Improved measurement of protein synthesis in human subjects using 2H-phenylalanine isotopomers and gas chromatography/mass spectrometry.
Sensitive methods to measure protein synthetic rate in vivo are required to assess changes in protein expression, especially when comparing healthy with infirm subjects. We have previously applied a 'flooding dose' procedure using (2)H(5)-phenylalanine ((2)H(5)-phe) and (2)H(8)-phe isotopomers as tracers, which has proven successful in measuring albumin and fibrinogen synthesis in response to feeding in cancer patients. Using tert-butyldimethylsilyl derivatives, we have observed that (2)H(7)-phe is formed with time in vivo from (2)H(8)-phe, probably during transamination. This increases errors when estimating the fractional synthetic rate (FSR) using the (2)H(8)-phe isotopomer compared with the (2)H(5)-phe isotopomer. We sought to improve this situation by use of an alternative derivative that overcomes this problem whilst also streamlining sample preparation. When using N-ethoxycarbonyltrifluoroethyl (ECTFE) amino acid esters, (2)H(8)-phe is effectively converted into (2)H(7)-phe through fragmentation under electron ionisation (EI), allowing both (2)H(8)-phe and (2)H(7)-phe isotopomers to be measured as a single intense C(7)(2)H(7)(+) fragment at 98 Th. To illustrate the improved situation, the mean RMS residual was calculated for all albumin data, for each isotopomer and for each derivative. Albumin-bound Phe was analysed as ECTFE-phe with improved precision, independent of the isotopomer used, confirming that the new derivative is superior.
Preston
Tom
T
Stable Isotope Biochemistry Laboratory, SUERC, Rankine Avenue, Scottish Enterprise Technology Park, East Kilbride, G75 0QF, UK. T.Preston@suerc.gla.ac.uk
Small
Alexandra C
AC
D000418:Albumins / Q000737:chemistry / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D003903:Deuterium / Q000008:administration & dosage / Q000737:chemistry*; D004952:Esters / Q000737:chemistry; D005466:Fluorocarbons / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D017646:Organosilicon Compounds / Q000737:chemistry; D010190:Pancreatic Neoplasms / Q000097:blood / Q000378:metabolism; D010649:Phenylalanine / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D000418:Albumins; D000596:Amino Acids; D004952:Esters; D005466:Fluorocarbons; D017646:Organosilicon Compounds; D011506:Proteins; C009660:t-butyldimethylsilyl compounds; D010649:Phenylalanine; D003903:Deuterium