We describe an automated, colorimetric determination of glucose in biological fluids that combines the specificity of glucose oxidase and of a new peroxide indicator reaction. In the presence of peroxidase, 3-methyl-2-benzothiazolinone hydrazone oxidatively couples with N,N-dimethylaniline to form a stable, intensely colored, water-soluble indamine dye, the concentration of which is proportional to that of the third reactant, hydrogen peroxide. This reaction, used earlier to determine uric acid [Clin. Chem. 17, 1154 (1971)], is substantially less affected by negative interference of reducing substances than are previously described peroxide indicators. Results from use of AutoAnalyzers I and II and this method were compared with those from a manual spectrophotometric hexokinase/glucose-6-phosphate dehydrogenase procedure, and showed good correlation for specimens from patients. The automated methods are suitable for measuring glucose in serum, plasma from fluoride-or iodoacetate-preserved blood, urine (without ion-exchange pretreatment), or cerebrospinal fluid. They eliminate the problem of falsely high results caused by medication or reducing metabolites associated with uremia, in methods in which alkaline ferricyanide or copper—neocuproine is used.
We describe a continuous-flow, automated determination of total cholesterol in serum, which is based on enzymatic hydrolysis of cholesterol esters, oxidation of cholesterol by cholesterol oxidase, and colorimetric measurement of liberated perioxide with 4-aminoantipyrine, phenol, and peroxidase. Free cholesterol is determined with the same AutoAnalyzer II manifold and reagents, except that cholesterol esterase is omitted from the reagent. Cholesterol-in-serum materials that have been assayed by an established method are used for calibration. We found this approach to be necessary because primary cholesterol standards in organic solvents are incompatible with the aqueous reagent. Results of the enzymatic total cholesterol method correlated well with those by an AutoAnalyzer II method which involves an extraction with isopropanol and the Liebermann-Burchard color reaction (total cholesterol, g/liter, yenz= 0991xlb +0.05;r=0.996). Results of the enzymatic free cholesterol procedure agreed satisfactorily with one in which free cholesterol is precipitated as the digitonide and subsequently analyzed colorimetrically with the Liebermann-Burchard reaction (free cholesterol, %, yenz = 0.982xdig -0.7;r= 0.956).
An automated colorimetric procedure is described for the determination of serum and urine uric acid with improved specificity. Hydrogen peroxide, formed when uric acid is oxidized by uricase, oxidatively couples with 3-methyl-2-benzothiazolinone hydrazone and N,N-dimethylaniline, in the presence of peroxidase, to produce a stable, sensitive indamine dye. The method is reproducible, and the results correlate well with those obtained by a manual spectrophotometric uricase procedure. Also, the automated procedure is resistant to many added reducing substances that cause positive interferences in alkaline phosphotungstate methods.
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