Improved measurement of protein synthesis in human subjects using 2H‐phenylalanine isotopomers and gas chromatography/mass spectrometry

Abstract

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.