for assays of total and direct bilirubin in serum. Bilirubin reacts with diazotized sulfanilic acid in an acidic medium to form a blue azopigment. Total bilirubin is assayed in the presence of reaction accelerators (caffeine, urea, and citric acid), direct bilirubin in their absence. The azo compound so formed is read at the same wavelength (570 nm) in both assays. A sample blank is run in parallel. Standard curves are linear for total and direct bilirubin concentrations up to 513.0 and 256.5 mumol/L, respectively. The method is characterized by (a) use of the same protocol for both assays, i.e., a one-step procedure with short reaction time (5 min at room temperature), and (b) use of a single working solution, which, refrigerated, is stable for one month. The method is reliable, yields results that compare closely with those of the classical Jendrassik--Gróf method, is suitable for routine use, and lends itself to automation.

Enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine.

The performance of an enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine is described. Phosphate ions react with inosine in the presence of purine nucleoside phosphorylase to form hypoxanthine; this is oxidized by xanthine oxidase to uric acid with production of hydrogen peroxide. The latter is determined with the aid of the chromogen system peroxidase/4-aminophenazone/N-ethyl-N-(3-methylphenyl)-N'-acetylethyl enediamine , the coloured product being measured at 555 nm. This series of reactions is completed in 5 min at 37 degrees C. The test is linear up to 240 mg/l. Analytical recovery in serum averaged 101.2 +/- 1.2% and in urine 101.9 +/- 3.2%. Within-run and between-run precision studies in serum and urine samples gave CVs less than or equal to 4.54% (at 22.0 mg/l). Results obtained by this method agree (r = greater than or equal to 0.983) with the molybdate UV and molybdenum blue methods. Interference by endogenous substances, including organic phosphate, was negligible.

A dry-reagent strip for quantifying carbamazepine evaluated.

We examined a new colorimetric homogeneous immunoassay for carbamazepine based on the apoenzyme reactivation immunoassay system (ARIS) principle. The test, in dry-reagent strip format, is to be used with the Ames Seralyzer reflectance photometer. Within-run CVs (n = 20) were 3.0%, 2.7%, and 2.8% at 3.0, 6.1, and 12.1 mg/L; between-run CVs (n = 15, in 15 days) were 4.1%, 2.7%, and 1.9% at 6.0, 9.1, and 12.1 mg/L. Mean analytical recovery was 99.9 (SD 2.3)%. Results by this test (y) for clinical plasma specimens (n = 96) compared very well with those obtained by fluorescence polarization immunoassay (y = 1.01 x - 0.02, r = 0.995) and by liquid chromatography (y = 0.99 x + 0.14, r = 0.990). Bilirubin (45 mg/L), uric acid (145 mg/L), and various anticoagulants at about fourfold the usual concentrations did not interfere. High concentrations of cholesterol (4.9 g/L), triglycerides (3.8 g/L), and hemoglobin (4 g/L) caused slight positive interference. Carbamazepine-10,11-epoxide cross reacted only at greater than or equal to 5 mg/L. The two-point calibration line was validly stored for at least three weeks. Free carbamazepine also can be measured. The test is convenient and rapid (test time 80 s), and thus is particularly useful in all clinical settings where prompt testing is needed.

Quantitative determination of phenobarbital and phenytoin by dry-phase apoenzyme reactivation immunoassay system (ARIS).

We assessed the performance of the apoenzyme reactivation immunoassay system (ARIS) reagent strip tests for determination of phenobarbital (PB) and phenytoin (PHT) with the Seralyzer reflectance photometer. In the assay, the drug of the sample competes with a flavine adenine dinucleotide (FAD)-drug conjugate for binding to a specific antibody; the unbound conjugate then activates apoglucose oxidase to reconstitute glucose oxidase, whose activity is kinetically monitored by a coupled chromogenic reaction. Within-run coefficients of variation (CVs) were less than or equal to 5.0% for PB and less than or equal to 5.6% for PHT; between-run CVs were less than or equal to 6.1% for PB and less than or equal to 6.5% for PHT. Mean analytical recoveries were 100.3% for PB and 100.2% for PHT. Test results were not significantly affected by bilirubin (5 mg/dL), hemoglobin (25 mg/dL), triglycerides (500 mg/dL), uric acid (15 mg/dL), or elevated levels of other antiepileptic drugs. Reagent strip tests correlated very well with substrate-labeled fluorescent immunoassay (r = 0.9923 and 0.9944 for PB and PHT, respectively), enzyme multiplied immunoassay technique (r = 0.9941 and 0.9919), and gas-liquid chromatography (r = 0.9980 and 0.9960). These homogeneous competitive colorimetric immunoassays are particularly suitable for emergency use, for testing small batches of samples, wherever prompt results are needed.

A kinetic colorimetric assay of gamma-glutamyltransferase.

We have explored a kinetic colorimetric method for measuring gamma-glutamyltransferase (EC 2.3.2.2) activity in serum, using L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide and glycylglycine as donor and acceptor substrates. The released product, 3,5-dibromo-4-hydroxyaniline, reacts with 2,5-dimethylphenol to produce a blue quinone monoimine in the presence of ascorbate oxidase (EC 1.10.3.3). This dye has peak absorption at 610 nm, whereas the donor substrate shows negligible absorption throughout the visible spectrum. The reaction can be run with all the reagents in a single working solution with serum as starter, or with the substrate solution as starting reagent. The sample/reagent volume ratio is 1:24. Adaptation of the method to several automated instruments gave good precision in all cases. Comparison with a method in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the donor substrate showed good correlation of results (r greater than or equal to 0.987). The dynamic range of the method exceeds the upper limits of the reference intervals for men (9-33 U/L) and women (8-25 U/L) by at least 18-fold.