Total parathyroidectomy without autotransplantation in the surgical treatment of secondary hyperparathyroidism of chronic kidney disease.

BACKGROUND: Subtotal parathyroidectomy (SP) and total parathyroidectomy (TP) with autotransplantation (TPai) are the most commonly adopted operations for the treatment of secondary hyperparathyroidism (2HPT). TP without autotransplantation had previously been confined to patients with advanced dialytic vintage, not eligible for kidney transplantation. Over the years, the procedure has gained more widespread use, but there is no precise knowledge on the immediate and long-term effects. METHODS: The authors analyzed the immediate and long-term results of TP without autotransplantation, that is after the systematic removal of at least four glands in 20 patients operated for 2HPT, which were compared with results from TPai in an equal number of cases. RESULTS: An improvement of the typical clinical symptoms was found in every patient undergoing surgery, and a significant reduction in intact PTH (iPTH) serum levels was achieved. Immediate normalization of iPTH level was observed in 11/20 TP cases, hypoparathyroidism in 4/20 and persistent HPT in 5/20 cases. One year of follow-up showed a slight increase in hypoparathyroidism, with 1/20 (5%) recurrence of the disease. One-year TPai results showed a similar percentage of euparathyroidism, as well as a higher longterm recurrence rate (4/20, 20%), although values do not reach statistical significance. CONCLUSIONS: TP may still be considered the operation of choice in patients with aggressive forms of 2HPT or of advanced dialytic vintage, with no access to renal transplantation, because of its low recurrence rate (5%). Post-operative aparathyroidism is rare, while hypoparathyroidism and hypocalcemia can be well controled by medical treatment.

[Hyperhomocysteinemia in chronic renal failure.]. / L'iperomocisteinemia nell'insufficienza renale cronica: aspetti clinici, nutrizionali e tossicità.

Chronic renal failure (CRF) is frequently associated with increased plasma levels of homocysteine (Hcy), an amino acid that can be considered a new uremic toxin according to recent evidence. Studies on Hcy described first homocystinuria, an inherited disease characterized by high plasma Hcy levels and premature cardiovascular disease, resulting in high mortal-ity rates. Hyperhomocysteinemia was then shown to be associated with cardiovascular events both in the general population and in CRF patients. Hcy is a sulfur amino acid derived from dietary methionine, an essential amino acid. Methionine is condensed with ATP to form S-adenosylmethionine (AdoMet), the universal methyl donor in transmethylation reactions. The AdoMet demethylated product is S-adenosylhomocysteine (AdoHcy), which is the direct precursor of Hcy in vivo. Hcy is toxic for the endothelium, it enhances vascular smooth muscle cell proliferation, increases platelet aggregation, and acts on the coagulation cascade and fibrinolysis. Several mechanisms have been discussed to explain Hcy toxicity. Hcy levels increase as renal function declines and progresses to ESRD; the causes of hyperhomocysteinemia are still unclear. Studies in humans show that renal metabolic extraction depends on renal plasma flow; in addition, an alteration of the extrarenal metabolic clearance, depending on uremic toxins, may occur. Among the consequences of hyperhomocysteinemia in renal failure are: impaired protein methylation, with altered protein repair processes; DNA hypomethylation, with an alteration in the allelic expression of genes regulated through methylation; and protein homocysteinylation. Further, this review is dealing with the 'reverse epidemiology' issue, outlining also the main Hcy-lowering strategies.

Increased plasma protein homocysteinylation in hemodialysis patients.

Hyperhomocysteinemia, an independent cardiovascular risk factor, is present in the majority of hemodialysis patients. Among the postulated mechanisms of toxicity, protein homocysteinylation is potentially able to cause significant alterations in protein function. Protein homocysteinylation occurs through various mechanisms, among which is the post-translational acylation of free amino groups (protein-N-homocysteinylation, mediated by homocysteine (Hcy) thiolactone). Another type of protein homocysteinylation occurs through the formation of a covalent -S-S- bond, found primarily with cysteine residues (protein-S-homocysteinylation). Scant data are available in the literature regarding the extent to which alterations in protein homocysteinylation are present in uremic patients on hemodialysis, and the effects of folate treatment are not known. Protein homocysteinylation was measured in a group of hemodialysis patients (n=28) compared to controls (n=14), with a new method combining protein reduction, gel filtration and Hcy derivatization. Chemical hydrolysis was performed, followed by high-pressure liquid chromatography separation. The effects of folate treatment on protein homocysteinylation, as well as in vitro binding characteristics were evaluated. Plasma Hcy, protein-N-homocysteinylation and protein-S-homocysteinylation were significantly higher in patients vs controls. Plasma Hcy and protein-S-homocysteinylation were significantly correlated. After 2 months of oral folate treatment, protein-N-homocysteinylation was normalized, and protein-S-homocysteinylation was significantly reduced. Studies on albumin-binding capacity after in vitro homocysteinylation show that homocysteinylated albumin is significantly altered at the diazepam-binding site. In conclusion, increased protein homocysteinylation is present in hemodialysis patients, with possible consequences in terms of protein function. This alteration can be partially reversed after folate treatment.

Homocysteine and oxidative stress.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease (ischemic disease, such as stroke and myocardial infarction, and arterial and venous thrombotic events) in the general population. We can assume that the association is causal, based on the example of homocystinuria, and on the evidence put forward by several basic science and epidemiological studies; however, the results of large intervention trials, which will grant further support to this hypothesis, are not yet available. In addition, the mechanisms underlying this relationship, and also explaining the several toxic effects of homocysteine, related or not to cardiovascular disease, are unclear. Oxidation is one of the most favored postulated mechanisms; others are nitrosylation, acylation, and hypomethylation. Regarding the relative importance of these mechanisms, each of these hold pros and cons, and these are weighed in order to propose a balance of evidence.

Metabolic consequences of hyperhomocysteinemia in uremia.

An elevated blood level of homocysteine (Hcy), a sulfur amino acid, is associated with increased cardiovascular risk. Hcy is generated from S-adenosylhomocysteine (AdoHcy), the demethylated product of S-adenosylmethionine (AdoMet) in transmethylation reactions. AdoHcy is a competitive inhibitor of AdoMet-dependent methyltransferases. AdoHcy accumulation is prevented by rapid metabolism of its products. Chronic renal failure (CRF) is almost constantly associated with hyperhomocysteinemia. It has been shown that: (1) AdoHcy concentration is significantly increased and the AdoMet-AdoHcy ratio is reduced in erythrocytes of patients with CRF; (2) erythrocyte membrane protein methyl esterification, catalyzed by the enzyme protein L-isoaspartyl O-methyltransferase (PCMT; EC 2.1.1.77), is reduced in CRF; PCMT catalyzes a repair reaction involved in the conversion of an isopeptide bond (detrimental to protein structure and function) into a normal peptide bond; (3) D-aspartate residues, a side product of protein methylation and repair, are significantly reduced in erythrocyte membrane proteins of patients with CRF; and (4) folate treatment significantly reduces plasma Hcy levels and improves AdoMet-AdoHcy ratios. Stable isotope studies recently confirmed that the rate of methyl transfer reactions is significantly reduced in uremia. Additional evidence, obtained by independent groups, is consistent with this interpretation. We recently found increased isoaspartyl content of circulating plasma protein levels, particularly albumin, which was only partially reduced after folate treatment, in uremia. This kind of molecular damage possibly is caused by protein increased intrinsic instability as a result of interference with the uremic milieu. In conclusion, Hcy is an uremic toxin involved in protein molecular damage through the inhibition of methylation reactions and protein PCMT-mediated repair.

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: implications for mechanism.

BACKGROUND: Several 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. METHODS: L-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. RESULTS: The 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. CONCLUSIONS: Plasma 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.

Homocysteine and transmethylations in uremia.

Homocysteine is regarded as a cardiovascular risk factor in both the general population and chronic renal failure patients. Among the mechanisms for homocysteine toxicity, its interference with transmethylation reactions, through its precursor/derivative S-adenosylhomocysteine, plays a multifarious role. In uremia, inhibition of S-adenosylmethionine methyl transfer reactions has been reported by independent investigators, using multiple approaches. This has several possible consequences, which can ultimately affect the patient's relative state of health.

Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.

Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release.

Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.

Plasma homocysteine and microvascular complications in type 1 diabetes.

BACKGROUND: Homocysteine is involved in a complex and dynamic system of vascular injury and repair and may thus contribute to the development of diabetic microangiopathy. This still debated issue has important scientific and clinical implications, since hyperhomocysteinemia can be corrected nutritionally. AIMS: 1) To evaluate the association between fasting plasma homocysteine, type 1 diabetes and its microvascular complications; 2) to elucidate the basis of this association by investigating the major determinants of plasma homocysteine in relation to diabetic microangiopathy. METHODS: We studied sixty-six consecutive patients with type 1 diabetes mellitus of > 10 years duration and normal serum creatinine (< 115 mumol/L, 1.3 mg/dL), and free from clinically detectable cardiovascular diseases. Forty-four non-diabetic controls were also studied. Plasma concentrations of homocysteine, folate and vitamin B12 were investigated together with the C677T mutation in the gene coding for methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism. Renal and retinal diabetic complications were evaluated as albumin/creatinine ratio on early-morning, urine spot collection and fundus photographs. FINDINGS: Fasting plasma homocysteine levels were very similar in patients and controls. Patients with microalbuminuria or proliferative retinopathy had significantly higher values than those without: 9.4 +/- 3.1 vs 7.4 +/- 2.8 mumol/L, p < 0.02 and 9.5 +/- 2.6 vs 7.3 +/- 3.0 mumol/L, p < 0.05. This difference was not attributable to confounders, such as age, sex and smoking, nor to dissimilar plasma folate and vitamin B12 concentrations. In contrast, homozygosity for the C677T mutation in the MTHFR gene--the commonest genetic defect linked to moderately increased plasma homocysteine--was significantly more frequent in patients with microalbuminuria and/or proliferative retinopathy (50% vs 13%, p < 0.004), odds ratio 6.7 (95% CI 1.7-27.6). CONCLUSIONS: Type 1 diabetes as such is not associated with increased plasma homocysteine levels, though patients with microalbuminuria and/or proliferative retinopathy display significantly higher values than those without. This difference is not attributable to obvious confounders, nor to differences in vitamin status, and may be partly mediated by genetic factors. Plasma homocysteine, together with other diabetes-related noxae, may thus be in a position to contribute to the development of nephropathy and the progression of retinopathy.

Homocysteine, a new cardiovascular risk factor, is also a powerful uremic toxin.

Homocystinuria, an inherited disease in which plasma levels of homocysteine are high, was discovered in the sixties and it soon became clear that the affected patients had striking features of generalized atherosclerosis. The most common causes of death were arterial and venous thrombosis, stroke, or myocardial infarction. Observations in this human model of hyperhomocysteinemia led to studies in the general population whose findings suggest - though not conclusively- that homocysteine is a cardiovascular risk factor. The same is true for patients with chronic renal failure who almost always have moderate to severe high blood homocysteine levels. Homocysteine accumulates in relation to the concentration of its precursor, S-adenosylhomocysteine, a powerful competitive transmethylation inhibitor. Inhibition of a methyltransferase required to repair damaged proteins has actually been detected in uremic patients' red blood cells. However, in view of the multiple, widespread metabolic roles of S-adenosylmethionine-dependent methyltransferases, in many organs and tissues including the vascular endothelium, hypomethylation is currently interpreted as one of homocysteine's most important mechanisms of action. Various biological compounds, including small molecules and nucleic acids, as well as proteins, which are involved in the pathophysiology of thrombosis and atherosclerosis, are all potential targets of hypomethylation. Epidemiological studies and experimental models tend to confirm that homocysteine is both a cardiovascular risk factor and a uremic toxin, acting through different mechanisms.

Impairment of endothelial functions by acute hyperhomocysteinemia and reversal by antioxidant vitamins.

CONTEXT: Increased levels of homocysteine are associated with risk of cardiovascular disease. Homocysteine may cause this risk by impairing endothelial cell function. OBJECTIVE: To evaluate the effect of acute hyperhomocysteinemia with and without antioxidant vitamin pretreatment on cardiovascular risk factors and endothelial functions. DESIGN AND SETTING: Observer-blinded, randomized crossover study conducted at a university hospital in Italy. SUBJECTS: Twenty healthy hospital staff volunteers (10 men, 10 women) aged 25 to 45 years. INTERVENTIONS: Subjects were given each of 3 loads in random order at 1-week intervals: oral methionine, 100 mg/kg in fruit juice; the same methionine load immediately following ingestion of antioxidant vitamin E, 800 IU, and ascorbic acid, 1000 mg; and methionine-free fruit juice (placebo). Ten of the 20 subjects also ingested a placebo load with vitamins. MAIN OUTCOME MEASURES: Lipid, coagulation, glucose, and circulating adhesion molecule parameters, blood pressure, and endothelial functions as assessed by hemodynamic and rheologic responses to L-arginine, evaluated at baseline and 4 hours following ingestion of the loads. RESULTS: The oral methionine load increased mean (SD) plasma homocysteine level from 10.5 (3.8) micromol/L at baseline to 27.1 (6.7) micromol/L at 4 hours (P<.001). A similar increase was observed with the same load plus vitamins (10.0 [4.0] to 22.7 [7.8] micromol/L; P<.001) but no significant increase was observed with placebo (10.1 [3.7] to 10.4 [3.2] micromol/L; P=.75). Coagulation and circulating adhesion molecule levels significantly increased after methionine ingestion alone (P<.05) but not after placebo or methionine ingestion with vitamins. While the mean (SD) blood pressure (-7.0% [2.7%]; P<.001), platelet aggregation response to adenosine diphosphate (-11.4% [4.5%]; P=.009) and blood viscosity (-3.0% [1.2%]; P=.04) declined in these parameters 10 minutes after an L-arginine load (3 g) following placebo, the increase after methionine alone (-2.3% [1.5%], 4.0% [3.0%], and 1.5% [1.0%], respectively; P<.05), did not occur following methionine load with vitamin pretreatment (-6.3% [2.5%], -7.9% [3.5%], and -1.5% [1.0%], respectively; P=.24). CONCLUSION: Our data suggest that mild to moderate elevations of plasma homocysteine levels in healthy subjects activate coagulation, modify the adhesive properties of endothelium, and impair the vascular responses to L-arginine. Pretreatment with antioxidant vitamin E and ascorbic acid blocks the effects of hyperhomocysteinemia, suggesting an oxidative mechanism.

Homocysteine, a new crucial element in the pathogenesis of uremic cardiovascular complications.

Most large observational studies available today establish that moderate hyperhomocysteinemia, either genetically or nutritionally determined, is an independent risk factor for myocardial infarction, stroke, and thromboembolic disease. This is also true for chronic renal failure patients, who exhibit a high prevalence of hyperhomocysteinemia (85-100%), which reaches high plasma concentrations (20-40 microM, while control values range between 8 and 12 microM). After a renal transplant, homocysteine levels decrease, but tend to be higher than normal. The cause of hyperhomocysteinemia in renal failure is still obscure, since recent data have questioned the previous notion that a net homocysteine renal extraction and/or excretion take place in man. No matter the cause of its increase, the sulfur amino acid homocysteine is thought to induce an increment in cardiovascular risk through three basic biochemical mechanisms: (1) homocysteine oxidation, with H2O2 generation; (2) hypomethylation through S-adenosylhomocysteine accumulation, and (3) protein acylation by homocysteine thiolactone. The final result is membrane protein damage, endothelial damage, and endothelial cell growth inhibition, among other effects. Hyperhomocysteinemia, in general, is susceptible of therapeutic intervention with the vitamins involved in its metabolism. Depending on the cause, vitamin B6, vitamin B12, betaine, and/or folic acid can be effectively utilized. Chronic renal failure patients benefit from folic acid in high dosage: 1-2 mg are usually not effective ('relative folate resistance'), while 5-15 mg reduce homocysteine levels to a 'normative' range (<15 microM) in a substantial group of patients. Good results are also obtained in transplant patients, best with a combination of folic and vitamin B6. The results of the interventional trials focusing on the possible reduction in cardiovascular risk after homocysteine-lowering therapy, both in the general population and in end-stage renal disease, are expected soon, as well as the genetic and biochemical studies in suitable models, with the aim to clarify the cause-effect link suggested by the numerous observational and basic science studies.

Homocysteine and chronic renal failure.

Homocysteine, a sulfur amino acid, is an important methionine derivative, which has been implicated in the pathogenesis of atherothrombosis. Although only observational, epidemiological studies are available at present, the evidence of an association between hyperhomocysteinemia and increased cardiovascular risk is quite strong and this is confirmed also in a population of chronic renal failure patients. From a biochemical standpoint at least three mechanisms have been summoned so far in order to explain homocysteine toxicity including: oxidation, hypomethylation, and acylation. Proteins are believed to play a crucial role as homocysteine molecular targets. Interference with the functions of several of such macromolecules has been so far described being mediated by any of the above mechanisms. Vitamins may positively influence homocysteine metabolism, thus facilitating the metabolic clearance of this compound. Therefore they are presently considered as potential means for reducing plasma levels of this amino acid and preventing vascular occlusions in hyperhomocysteinemic patients. These compounds, with special regard to folate, are eligible for interventional clinical trials, from which the definitive answer on the role of homocysteine in atherothrombosis is expected.

D-amino acids in aging erythrocytes.

Mature human erythrocytes are highly differentiated cells which have lost the ability to biosynthesize proteins de novo. During cell aging in circulation, erythrocyte proteins undergo spontaneous postbiosynthetic modifications, regarded as "protein fatigue" damage, which include formation of isomerized and/or racemized aspartyl residues. These damaged proteins cannot be replaced by new molecules; nevertheless, data support the notion that they can be repaired to a significant extent, through an enzymatic transmethylation reaction. This repair reaction has therefore been used as a means to monitor the increase of altered aspartyl residues in erythrocyte membrane proteins during cell aging. The relationship between protein repair and aspartyl racemization in red blood cell stress and disease is discussed.

Metabolic consequences of folate-induced reduction of hyperhomocysteinemia in uremia.

Plasma homocysteine, a well-recognized risk factor for cardiovascular disease, is elevated in uremic patients on hemodialysis. The authors have recently demonstrated that one consequence is the reduction in red cell membrane protein methylation levels, caused by a rise of intracellular adenosylhomocysteine, a potent inhibitor of methyltransferases. Protein methylation is involved in a repair mechanism of damaged membrane proteins, and an impairment in methylation leads to the accumulation of altered proteins. Therapy with folates, cofactors in the transformation of homocysteine to methionine, is effective in lowering plasma homocysteine. This article details a study on the metabolic effects of oral methyltetrahydrofolate, the active form of folic acid, on 14 uremic hemodialysis patients. Two months of therapy led to a significant reduction of plasma homocysteine levels, with a proportional response to pre-folate levels. In five of 13 patients with homocysteine levels above 20 microM, plasma homocysteine level was reduced to less than 15 microM. After treatment, levels of adenosylmethionine, the methyl donor in transmethylations, had significantly increased; levels of adenosylhomocysteine had increased to a smaller extent. Therefore, the ratio between the two compounds, an excellent indicator of the presence and the degree of methylation inhibition, was significantly ameliorated. Methionine plasma levels increased after treatment in all patients and were correlated with posttreatment adenosylmethionine levels. It was concluded that treatment with methyltetrahydrofolate brings the plasma homocysteine concentration back to an "acceptable" level, and the metabolic consequences are in the direction of an increase in the normal flow of transmethylations, as monitored by an increase in the [adenosylmethionine]/[adenosylhomocysteine] ratio.

D-aspartate content of erythrocyte membrane proteins is decreased in uremia: implications for the repair of damaged proteins.

The authors of this article have demonstrated that erythrocytes from patients affected by either chronic renal failure or ESRD, conditions associated with erythrocyte membrane disorders, show reduced levels of methyl esterified membrane proteins because of elevated S-adenosylhomocysteine concentration. The enzyme protein L-isospartyl (D-aspartyl) O-methyltransferase, responsible for the bulk of this methyl esterification, is implicated in the repair of proteins containing isomerized and racemized aspartyl residues, which arise from L-asparaginyl and L-aspartyl residues. The presence of these altered residues, spontaneously generated during protein aging, can adversely affect protein function. The amount of D- and L-aspartyl residues (and their isomerized derivatives) in erythrocyte membranes from hemodialysis patients was determined. The total level of D-aspartyl derivatives (D-Asx) actually was found to be lower than in controls. In contrast, neither the abundance of several other amino acids, nor of total non-Asx D-amino acids, differs between patients and controls. Mathematical simulation of relevant reactions supports the hypothesis that these effects reflect the lessening of the normal D-isoaspartyl residue accumulation that occurs as a side reaction in the methyltransferase-induced repair process. This evidence is the first that D-Asx content is influenced in vivo by L-isoaspartyl (D-aspartyl) O-methyltransferase activity and can be significantly altered in a disease where this activity is inhibited, thus representing a red flag in a disrupted circuit.

Adverse effects of hyperhomocysteinemia and their management by folic acid.

A moderate increase in plasma homocysteine is an independent risk factor for cardiovascular disease. Plasma homocysteine is frequently elevated in chronic renal failure and in uremic patients, and the major causes of death in these patients are cardiovascular accidents. Homocysteine metabolism and mechanisms of toxicity are reviewed. Homocysteine elevation in blood leads to the intracellular increase of its precursor, adenosylhomocysteine, a powerful inhibitor of adenosylmethionine-dependent transmethylations. In vitro evidence shows that this increase is reversible upon homocysteine removal. Membrane protein methylation levels are consistently reduced in erythrocytes of both chronic renal failure and hemodialysis patients. This widespread enzymatic methylation is a key step for the repair of molecular damage resulting from the spontaneous deamidation and isomerization reactions of susceptible residues in proteins. In agreement with these findings is the observation that the concentration of a stable side product, D-Asx, of the repair process is significantly lower in erythrocyte membrane proteins from hemodialysis patients than from controls, showing that the repair of damaged membrane proteins is actually defective. It has been shown that treatment with folates dramatically lowers plasma homocysteine, presumably by improving remethylation to methionine. This indicates that folates and/ or their active derivative, i.e., methyltetrahydrofolate, could be effective in ameliorating transmethylations as well.