The application of liquid chromatography/mass spectrometry (LC/MS) followed by principal components analysis (PCA) has been successfully applied to the screening of rat urine following the administration of three candidate pharmaceuticals. With this methodology it was possible to differentiate the control samples from the dosed samples and to identify the components of the mass spectrum responsible for the separation. These data clearly show that LC/MS is a viable alternative, or complementary, technique to proton NMR for metabonomics applications in drug discovery and development.

Induction of apoptosis in HL-60 cells, using a variety of cytotoxic drugs, resulted, in all cases, in inhibition of CDP-choline:1,2-diacylglycerol choline phosphotransferase, leading to an accumulation of its substrate, CDPcholine, and inhibition of phosphatidylcholine biosynthesis. Incubation of the cells with phosphatidylcholine reduced the number displaying an apoptotic morphology following drug treatment, and this was inversely related to the degree to which the drugs inhibited phosphatidylcholine biosynthesis. Inhibition of choline phosphotransferase by two of the drugs, farnesol and chelerythrine, was shown to be due to direct inhibition of the enzyme, while inhibition by the other drugs, etoposide and camptothecin, could be explained by the intracellular acidification that followed induction of apoptosis.

Programmed cell death or apoptosis was induced in human promyelocytic leukemia (HL-60) and Chinese hamster ovary (CHO-K1) cells using several cytotoxic drugs that have different modes of action, including camptothecin, ceramide, chelerythrine, etoposide, farnesol, geranyl geraniol, and hexadecylphosphocholine. The consequent changes in cellular metabolism were monitored using 31P MRS measurements on intact cells and cell extracts. Cells undergoing programmed cell death exhibited characteristic changes in the levels of glycolytic and phospholipid metabolites. The most significant changes were increases in the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate and in the concentration of CDP-choline, which is an intermediate in phosphatidylcholine biosynthesis. In HL-60 cells, the increase in fructose-1,6-bisphosphate levels could be explained by depletion of cellular NAD(H) levels. All of the agents used to induce apoptosis caused the accumulation of CDP-choline. Since the resonances of this compound occur in a relatively well resolved region of tissue spectra, it could provide a marker for apoptosis that would allow the noninvasive detection of the process in vivo using 31P MRS measurements.

High resolution 1H NMR spectroscopy of urine and plasma, conventional clinical chemical methods and histopathology have been applied to investigate the effects of uranyl nitrate (UN) on renal function and biochemistry in the Fischer 344 (F344) rat. Administration of UN (5-20 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy assessed conventionally by histopathology and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), and related to changes in the patterns of low MW metabolites observed in 400 MHz 1H NMR spectra of urine. The changes in urinary metabolite profiles included elevations in glucose accompanied by minor elevations in certain amino acids (alanine, valine and glutamate). 1H NMR urinalysis also revealed altered excretion of low MW metabolites which are not routinely measured, such as L-lactate, acetate, citrate, succinate and 2-oxoglutarate (2-OG). In addition, the striking appearance of high concentrations of 3-D-hydroxybutyrate (HB) in the urine was noted, in the absence of acetoacetate or acetone, and it is suggested that this may provide a new marker of proximal tubular damage for certain types of nephrotoxic mechanism. Broadening of the 1H NMR signals of citrate following 10 mg/kg UN was shown to be due to a dynamic exchange process involving chelation with urinary Ca2+ and Mg2+ ions. Conventional biochemical analysis of plasma from UN-treated rats revealed dose-related increases in creatinine, urea and HB concentrations. 1H NMR-detected evidence of raised alanine amino-transferase (ALT) levels in rats administered the highest dose of UN was indicated by the partial deuteration of alanine in lyophilised plasma reconstituted in 2H2O. The degree of 1H NMR-detected abnormalities agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity and more fully characterised the renal changes produced by UN. The significance of HB-uria in UN-induced proximal nephropathy is discussed in relation to biochemical observations on other proximal nephrotoxins.

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