Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver

San Ching, Donald C. Mahan, Regis F Moreau, Konrad Dabrowski

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Modifications of the analytical method to determine L-gulono-γ-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-γ-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-γ-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-γ-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-γ-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-γ-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-γ-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.

Original languageEnglish (US)
Pages (from-to)139-146
Number of pages8
JournalJournal of Nutritional Biochemistry
Volume14
Issue number3
DOIs
StatePublished - Mar 1 2003

Fingerprint

L-Gulonolactone Oxidase
Enzyme activity
Lactones
Liver
Ascorbic Acid
Swine
Oxidoreductases
Enzymes
Tissue
Buffers
Weights and Measures
Glutathione
Dehydroascorbic Acid
Ice
Liquid nitrogen
Substrates
Nitrogen
Sampling
Cooling

Keywords

  • Ascorbic acid
  • L-gulono-γ-lactone oxidase
  • Pigs
  • Vitamin C

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Nutrition and Dietetics
  • Clinical Biochemistry

Cite this

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver. / Ching, San; Mahan, Donald C.; Moreau, Regis F; Dabrowski, Konrad.

In: Journal of Nutritional Biochemistry, Vol. 14, No. 3, 01.03.2003, p. 139-146.

Research output: Contribution to journalArticle

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