Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry

Abstract

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.

Integrated compensatory network is activated in the absence of NCC phosphorylation

“…But unlike acidosis, which also activates phosphoenolpyruvate carboxykinase (PEPCK) to consume α-KG and generate glucose and bicarbonate (69,87), PEPCK is not induced in SPAK KO mice, and, as a result, α-KG levels increase rather than diminish.…”

Operation of the Purine Nucleotide Cycle in Animal Tissues

“…g. After addition of fluorocitrate, ammonia production and renal uptake of aspartate showed a parallel decline, but tissue levels and uptake rates of glutamine and glutamate were unaffected. Nissim et al (1985Nissim et al ( , 1986 and Tornheim et al (1986) studied the metabolism of ["N]glutamine, [lSN]glutamate and [16N]aspartate in isolated kidney tubules. g, which is equal to the rate of ammonia formation by glutamate dehydrogenase.…”

“…In tubules from normal rats incubated with [2-"N]glutamine, however, the isotopic enrichment of the 6-amino group of purine nucleotides was higher and faster than that of free ammonia. In normal acid-base status, the turnover of the adenine nucleotides seems therefore to be able to account for the ammonia formed from glutamate (Nissim et al, 1985).…”

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

“…By using gas chromatography-mass spectrometry (GC-MS) to determine I5N isotopic abundance in ammonia and selected amino acids, rigorous definition of important precursor-product relationships and measurement of rates of flux along the primary pathways of nitrogen metabolism were possible. This technique has been used successfully in earlier investigations of amino acid and ammonia metabolism in cultured astrocytes and cerebellar explants (Yudkoff et al, 1982(Yudkoff et al, , 1983(Yudkoff et al, , 1984(Yudkoff et al, , 1986(Yudkoff et al, , 1987 and renal tissue (Nissim et al, 1985). We now apply GC-MS to determine how glutamate nitrogen is metabolized in synaptosomes and how this process is affected by omission of glucose from the incubation medium.…”