Biomedical Sciences ETDs

Publication Date

5-1-1979

Abstract

An individual's thiamine status can be assessed by the measurement of erythrocyte transketolase (ETK) activity and a new method for this type of measurement in rat and human whole blood hemolysate samples has been developed and completely automated. This assay is based on enzymatic activity of the hexose monophosphate shunt which involves interconversion of hexoses and pentoses and production of NADPH reducing power. All of the enzymes of the hexose monophosphate shunt (HMS) are present in erythrocytes, including transketolase (TK) which appears to be the rate-Iimiting enzyme in this reaction series. Reaction conditions are established to ensure a direct relationship between ETK activity and NADPH produced in the HMS. The NADPH reduces oxidized gluthathione (GSSG) to produce two moles of reduced gluthathione (GSH) which in turn reduces DTNB (5,5 dithiobis-2-nitrobenzoic acid) to a measurable chromogen TNB (5-thio-2-nitrobenzoic acid). Two factors increase the sensitivity of the method; the production of two moles of TNB per mole of NADPH and the high molar absorptivity coefficient of TNB at 410 nm. An experimental rat feeding study was performed to determine the affect of dietary thiamine intake on ETK activity and to test the ability of this system to measure changes in ETK activity. ETK activity is measured in both the presence and absence of exogenous thiamine pyrophosphate (TPP) to determine the degree of in vitro stimulation of TK by its coenzyme. The results of this study indicate that this new approach provides a sensitive, reproducible and technically feasible method for measurement of ETK activity.

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Masters

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Philip J. Garry

Second Committee Member

Jimmy Clayton Standefer

Third Committee Member

Leslie Frank Smith

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