Turnover Rates and Intracellular Pool Size Distribution of Citrate Cycle Intermediates in Normal, Diabetic and Fat‐Fed Rats Estimated by Computer Analysis from Specific Activity Decay Data of ¹⁴C‐Labeled Citrate Cycle Acids

Abstract

Specific radioactivity curves of citrate, malate, glutamate and aspartate were measured i ?~ vivo in liver from normal, fat-fed and diabetic rats with [l-14C]acetate as precursor a t time intervals of 0.1 to 20 min. A mathematical model of 14 pools and 40 interconnecting flux rates been constructed and by computer analysis on the Univac 1108 flux rates and intra-and extramitochondrial metabolite pool distribution calculated from the measured spec& radioactivity curves. Flux rates and pool distributions were varied by using an automatic minimization routine to minimize the difference between observed and theoretical values until an acceptable fit of computer generated specific radioactivity time curves to experimentally observed specific radioactivities was obtained. Steady state kinetics were assumed, i.e. the model was fixed with no change in pool sizes over the measured time interval. The main conclusions were :1. A state of non-equilibration of isotope exists for the acetyl-CoA and acetylcarnitine, citrate, malate, aspartate, glutamate and a-ketoglutarate pools distributed between cytoplasmic and mitochondrial compartments.2. Cytoplasmic pools of citrate, acetyl-CoA and acetylcarnitine account for the slow rate of radioactivity decay observed for these metabolites in vivo.3. Key flux rates were acetate entry into the citrate cycle of 0.36, 0.28 and 1.05 ymoles x min-l x g liver-l in normal, fat-fed and diabetic rats. Citrate turnover rates were 1.21, 0.37 and 1.94 ymoles x min-l x g liver-l for normal, fat-fed and diabetic rats. The acetate pool was 0.23 ymoleslg liver in normal, 0.44 pmoles/g liver in fat-fed and 0.38 pmoles/g liver in diabetic rats. Metabolite pool distribution was greater in the extra-than intramitochondrial space: the shift was most marked in the diabetic rat where the intra-to extramitochondrial malate distribution was in the ratio of 1 : 21.5.

[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate

“…The oxidation rate was then equivalent to 8.0 +0.8punit of CH2/min per IOOg body wt., and R, the rate of conversion of 2-oxoglutarate into malate in the cycle, can be shown to have been 4.0-RCIT/3-RGN/2umol/min. This rate is a little less than that calculated in a kinetic isotope study in vivo (Sauer et al, 1970), but, since it is at least twice as much as most values measured in vitro, a lower value was sometimes used.…”

The effects of the stress caused by experimental procedures on alanine aspartate, glutamate and glutamine in rat liver

“…Glutamate is formed by the very general transamination reaction: amino acid+ 2-oxoglutarate oxo acid+glutamate, and by deamidation of glutamine. Both amino acids and 2-oxoglutarate are supplied considerably faster than the net rate of increase in glutamate, maximally about 0.2,umol/ min per g. 2-Oxoglutarate is formed from citrate at about 1.25,umol/min per g in rat liver in vivo (Sauer, Erfle & Binns, 1970), i.e. about 6 times as fast.…”

Determination of pools of tricarboxylic acid cycle and related acids in bacteria

“…In B. licheniformis celLs growing logarithmically on glucose, the pool sizes were unique but in the same range as in E. coli. Pools of several of the acids differed significantly (pyruvate, succinate, malate) from those of E. coli and liver (11,18). In confirmation of the work of Lowry I I et al (11) reporting on the pools of several of these acids, growth of cells on alternate carbon sources led to altered pools of at least some tricarboxylic acid cycle intennediates.…”

“…The measurement of the intracellular pools of the tricarboxylic acid cycle and related acids in bacteria has been an elusive problem, because the levels of these acids are relatively low and exist in pools that are rapidly turning over (5,18). Therefore, large quantities of cells must be harvested rapidly by a method that ensures the complete removal of the extracellular medium, since significant quantities of metabolic intermediates have been found in the extracellular medium of growing bacteria (5,14).…”