Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions.

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the L-arginine/NO pathway in health and disease.

Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid L-arginine by the action of nitric oxide synthases (NOS) in what is called the L-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, L-arginine and its simultaneously generated product, L-citrulline. Considering only a fraction of the endogenous L-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of L-arginine and/or L-citrulline alone. Instead, to estimate NO-related changes in the L-arginine and/or L-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of L-arginine to NO and L-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.

Plasma protein synthesis measurements using a proteomics strategy.

The analysis of the synthesis of proteins has been the subject of many studies in animals and humans. Plasma proteins can be used as an easy accessible source of specific proteins. In this paper, an innovative method to study the synthetic rate of plasma proteins is described. This methodology, based on the proteomics approach, enables the direct observation of the effects of posttranslational modifications of protein synthesis and/or degradation. The methodology is based on 1D or 2D electrophoresis and subsequent electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS). Protein synthesis is measured in isotopically labeled peptides of the identified proteins. This innovative method can be used to assess amino acid adequacy and safety by studying protein synthesis and posttranslational modification of plasma proteins in more detail.