Glycosylated hemoglobin by colorimetry in normal subjects, diabetics, and nondiabetics.
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Hemoglobin in plasma can be determined by the color-developing action of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid), which is oxidized to a colored form by a peroxidase-like effect of hemoglobin in the presence of hydrogen peroxide. Sensitivity, precision, and accuracy are discussed. The calibration curve is linear for hemoglobin concentrations up to 1 g/L; the minimum detectable concentration is 20 mg/L. The within-run precision (CV) was 2.39%, analytical recovery 101.8%. Interference from plasma proteins and lipids was eliminated by centrifuging the reaction mixture before measuring its absorbance at 410 nm.
A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid.
We describe a procedure for assay of diaphorase activity in commercial purified preparations and in clinical chemical reagents by use of iodonitrotetrazolium chloride or other tetrazolium salts. The method is based on measurement of the formazan produced by enzymic reduction of tetrazolium salts in the presence of NADH. The assay procedure has been optimized for linear kinetics, simplicity of operation, nondetectable blank rates, and extended activity/enzyme concentration proportionality. The proposed method has several advantages over the older assay by use of dichlorophenolindophenol.
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A popular colorimetric analysis for serum acetaminophen, based on ring-nitration of the drug, is demonstrated to yield both "false positives" and erroneously high results for serum from uremic patients. The interference appears to be anionic at physiological pH, and correlates significantly with serum creatine concentration and with the magnitude of the "anion gap." A modification of the analysis involving extraction of the acetaminophen with ether eliminates the interference. As little as 10 mg of drug per liter can be accurately measured in serum by the proposed procedure. Analytical recovery was 97% at a concentration of 1 g/L. Coefficients of variation for the analysis at respective concentrations of 100 and 500 mg/L were: within-run, 2.7% and 2.0%; between-run, 3.1% and 4.2%. I encountered no serious interferences from other drugs. The proposed method, rapid and reliable, is recommended for routine use in the clinical laboratory.
We describe the application to the Technicon SMAC system of a micellar-improved calmagite method for the measurement of magnesium. In this continuous-flow method a dialyzer is not needed, because protein interference is negligible. Sample interaction was estimated to be 3%. The increased sensitivity of the method allows for a small sample volume (37 microL/min). Results obtained agreed well with those by an atomic absorption procedure (x): SMAC magnesium - 0.971 x + 0.030 mmol/L (n = 58). The estimated total error for the SMAC procedure was less than 50 mumol/L at two analyte concentrations: 0.50 and 1.50 mmol/L. The inclusion of magnesium estimation on SMAC should extend the screening function of this analyzer.
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Reportedly, levodopa (L-DOPA) administration produces spuriously high values for plasma uric acid as measured by the commonly used phosphotungstic acid-hydroxylamine colorimetric method. We confirm this interference, not only by L-DOPA but also by three of its major metabolites: dopamine, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. However, at therapeutic concentrations in plasma (less than 5 mg/L), the maximum spurious uric acid concentration due to L-DOPA is less than 2 mg/L. Also, at reported peak plasma concentrations of L-DOPA plus three of its major metabolites, the maximum spurious uric acid concentration due to all four compounds combined is less than 8.5 mg/L. Therefore, the hyperuricemia observed with this method in some patients who are chronically receiving L-DOPA cannot be attributed only to interference by L-DOPA and its metabolites in the colorimetric determination of uric acid. Evidently L-DOPA may increase laboratory values for plasma uric acid concentrations, both by pharmacological and chemical mechanisms.
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We studied conditions affecting a colorimetric assay of total orotic acid (orotic acid plus orotidine) in urine. Most interfering substances can be conveniently removed on a small column of cation-exchange resin, and an improved control reaction corrects for residual background color. Analytical recovery from urine is nearly complete (greater than 95%) and the absorption spectrum for analyte eluted from the column closely resembles that for an orotic acid standard. We determined reference intervals for total orotic acid, expressed as a molar ratio to creatinine, for neonates, children, and adults, and assessed the effect of age, protein intake, and pregnancy. The method is simple enough to use as a reliable and accurate urine-screening test.
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