Allan M. Rofe

Hepatic oxalate production: The role of hydroxypyruvate

The metabolism of hydroxypyruvate to oxalate was studied in isolated rat hepatocytes. [14C]Oxalate was produced from [2-14C]- and [3-14C]- but not [1-14C]hydroxypyruvate. No oxalate was produced from similarly labeled pyruvate. The mechanism by which hydroxypyruvate is metabolized to oxalate involves decarboxylation at the carbon 1 position as the initial step. This activity was distinct from that which produced CO2 from the carbon 1 position of pyruvate. Hydroxypyruvate decarboxylase activity was found mainly in the mitochondria, with the remainder (25%) in the cytosol. No activity was present in the peroxisomes, the probable site of oxalate production from glycolate and glyoxylate. Hydroxypyruvate, but not pyruvate stimulated [14C]oxalate production from [U-14C]fructose, suggesting that hydroxypyruvate is either an intermediate in the fructose-oxalate pathway, or that it prevents carbon from leaving that pathway. The lack of effect of pyruvate in this regard is evidence against redox being the primary effect of hydroxypyruvate and focuses attention on hydroxypyruvate and its precursors as important sources of carbon for oxalate synthesis from both carbohydrate and protein.

Investigations into the Effect of Glyoxylate Decarboxylation and Transamination on Oxalate Formation in the Rat

The decarboxylation and transamination reactions of glyoxylate, which divert this precursor from oxalate formation, have been investigated. Decarboxylation of glyoxylate is synergistic with 2-oxoglutarate and catalysed by 2-oxoglutarate:glyoxylate carboligase which co-chromatographs with the 2-oxoglutarate dehydrogenase complex. The activity is located in the mitochondrial fraction and is probably due to the E1 subunit of the complex. A greater amount of decarboxylation occurs from 2-oxoglutarate than from glyoxylate but the presence of 2-oxoglutarate does not affect oxalate formation from glyoxylate. There is no oxalate formation from 2-oxoglutarate. Studies with rat liver homogenates showed that a number of amino acids can participate in glyoxylate transamination. However, using isolated rat hepatocytes, these reactions did not have a significant effect on oxalate formation from glyoxylate with the exception of cysteine which caused an 80% reduction in oxalate formation. Investigation of this inhibition indicated that it was most likely due to the formation of a cysteine-glyoxylate adduct which makes glyoxylate unavailable for oxidation to oxalate. This cysteine inhibition of oxalate formation was also demonstrated in normal rats and rats made hyperoxaluric by injecting them with either glyoxylate or glycolate. The results indicate that sulphydryl compounds, which can have a therapeutic role as oxalate-lowering agents, may be able to be developed.

Biochemical manifestations of a rat mammary adenocarcinoma producing cachexia: In vivo and in vitro studies

The physical and metabolic characteristics of a Dark Agouti rat mammary adenocarcinoma and its effects on host metabolism arc described. The tumour was characterized by a lack of glandular differentiation, tetraploidy, a rapid mitotic index and a high rate of glycolysis. The adenocarcinoma was readily maintained in tissue culture and could be passaged through the host by inoculating either cell suspensions or tissue explants. In the rat, tumour growth resulted in a loss of adipose tissue at a tumour mass of less than 5% body weight indicating that increased energy expenditure was already present at that stage. In addition the tumour caused anaemia, hypercalcaemia and hypoglycaemia. Hyperketonaemia was also observed in fasted tumour‐bearing rats. Methotrexate arrested tumour growth in vivo. These aspects of the tumour model make it useful for investigations into host‐tumour competition and mechanisms of cachexia.

The Inhibition of Metabolic Oxalate Production by Sulfhydryl Compounds

A number of sulfhydryl compounds were shown to inhibit CO2 and oxalate formation from glyoxylate by rat liver homogenates and hepatocytes. The most significant inhibition occurred with cysteine and this inhibition was concentration-dependent. In rats made hyperoxaluric by administering ethylene glycol in their drinking water, daily intraperitoneal injections of cysteine caused a rapid and marked decrease in urinary oxalate excretion which was maintained over the duration of the treatment (28 days). Over this time period, the level of urinary oxalate excretion in these ethylene glycol-treated rats was reduced to that of the controls. It is postulated that the decrease is due to the formation of a cysteine-glyoxylate adduct, 2-carboxy-4-thiazolidine carboxylate, which prevents glyoxylate being further oxidized to oxalate. Cysteine or similar sulphydryl compounds may therefore have potential as therapeutic agents in the prevention of renal stones.

Beneficial effects of endotoxin treatment on metabolism in tumour-bearing rats.

The effects of endotoxin treatment on host metabolism in tumour‐bearing investigated. Metabolism in control rats (non‐tumour‐bearing) was slightly altered by endotoxin treatment, whereas in tumour‐bearing rats a number of biochemical parameters that were intially perturbed by the presence of the tumour had returned to normal at 48 h post‐treatment. The beneficial effects included increased blood glucose and insulin concentrations, and decreased ketone body, triglyceride and lactate concentrations. Potentially non‐beneficial effects of endotoxin observed in both tumour‐bearing and control rats included decreased plasma cholesterol, and increased plasma phosphate, potassium and alkaline phosphatase levels.

Metabolic consequences of methotrexate therapy in tumour-bearing rats

The metabolic response of the tumour‐bearing host to methotrexate (MTX) therapy was investigated with particular attention to effects resulting from MTX‐induced anorexia. Biochemical changes in female Dark Agouti rats bearing mammary adenocarcinomas and treated with MTX (0.5 mg/kg. 2 i.m. injections, 24 h apart) were compared with untreated (CON) tumour‐bearing rats, and tumour‐bearing rats pair‐fed (PF) to the MTX group. MTX treatment halted progression of the tumour (tumour 6% of bodyweight) while the tumour burden doubled in the CON and PF groups.

The effect of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) on urinary oxalate excretion: studies using a hyperoxaluric rat model.

PURPOSEnTo determine the efficacy of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) in reducing urinary oxalate excretion under hyperoxaluric conditions and to determine whether by inclusion of glycolate in a standard diet, cysteine:glyoxylate adduct can be detected in hyperoxaluric rats given either compound.nnnMATERIALS AND METHODSnHyperoxaluria (200% above basal) was induced 2 days prior to commencement of the studies and maintained throughout. After a 3 days baseline, animals were randomly allocated to a control or treatment group. Standard diet containing either (L)-cysteine or OTZ was then fed to the treatment groups for 5 days while standard diet alone was fed to the control groups. Urinary oxalate excretion was subsequently monitored and average daily rates were then compared with basal values. Plasma and urine were analyzed for adduct.nnnRESULTSnBoth (L)-cysteine and OTZ significantly reduced urinary oxalate excretion relative to the basal hyperoxaluric level (28.6 +/- 1.5 micromol./day). While (L)-cysteine reduced oxalate excretion over the 5 day treatment period by only 7.82 +/- 1.39 micromol./day (27%), OTZ reduced it by 12.34 +/- 1.58 micromol./day (43%). Adduct could not be detected in plasma or urine in this study.nnnCONCLUSIONSnThis study confirms that both (L)-cysteine and OTZ are effective in reducing urinary oxalate excretion under hyperoxaluric conditions, with OTZ being more effective than (L)-cysteine. These compounds were shown to be 3- to 4-fold more effective in reducing urinary oxalate excretion under hyperoxaluric conditions when compared with the results from previous studies under normooxaluric conditions.

The efficacy of (L)-2-oxothiazolidine-4-carboxylate (OTC) and (L)-cysteine in reducing urinary oxalate excretion.

The effects of orally administered (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylate (OTC) on urinary oxalate excretion were investigated in male Porton rats, as (L)-cysteine has been shown to form an adduct with glyoxylate in vitro. Feeding of OTC (204 +/- 1 mg. per day) for 5 days increased urinary cyst(e)ine, p < 0.001; sulphate, p < 0.001; phosphate, p < 0.05; and calcium, p < 0.05; and decreased urinary pH, p < 0.001. In addition, OTC feeding significantly decreased urinary oxalate excretion when compared with controls, p < 0.05 (delta OTC-delta Control -4.26 +/- 1.55 nmol./day/gm.). In the 5-day period after cessation of OTC feeding, all urinary parameters returned to control levels. At the completion of this recovery period there were no significant differences in any of the clinically significant plasma parameters. When fed for 22 days (191 +/- 3 mg. per day) OTC decreased urinary oxalate compared with controls, p < 0.05 (delta OTC-delta Control -9.47 +/- 4.24 nmol./day/gm.). Other urinary parameters (uric acid, magnesium, calcium, phosphate, creatinine, pH and volume) were not significantly altered by OTC feeding. Again, at the completion of this feeding period there were no significant differences in any of the clinically significant plasma parameters. (L)-cysteine feeding for 5 days (184 +/- 10 mg. per day) increased urinary sulphate, p < 0.001; and magnesium, p < 0.05, and decreased urinary pH, p < 0.001. In addition, (L)-cysteine feeding did not significantly change urinary oxalate excretion when compared with the controls (delta(L)-Cysteine-delta Control -2.94 +/- 2.14 nmol./day/gm.). However, at the completion of this feeding period, plasma urate, p < 0.02; and glucose, p < 0.05, were decreased, and plasma potassium, p < 0.01, was increased. these results indicate that orally administered OTC is effective in reducing urinary oxalate excretion without altering plasma biochemistry. It is suggested that (L)-cysteine-glyoxylate adduct formation is the mechanism by which OTC reduces urinary oxalate excretion through a reduction in endogenous oxalate production.

Idiopathic Calcium Oxalate Urolithiasis and Endogenous Oxalate Production

Despite the great effort that has gone into investigating urolithiasis, this condition still persists as one of the major ailments of the urinary tract. Calcium oxalate urolithiasis is the most common form, accounting for some 60 to 80% of total stones. This review examines the elements (i.e., urine volume and pH and urinary excretion of calcium, oxalate, citrate, urate, magnesium, pyrophosphate, and glycosaminoglycans) that give rise to idiopathic calcium oxalate urolithiasis. Treatment strategies for idiopathic calcium oxalate urolithiasis, including lithotripsy, also are discussed. Urinary oxalate excretion is a major risk factor for calcium oxalate urolithiasis, with 85 to 95% of the urinary load derived endogenously. The factors controlling endogenous oxalate production are reviewed, including pathways for the diversion of glyoxylate from oxalate production. The use of beta-aminothiols and other substances to reduce endogenous oxalate production in subjects with idiopathic calcium oxalate urolithiasis is also discussed. A review of current methodologies for the determination of urinary oxalate is also included.

Creatine Kinase Reference Intervals Determined from a Multi-Centre Data Pool

&NA; Reference intervals for creatine kinase assayed at 37°C using N‐acetyl cysteine‐activated methods have been determined on data obtained from 10 laboratories throughout Australia. The pooled distributions for males and females are skewed towards higher values and cannot be transformed to Gaussian distributions. The reference interval for females was calculated to be 34 to 180 U/l and for males it was 46 to 300 U/l. However, if creatine kinase is to be used in the diagnosis of myocardial infarction, the upper limit of the reference interval for males is considered to be too high. It is concluded that for males, the upper limit may need to be determined on specific populations such as hospital inpatients.