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Amino Acid and Thiol Redox Analysis in Human Plasma and Red Blood Cells (AAA/GSH)

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Aminoacids, the building blocks of proteins, play important roles in many aspects of physiology. Arginine is used by nitric oxide synthase to generate nitric oxide (NO), a compound essential for normal blood flow. In the generation of NO from arginine, Citruline is formed. Arginase, the enzyme that breaks down arginine to ornithine is released from red blood cells when they hemolyze and limits the availability of arginine for nitric oxide synthase, and leads to the formation of downstream products such as proline. Increase of arginase in the circulation correlates with measures of hemolysis, cell free hemoglobin and LDH. Oxidative stress plays an important role in the the hemoglobinopathies and thiol amino acids are important antioxidants. Sulfur-containing antioxidants, such as glutathione, cysteine, and methionine (aminothiols) are the first line of defense against the insidious rise in oxidants. The imbalance between oxidative and antioxidative forces is reflected by the low ratios of reduced and oxidized aminothiols. Major cellular aminothiols are methionine, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, cysteine, glutathione, and cysteinylglycine. These amino acids are metabolically linked via the transmethylation, transsulfuration and glutathione (GSH) synthesis pathways.  Measuring these parameters will provide a sensitive and comprehensive means to detect potential increases in oxidative stress in patients.

Our aminoacid panels measure aminoacid composition in plasma and red cells and include both the Thiol amino acid panel and the Arginine metabolite panel.

The comprehensive analysis of the complete array of thiol amino acids and the arginine metabolites in plasma or peripheral blood cells sensitively measures amino acid concentration as determined by liquid chromatography and tandem mass-spectrometry: LC/MS/MS detection (Suh, et. al. 2009). A small aliquot (0.1 ml) will be used to quantitate levels of these amino acids using an alkylchloroformate derivatization technique. A Micromass Quattro LCZ atmospheric pressure ESI-LC/MS will be employed.

1. Suh, J. H., Kim, R., Yavuz, B., Lee, D., Lal, A., Ames, B. N., and Shigenaga, M. K. (2009) Clinical assay of four thiol amino acid redox couples by LC-MS/MS: Utility in thalassemia. J Chromatogr B Analyt Technol Biomed Life Sci 877, 3418-3427
Analytes included are:

Thiol amino acid panel:                                    Arginine metabolite panel:
            Methionine                                                Arginine
            Methionine sulfoxide                                   Citrulline
            S-adenosylmethionine                                 Ornithine
            S-adenosylhomocysteine                             Putrescine
            Homocysteine                                            Spermidine
            Homocystine                                              Spermine
            Cystathionine                                             Proline
            Glutathione disulfide          
            Cysteinylglycine disulfide