Date of Award
Chemistry and Biochemistry
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Part I. A new fluorometric, coupled, enzymatic method for the determination of adenosine triphosphate is described. The procedure uses the hexokinase reaction coupled with three other enzymes in a system of phosphorylation and double reduction to form NADPH. The NADPH is measured in a reaction catalyzed by diaphorase in which the nonfluorescent material, resazurin, is converted to the highly fluorescent compound, resorufin. This kinetic method can determine adenosine triphosphate at the picomole level. Intra- and inter-assay CV's were both less than 3% and recoveries were quantitative. No significant interference was observed from EDTA or heparin. Correlation with an established method was significant (r = 0.99). Adenosine triphosphate was measured in platelets. Mean ATP levels in the platelets of "normal" subjects were found to be 2.17 (+OR-) 0.76 nmol/10('8) platelets (14.22 (+OR-) 6.32 nmol/mg protein). Part II. A new procedure was developed for the determination of non-enzymatically glycated albumin (GA). Albumin was separated from serum or plasma using Sepharose-blue dextran affinity chromatography. The "fructosamine assay" was improved and used to determine GA. The stable ketoamine linkage in GA reduced 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide (MTT) to a colored formazan. Optimum conditions for the assay were established using the simplex optimization technique. The reagent blank value was minimal and MTT had a 3-fold greater molar absorptivity than nitroblue tetrazolium used in the original "fructosamine assay". The assay was adapted for use in a centrifugal analyzer (Flexigem('tm)) and GA was used as the standard. The within and between run CV's were 4.6% and 8.5%, respectively. Recovery of GA was quantitative. The reference range for this method was 8.94 - 11.19% GA and normal and diabetic populations can be clearly discriminated (p < 0.005). Values obtained with this method correlate well with a thiobarbituric acid assay (r = 0.974) but not with those for glycated hemoglobin (r = 0.350). Source: Dissertation Abstracts International, Volume: 46-08, Section: B, page: 2649. Thesis (Ph.D.)--University of Windsor (Canada), 1985.
CAINES, PATRICK STEPHEN MICHAEL., "I. A FLUOROMETRIC COUPLED ENZYMATIC METHOD FOR THE DETERMINATION OF ADENOSINE TRIPHOSPHATE IN PLATELETS. II. COLORIMETRIC DETERMINATION OF NON-ENZYMATICALLY GLYCATED ALBUMIN." (1985). Electronic Theses and Dissertations. 2349.