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Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: Implications for mechanism.BackgroundSeveral alterations of protein structure and function have been reported in uremia. Impairment of a transmethylation-dependent protein repair mechanism possibly related to a derangement in homocysteine metabolism is also present in this condition, causing erythrocyte membrane protein damage. Homocysteine may affect proteins via the accumulation of its parent compound S-adenosylhomocysteine (AdoHcy), a powerful in vivo methyltransferase inhibitor. However, since plasma homocysteine is mostly protein bound, a direct influence on protein structures cannot be ruled out. We measured the levels of L-isoaspartyl residues in plasma proteins of uremic patients on hemodialysis. These damaged residues are markers of molecular age, which accumulate when transmethylation-dependent protein repair is inhibited and/or protein instability is increased.MethodsL-isoaspartyl residues in plasma proteins were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). Plasma concentrations of homocysteine metabolites were also measured under different experimental conditions in hemodialysis patients.ResultsThe concentration of damaged plasma proteins was increased almost twofold compared to control (controls 147.83 ± 17.75, uremics 282.80 ± 26.40 pmol of incorporated methyl groups/mg protein, P < 0.003). The major protein involved comigrated with serum albumin. Although hyperhomocysteinemia caused a redistribution of thiols bound to plasma proteins, this mechanism did not significantly contribute to the increase in isoaspartyl residues. The S-adenosylmethionine (AdoMet)/AdoHcy concentration ratio, an indicator of the flux of methyl group transfer, was altered. This ratio was partially corrected by folate treatment (0.385 ± 0.046 vs. 0.682 ± 0.115, P < 0.01), but protein L-isoaspartate content was not.ConclusionsPlasma protein damage, as determined by protein L-isoaspartyl content, is increased in uremia. This alteration is to be ascribed to an increased protein structural instability, rather than the effect of hyperhomocysteinemia.

Mechanism of erythrocyte accumulation of methylation inhibitor S-adenosylhomocysteine in uremia. We have recently demonstrated that methyl esterification of erythrocyte membrane proteins, a reaction involved in recognition and repair of specifically damaged proteins, is impaired in uremia. This is accompanied by a significant increase in intracellular S-adenosylhomocysteine (AdoHcy), a potent inhibitor of methyltransferases. AdoHcy accumulation is normally prevented by its enzymatic hydrolysis to homocysteine (Hey) and adenosine, a reversible reaction catalyzed by AdoHcy hydrolase. To assess the contribution that Hey offers in the elevation of AdoHcy, we measured plasma and red blood cell Hey, AdoHcy, adenosine, and S-adenosylmethionine (AdoMet) intracellular concentrations, as well as RBC AdoHcy hydrolase specific activity, in standard hemodialysis patients and normal subjects. Plasma and red blood cell Hey levels are significantly higher in the dialysis group, and are positively correlated to AdoHcy levels. Adenosine and AdoMet levels, and AdoHcy hydrolase specific activity are not significantly different between the two groups. The enzymatic formation of labeled AdoHcy from Hey and tracer adenosine appears to be significantly increased, in vitro, in erythrocytes from both control and uremic patients, when 50 µM Hey (concentration comparable to plasma levels actually found in vivo in uremic patients) is added to the incubation medium. When erythrocytes from uremic patients are incubated in vitro in absence of Hey, a significant reduction of intracellular AdoHcy is observed with time compared to identical samples incubated in presence of 50 µM Hey, with a T1/2 of approximately 270 minutes. The results allow us to conclude that plasma and red cell Hey levels actually found in uremia can be effectively responsible for the intracellular accumulation of the toxic compound AdoHcy.

To the Editor: Hydrogen sulfide, H2S, the third endogenous gas with cardiovascular properties after nitric oxide and carbon monoxide, is a newly recognized vasorelaxant, and H2S deficiency is involved in the pathogenesis of hypertension and atherosclerosis.1 We demonstrated that in hemodialysis patients, H2S levels are decreased through transcriptional deregulation of genes encoding H2S-producing enzymes.2 In fact, gene expression of cystathionine γ-lyase (CSE), an enzyme implicated in H2S formation, is downregulated in these patients.2 We measured H2S levels before and after a single standard 4-h hemodialysis session in 131 patients, in two dialysis sessions separated by 1 month. Measurement was performed by a slight modification of our previously published method.2 Basically, a 20-min incubation period was added before deproteinization. Homocysteine is a main direct H2S precursor in vivo, which functions through the catalytic activity of either cystathionine β-synthase or CSE (see Perna et al.3 for a review). Plasma total homocysteine was also measured by using high-performance liquid chromatography separation and fluorescence detection.4