Effect of alanine supplementation on oxalate synthesis

Abstract

The Primary Hyperoxalurias (PH) are rare disorders of metabolism leading to excessive endogenous synthesis of oxalate and recurring calcium oxalate kidney stones. Alanine glyoxylate aminotransferase (AGT), deficient in PH type 1, is a key enzyme in limiting glyoxylate oxidation to oxalate. The affinity of AGT for its co-substrate, alanine, is low suggesting that its metabolic activity could be sub-optimal in vivo. To test this hypothesis, we examined the effect of L-alanine supplementation on oxalate synthesis in cell culture and in mouse models of Primary Hyperoxaluria Type 1 (Agxt KO), Type 2 (Grhpr KO) and in wild-type mice. Our results demonstrated that increasing L-alanine in cells decreased synthesis of oxalate and increased viability of cells expressing GO and AGT when incubated with glycolate. In both wild type and Grhpr KO male and female mice, supplementation with 10% dietary L-alanine significantly decreased urinary oxalate excretion ~30% compared to baseline levels. This study demonstrates that increasing the availability of L-alanine can increase the metabolic efficiency of AGT and reduce oxalate synthesis.

Keywords: Alanine; Alanine:glyoxylate aminotransferase; Kidney stones; Oxalate; Primary hyperoxaluria.

Fig 1.. Metabolism of glyoxylate to oxalate in mouse hepatocytes.

AGT: alanine:glyoxylate aminotransferase, GO: glycolate oxidase, GRHPR: glyoxylate reductase, LDH: lactate dehydrogenase. The conversion of hydroxyproline to glyoxylate involves several enzymatic steps: (hydroxyproline dehydrogenase, 1P5C dehydrogenase, aspartate aminotransferase, 4-hydroxy-2-oxo-glutarate aldolase (HOGA)). In mice AGT is expressed both in peroxisomes and in mitochondria, unlike humans in which AGT and GO are only localized in peroxisomes. AGT is the enzyme deficient in primary hyperoxaluria type 1 and GRHPR in type 2. PH3 results from HOGA deficiency. (*) denotes the enzymes expressed (LDH, GRHPR) and overexpressed (AGT, GO) in the Chinese Hamster Ovary cells in their respective subcellular compartments.

Fig. 2.. Alanine intracellular content in cells incubated with L-alanine.

CHO cells were incubated with L-alanine 0–10mM and intracellular alanine content was measured by HPLC and normalized to total cell protein. (results mean ± SEM, n=3). p < 0.01 with one-way ANOVA analysis. L-alanine is present in the Ham’s F12 cell culture media at low concentration (0.1 mM).

Fig. 3.. Effect of L-alanine supplementation on CHO cell viability in a glycolate toxicity assay.

CHO cells were preincubated for 24h with different concentrations of L-alanine before being challenged with glycolate (0–0.75mM) and L-alanine for 24h. Cells expressing GO metabolize glycolate to glyoxylate and oxalate, resulting in toxicity that is dependent on the metabolic activity of AGT. A: CHO GO AGT-MA; B: CHO GO AGT-mi; C: CHO GO AGT-170; D: CHO GO. Glycolate 0 mM (dotted line), 0.25 mM (semi dashed line), 0.5 mM (dashed line), 0.75 mM (full line). There is no change in viability at any glycolate and L-alanine level in CHO wt cells (not shown, p=0.6). Results mean ± SEM (n=5–9). Effect of L-alanine and glycolate assessed by 2-way ANOVA, p< 0.001 for both factors in CHO GO AGT-MA, AGT-mi, AGT-170 and only for the effect of glycolate for CHO GO.

Fig. 4.. Effect of L-alanine on oxalate concentration in cell culture media following glycolate metabolism.

CHO cells (wt, GO, AGT-MA [MA], AGT-mi [mi], AGT-170 [170]) were pre-incubated with L-alanine (“−”: 0; “+”: 2 mM) for 24h before addition of 0.75 mM glycolate to the media. The content of oxalate in the cell culture media (extracellular oxalate) was normalized to total cell protein. Results expressed as mean ± SEM (n=2–4). **/***p<0.01/0.001, with t-test.

Fig. 5.. Effect of L-alanine on urinary oxalate excretion in mice.

Male (A, B) and female (C) mice from Wt, Grhpr (Gr) KO and Agxt KO strains were fed a controlled low oxalate diet with 0 (baseline) followed by 10% (A, C) or 5% (B) additional L-alanine (treatment) for 10 days. Urinary oxalate excretions were measured on 24h urine collections in metabolic cages and expressed as μg oxalate / mg creatinine. Results expressed as mean ± SD. (*/**/***): data analyzed with paired t-test, p<0.05/0.01/0.001 compared to pre-treatment baseline.