Gas chromatography/mass spectrometry analyses of [2,3,3-d3] serine, [2,3,3-d3] cysteine and [3-13 C] cysteine in plasma and skin protein: measurement of transsulphuration in young sheep.
A new procedure using stable isotope labelled serine (L-[2,3,3-d3] serine) and cysteine (L[13-3-13 C] cysteine) and analysis by gas chromatography/mass spectrometry (GC/MS) has been developed to measure transsulphuration in sheep. The enrichments of the tracers in plasma and skin biopsy samples were measured by GC/electron impact MS analysis of the t-butyldimethylsilyl derivatives. The measured recoveries of the standards enriched with [3-13 C] cysteine from 0.1% to 8%, or with [2,3,3-d3] serine from 0.14% to 14% were greater than 99% of the theoretical values, and the variation coefficients were less than 3% when the enrichment was higher than 0.5%. The use of dithiothreitold (DTT) as a reducing agent before deproteinization of the sample and during the derivatizations successfully increased the cysteine peak area and significantly improved reproducibility in the analysis. The cysteine residues in protein from the skin biopsy were also during the protein hydrolysis with DTT in 6 n HCI. The method was applied to measure transsulphuration of methionine in young sheep. The amount of cysteine derived from transsulphuration accounted for 17% to 21% of the irreversible loss rate of cysteine, depending on the substrate supplies. The results are consistent with other reports. Compared with conventional methods of measuring transsulphuration using radioactive isotopes, the processes of animal experimentation was sample analysis were simple, and there were no radiation hazards. The method should prove useful in studies on the metabolism of methionine and cysteine in human and animals.
Liu
S M
SM
CSIRO Animal Production and Cooperative Research Centre for Premium Quality Wool, Wembley, Australia.
Figliomeni
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D000603:Amino Acids, Sulfur / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000032:analysis*; D003545:Cysteine / Q000032:analysis* / Q000097:blood; D004032:Diet; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D007202:Indicators and Reagents; D011506:Proteins / Q000032:analysis*; D012694:Serine / Q000032:analysis* / Q000097:blood; D012756:Sheep; D012867:Skin / Q000737:chemistry*
D000603:Amino Acids, Sulfur; D001798:Blood Proteins; D007202:Indicators and Reagents; D011506:Proteins; D012694:Serine; D003545:Cysteine; D004229:Dithiothreitol