Nuclear magnetic resonance timecourse studies of glyphosate metabolism by microbial soil isolates.

1. Triple Resonance Isotope EDited nmr spectroscopy (TRIED) has been developed to detect and examine minute levels of glyphosate metabolites in microbial soil isolates. Using stable isotopic labelling (13C and 15N), TRIED allows the simultaneous detection of multiple metabolites in crude matrices at submicrogram levels. An improvement over earlier techniques where milligrams are needed, TRIED can detect 500 ng of triply labelled compound in a crude sample (1:14,000 mass ratio) in just hours. 2. TRIED is used here to compare the kinetics and metabolic pathways of glyphosate metabolism by two strains of Ochrobactrum anthropi, LBAA and S5. Both LBAA and S5 appear to metabolize glyphosate primarily via the aminomethylphosphonate (AMPA) pathway, since no detectable levels of glycine or sarcosine are observed in the media or lysates of either microbe. The formation of N-methylAMPA is common to the metabolism of both microorganisms, but N-acetylAMPA is observed only in LBAA. N-methylacetamide is detected predominantly in media and lysates of S5, although some evidence also points to the formation of this metabolite in LBAA. 3. Results are consistent with conventional radioactive tracer studies. TRIED nmr provides more specific structural information complementary to radiolabel methods. Both nmr and radioactivity studies show S5 glyphosate metabolism to be much slower than that of LBAA.

Quantitative 31P nuclear magnetic resonance analysis of metabolite concentrations in Langendorff-perfused rabbit hearts.

The quantitative analysis of the mobile high-energy phosphorus metabolites in isovolumic Langendorff-perfused rabbit hearts has been performed by 31P NMR utilizing rapid pulse repetition to optimize sensitivity. Absolute quantification required reference to an external standard, determination of differential magnetization saturation and resonance peak area integration by Lorentzian lineshape analysis. Traditionally accepted hemodynamic indices (LVDP, dp/dt) and biochemical indices (lactate, pyruvate) of myocardial function were measured concomitantly with all NMR determinations. Hemodynamically and biochemically competent Langendorff-perfused rabbit hearts were found to have intracellular PCr, ATP, GPC, and Pi concentrations of 14.95 +/- 0.25, 8.08 +/- 0.13, 5.20 +/- 0.58 and 2.61 +/- 0.47 mM respectively. Intracellular pH was 7.03 +/- 0.01. Cytosolic ADP concentration was derived from a creatine kinase equilibrium model and determined to be approximately 36 microM. Reduction of perfusate flow from 20 to 2.5 ml/min demonstrated statistically significant decreases in PCr, ATP, and pH as well as an increase in Pi that correlated closely with the independent hemodynamic and biochemical indices of myocardial function. The decrease in ATP and PCr concentrations precisely matched the increase in Pi during reduced flow. These results constitute the first quantitative determination of intracellular metabolite concentrations by 31P NMR in intact rabbit myocardium under physiologic and low flow conditions.

Determination of intact-tissue glycerophosphorylcholine levels by quantitative 31P nuclear magnetic resonance spectroscopy and correlation with spectrophotometric quantification.

A method for the dynamic quantification of glycerophosphorylcholine (GPC) levels in intact tissue by 31P nuclear magnetic resonance spectroscopy was developed and verified by a spectrophotometric technique. Intact tissue nuclear magnetic resonance areas were quantified utilizing an external standard and were corrected for magnetization saturation. Interactive computerized spectral fitting through Lorentzian lineshape analysis and subsequent integration with normalization to the external standard was utilized for the absolute quantification of GPC concentration. Hemodynamically and metabolically uncompromised Langendorff perfused rabbit hearts contained 1.70 +/- 0.23 mumol GPC/g wet wt. This value was not statistically significantly different from the value of 1.45 +/- 0.23 mumol GPC/g wet wt determined by an analytical technique employing glycerophosphoryl-[Me-3H]choline as an internal standard with spectrophotometric quantification. Both methods were accurate with a standard error of 11 and 10%, respectively. The recovery of internal standards utilizing the spectrophotometric technique was 95 +/- 8%. The application of these methods should facilitate the quantification of changes in tissue levels of glycerophosphorylcholine noted in several disease states.