An automated procedure for measuring biotinidase activity in serum

In this automated procedure for quantifying biotinidase activity in human serum, a manual colorimetric method that measures conversion of the enzyme's artificial substrate N-biotinyl p-aminobenzoate was modified for use with a Technicon AutoAnalyzer II. The intra-run replicate precision (CV) was 2.1% and the day-to-day CV was 4.6% for quality-control sera. Results were linearly related to biotinidase activity in serum over the complete range of clinically relevant values, 0.2 to 11.0 U/L. Moreover, results of the automated assay were not significantly different from those of the manual assay. Because the automated procedure is faster and more precise, we recommend it for population-based studies and some screening studies.     

An automated, direct method for measuring adipocyte cell size.

Current methods for direct determination of fat cell size are optical and non-automated. They are thus tedious, but have the advantage of providing estimates of the variance of a cell size distribution, as well as a measure of the mean cell size. An indirect method based on counting the number of osmium-fixed fat cells derived from a known wet weight of adipose tissue is also available. This method is automated and thus rapid, but does not provide information about the variance of the cell size distribution. In the present paper we describe a direct, automated method of fat cell sizing that provides estimates for both mean cell size and the variance of the cell size distribution. The correlation between our method and in indirect method based on counting osmium-fixed cells was 0.96. Transformation of the volume distribution to diameters indicated that cell diameters appeared to be normally distributed, confirming the observations of others.     

An automated computerized method using Finapres for measuring cardiovascular reflexes.

1. The major drawback of the cardiovascular reflex tests used to study autonomic failure is the time involved in calculating the results. To overcome this disadvantage, we have developed an automated computerized program using a FINger Arterial PRESsure instrument for the measurement of beat-to-beat heart rate and blood pressure on a finger. 2. This program calculates heart rate variability during three standardized tests, forced breathing, standing up and the Valsalva manoeuvre, and records blood pressure values in response to standing up and sustained handgrip. The time taken to perform the test and to calculate the results is usually 25 min. 3. The reproducibility of the tests in 21 normal subjects was comparable with the reproducibility obtained with conventional test methods using an ECG and a sphygmomanometer. 4. In addition, we determined the age-dependent normal values of the seven test parameters in 124 subjects aged 20-90 years. 5. Using this program in 10 patients with longstanding (14-50 years) complicated diabetes, in each of them four or more abnormal test results were found.     

Screening for plasma cholinesterase deficiency: an automated succinylcholine based assay

We describe an automated kinetic method that uses a single aqueous reagent to measure the in vitro hydrolysis of the muscle relaxant succinylcholine. The substrate succinylcholine is hydrolyzed by plasma cholinesterase (EC 3.1.1.8), and the choline produced is oxidized by choline oxidase (EC 11.3.17) in the presence of peroxidase, 4-aminophenazone and phenol, to yield a chromagen with maximum absorbance at 500 nm. The method is reproducible (CV 1.3%), correlates well with a manual procedure using the same substrate (r = 0.994, y = 0.99x - 0.25), and is linear to 150 U/L. The method is well suited to pre-operative screening and detection of "at-risk" individuals, as illustrated by the family of one patient who had a prolonged succinylcholine apnea.     

An automated colorimetric method for the estimation of lactate dehydrogenase activity in serum

An automated method for estimating lactate dehydrogenase (LDH) in serum is presented. Fe3+ is reduced to Fez+ by NADH formed when lactate is oxidized to pyruvate. Fe2+ complexed with 2,2-bipyridyl is measured colorimetrically. Solutions of Fe2+ salt are used as standards. Nicotinamide is used to stabilize NAD+ in solution and is shown to enhance enzyme activity. The method is less expensive than most automated methods published and reagents are stable for at least 4 weeks. Results correlate well with a kinetic spectrophotometric method.     

An automated method for lysozyme assay

An automated method for the estimation of lysozyme concentration in serum and urine is described. The method has proved to be satisfactory in routine use when compared with a manual method.     

A new automated colorimetric method for measuring total oxidant status

OBJECTIVES: To develop a new, colorimetric and automated method for measuring total oxidation status (TOS). DESIGN AND METHODS: The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined. RESULTS: There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r=0.99, P<0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 micromol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 micromol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23+/-3.11 micromol H2O2 Equiv./L) than in healthy subjects (14.19+/-3.16 micromol H2O2 Equiv./L, P<0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r=-0.66 P<0.01). CONCLUSIONS: This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.     

Test-strip method for measuring lactate in whole blood

We have developed a dry reagent strip system for measuring lactate in whole blood. The test strip contains lactate oxidase (no EC number assigned), horseradish peroxidase (EC 1.11.1.7), and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine. The system is designed to measure with a reflectometer the color that developed in the test strip, although the lactate concentration can be estimated without the reflectometer. The between-run coefficients of variation for controls at three concentrations were 2.9-5.3%. The lactate concentrations in blood samples from healthy subjects before and after exercise correlated well (r = 0.97) with the results measured by the comparison method with the use of lactate oxidase. This dry reagent strip system provides a convenient and rapid test for measuring blood lactate in clinical and sports medicine.