Amino acid quantitation in aqueous matrices via trap and release membrane introduction mass spectrometry: homocysteine in human plasma.

Trap and release membrane introduction mass spectrometry (T&R-MIMS) using a removable direct insertion membrane probe (DIMP) is employed to determine the total homocysteine concentration (tHcy) directly from human plasma after derivatization with ethyl chloroformate. The method uses no chromatographic separation, is linear, reproducible, and displays limit of quantitation (2 pM) sufficiently below the threshold concentration of tHcy in plasma. It also combines chemical, membrane, and mass spectrometric discrimination, and can be used to determine selected amino acids in human plasma simultaneously. After derivatization with ethyl chloroformate, many amino acids in aqueous solution are observed to be efficiently detected; hence T&R-MIMS is promising as a simple and sensitive technique for simultaneous quantitation of selected amino acids in plasma and urine, and in other aqueous matrices.

Development of an amperometric biosensor based on glutathione peroxidase immobilized in a carbodiimide matrix for the analysis of reduced glutathione from serum.

The development of an amperometric biosensor for the reduced glutathione determination in serum is described. The biosensor is based on glutathione peroxidase (GSH-Px, EC 1.11.1.9) immobilized onto a pyrolytic graphite-working electrode using carbodiimide as enzymatic condensing reagent. This resulted in an amperometric biosensor with good sensitivity and stability. The reduced glutathione (GSH) was enzymatically converted to glutathione disulfide (GSSG) in the presence of hydroperoxide, which was monitored amperometrically by its electrooxidation at +0.65 V vs. SCE (saturated calomel electrode). Glutathione measurement was carried out by maintaining the ratio between GSH and hydrogen peroxide at 2:1 (25 degrees C). The amperometric response of the biosensor was linearly proportional to the GSH concentration between 1.9x10(-5) and 1.4x10(-4) mol/l, in 0.1 mol/l phosphate buffer (pH=7.8), containing 0.1 mol/l KCl and 0.5 mmol/l Na(2)H(2)EDTA, as the supporting electrolyte. In presence of interfering compounds, the recoveries ranged between 97.2% and 110.7%. The biosensor useful lifetime was at least 2 months when it was evaluated after continuous use. Serum samples analyzed by this biosensor showed a good correlation with the results from the spectrophotometric method (Ellman's reagent) used as reference, presenting relative deviations lower than 7.0%. The low apparent Michaelis-Menten constant value, K(M)(app)=1.6 mmol/l, demonstrated that GSH-Px immobilized on pyrolytic graphite exhibited a high affinity to GSH, without loss of enzymatic activity.

Defective neutrophil function in workers occupationally exposed to lead.

Chemotaxis and nitroblue tetrazolium reduction were measured in peripheral blood neutrophils of workers occupationally exposed to lead. These two neutrophil functions were significantly reduced, as compared to controls, even in those workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations below the currently accepted biological limit values of 60 micrograms/dl and 6 mg/l, respectively. The immunosuppressive effects of relatively low level lead absorption suggests that immune dysfunction may be a sensitive indicator of lead exposure.