Department of Nephrology, Hôpitaux Universitaires de Strasbourg, 1 University Laboratories of Biochemistry, Faculty of Medicine of Strasbourg and 2 Department of Haemostasis and Thrombosis, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg, France
Correspondence and offprint requests to: Professor T. Hannedouche, Department of Nephrology, Hôpitaux Universitaires de Strasbourg, PO Box 426, 67091 Strasbourg Cedex, France.
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Abstract |
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Methods. In a cohort of 63 stable chronic haemodialysis patients, we examined the causal relationship between hyperhomocyst(e)inaemia and vascular endothelial and haemostatic function. All their markers were determined before and after an 8-week course of a 10 mg per day oral folate supplementation, a manoeuvre known to decrease hyperhomocyst(e)inaemia in uraemic patients.
Results. History of at least one cardiovascular atherothrombotic event was present in 47.6% of the haemodialysed patients, and radiographic evidence of vascular calcifications in 70%. Hyperhomocyst(e)inaemia was found in all patients, averaging 3.5-fold the upper limit of normal values (P<0.001), despite the lack of clinical and biological evidence of malnutrition. Fibrinogen, von Willebrand factor and plasminogen activator inhibitor type 1, but not endothelin 1, were significantly higher in haemodialysis patients than in controls. After adjustment for all variables, past history of cardiovascular events was independently associated with higher levels of homocyst(e)inaemia only (odds ratio (OR) 1.06; 95% confidence interval (CI) 1.011.12; P<0.026). The presence of aortic calcifications was independently and significantly associated with age (OR 1.37; 95% CI 1.071.75; P<0.025), homocyst(e)inaemia (OR 1.14; 95% CI 1.021.27; P<0.05) and fibrinogen concentration only (OR 9.74; 95% CI 1.2575.2; P<0.05). None of the endothelialhaemostatic factors was, however, related to homocyst(e)ine levels. Mid-term folate supplementation decreased plasma homocyst(e)ine levels significantly without achieving normal values. No significant change of endothelialhaemostatic markers was observed, however, despite the drop in plasma homocyst(e)ine.
Conclusions. Hyperhomocyst(e)inaemia is associated with increased cardiovascular risk in haemodialysis patients. Folate supplementation was partially effective in lowering hyperhomocyst(e)inaemia, but its usefulness in terms of reduction in cardiovascular morbidity and mortality remains to be determined in prospective trials.
Keywords: homocyst(e)ine; haemostatic factors; endothelium; haemodialysis; chronic renal failure
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Introduction |
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Arterial lesions in uraemia consist of typical atherosclerosis in a large percentage of dialysis patients studied before renal transplantation [2]. These atherosclerotic lesions can be associated, although not consistently, with diffuse `pipe-stem'-type vascular calcifications located in the tunica media or internal elastic lamina without foam cells or macrophages [3]. Atherosclerosis is initiated through endothelial abnormalities [4] and elevated plasma concentrations of fibrinogen and of putative markers of endothelial dysfunction including von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1). All of these have been shown to predict the risk of cardiovascular events [5,6], especially coronary accidents.
Homocysteine is a sulfur-containing amino acid that results from the demethylation of methionine. Patients with homocystinuria, a rare inherited metabolic defect (defective cystathionine-ß-synthase) leading to extraordinarily high blood homocyst(e)ine levels, develop widespread and severe atherosclerosis and generally die before the age of 20 years from cerebral and coronary occlusive accidents [7].
Recently, milder increases in homocyst(e)ine have been recognized as an independent risk factor for stroke and other cardiovascular diseases [8,9]. More than 20 casecontrol and cross-sectional studies of >2000 subjects consistently have indicated that patients with cardiovascular diseases tend to have higher blood levels of homocyst(e)ine than subjects without disease. It is of interest to note that most such patients have homocyst(e)ine levels within what is considered the normal range [10]. The direct causal role of homocyst(e)ine in these cardiovascular events is suggested by reports of vascular lesions induced in baboons infused with homocysteine for 3 months and of the toxic effect of homocyst(e)ine on vascular endothelium [7].
Hyperhomocyst(e)inaemia can be acquired in several conditions including cobalamin or folate deficiency, and uraemia, where the increase in homocyst(e)ine is positively correlated to plasma creatinine and culminates in dialysis patients [11]. In a cohort of uraemic patients not requiring dialysis, we previously have found higher homocyst(e)ine levels in patients with cardiovascular complications than in patients without such complications [11], suggesting that part of the `accelerated atherosclerosis' could result from hyperhomocyst(e)inaemia. However, the specific role of high homocyst(e)ine levels in inducing endothelial dysfunction, atherosclerosis and cardiovascular morbidity in dialysis patients remains largely unknown.
The aims of the present study were: (i) to determine the prevalence and magnitude of hyperhomocyst(e)inaemia in a large cohort of haemodialysis patients and to assess the potential role of nutritional or folate deficiencies; (ii) to establish in these patients the potential link between hyperhomocyst(e)inaemia, past history of cardiovascular events and present markers of atherosclerosis such as vascular calcinosis; and (iii) to elucidate the link between hyperhomocyst(e)inaemia and endothelialhaemostatic dysfunction.
This was evaluated by measuring plasma markers of endothelial function before and after folate supplementation, a manoeuvre known to lower plasma homocyst(e)ine [12].
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Subjects and methods |
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Sixty three Caucasian patients met the following criteria and were enrolled in the study. Patients were characterized as follows: 44 men, 19 women; mean age 59±13 years, mean duration of dialysis treatment 45±40 months. All patients were dialysed for 4 h three times a week with standard bicarbonate buffer and heparin anticoagulation. Initial nephropathy was chronic glomerulonephritis (n=29), diabetic nephropathy (n=9), polycystic kidney disease (n=6), interstitial nephritis (n=6), vascular nephropathy (n=4) and others (n=9).
All patients (n=63), as well as 40 age-matched healthy volunteers, serving as controls, were evaluated for measurement of the following variables: serum albumin, insulin-like growth factor 1 (IGF-1), IGF-BP3, vitamin B12, serum and intra-erythrocyte folate, homocyst(e)ine, oxalic acid, total and high-density lipoprotein (HDL) cholesterol, triglycerides, lipoprotein (a) [Lp(a)], fibrinogen, vWF, PAI-1, endothelin 1 (ET-1). Blood samples were drawn in the fasting state before a dialysis session. Samples were drawn on ice, immediately centrifuged and stored at -80°C before specific analysis. Calciumxphosphorus product and intact parathyroid hormone (iPTH) were calculated as the average of monthly and tri-monthly determinations, respectively, over the 12 months preceding the study.
Traditional vascular risk factors such as hypertension, diabetes and smoking habits were also evaluated. Patients were considered as hypertensive when they were receiving treatment with at least one antihypertensive drugs, as diabetics when hyperglycaemic and requiring insulin treatment, and as a smoker when currently smoking more than five cigarettes or equivalent per day.
Past history of cardiovascular events was evaluated from in-centre medical records of each uraemic patients. Myocardial infarction was diagnosed if the symptoms met the WHO criteria and they were associated with either elevated plasma concentrations of enzymes or characteristic electrocardiographic changes. Other acute coronary syndromes were diagnosed according to clinical symptoms and typical electrocardiographic or angiographic changes [13]. Strokes were diagnosed on the basis of medical records showing a neurological deficit of sudden or rapid onset that persisted for >24 h. Strokes, either ischaemic or haemorrhagic, were confirmed on abnormal CT scans, which were available in all cases of clinical strokes. Aortic aneurysms and renal artery stenoses were documented by CT scan and/or angiograms. Peripheral vascular disease was diagnosed based on clinical history (claudication, rest pain of the extremities, arterial bypass surgery or amputation) and characteristic angiographic abnormalities.
Aortic calcinosis was assessed by radiographic detection of calcified deposits in the abdominal aorta [14]. Lateral abdominal films (T12S1) were made from a fixed distance with the subject seated. Calcifications in the abdominal aorta were identified as linear densities in an area parallel and anterior to the lumbar spine (L1L4) and were scored according to the length of the involved area. Nil calcification or <2 cm patchy calcifications were classified as absent, whereas larger streaks of calcification longer than 2 cm, generally found on both the anterior and posterior wall of the aorta, were considered as present. X-rays of the hand were performed and examined to confirm the absence or presence of calcinosis on small digital arteries.
To assess further the relationship between homocyst(e)inaemia, lipids and haemostasisendothelial markers including fibrinogen, vWF, PAI-1 and ET-1, all these variables were determined before and after folate supplementation for 2 months, a manoeuvre reported to lower homocyst(e)ine levels in this group of patients [12].
Patients were randomized into two groups treated either by placebo or by folic acid 10 mg administrated orally for a period of 8 weeks. After 8 weeks, homocyst(e)ine levels, endothelial markers, lipids and haemostatic factors were again determined.
In addition, 10 patients of each group were chosen randomly to study the effect on endothelial activation of lowering homocyst(e)inaemia. To do so, we used the property of an angiotensin-converting enzyme (ACE) inhibitor that, besides blocking generation of angiotensin II, also shows the catabolism of active bradykinin, a potent vasodilator acting through binding to an endothelial B2 receptor and stimulation of nitric oxide (NO) synthase. One hour before the start of the dialysis session, we determined blood pressure (oscillometric automatic device), plasma active renin concentration, plasma cGMP and plasma NO2NO3 (NOx), serving as putative terminal metabolites of NO), before (T0) and 30 min after (T30) the intake of a single oral dose of 25 mg of captopril.
Measurements
Plasma total homocyst(e)ine was determined by high-performance liquid chromatography coupled with a fluorometric detection method [15]. For the quantitative determination of folates in plasma and red blood cells (after haemolysis), we used the CIBA-Corning automated Chemiluminescence System (ACS). IGF-1 and IGF-BP 3 were determined by competitive radioimmunoassay (RIA) (Nichols Institute, San Juan Capistrano, CA). ET-1 was quantified after acidification and extraction using RIA (Nichols Institute). Oxalic acid was measured by a colorimetric enzymatic assay. Albumin was determined by a colorimetric assay, cholesterol by an enzymatic assay, HDL cholesterol and Lp(a) by a nephelometric assay, and PAI-1 activity was measured by a chromogenic substrate assay (Biopool, Umea, Sweden). Fibrinogen determination was measured by the method of Clauss [16]. vWF was determined by a ristocetin agglutination technique. Active renin concentration was measured by the RENIN III kit (Institut Pasteur, Paris, France), cGMP was measured by RIA (Immunotech Int., Marseilles, France). Plasma nitrites and nitrates (NOx) were determined according to Green et al. [17] by a continuous flow method adapted on Alpkem analyser (ALPKEM Corp., Seattle, OR).
Statistical analysis
All results are expressed as mean values±SD unless stated otherwise. Although all variable and, specifically, homocyst(e)ine concentrations were distributed normally in controls and the haemodialysis population, non-parametric Wilcoxon's test was used for respective comparisons of controls vs patients; and patients with vs without cardiovascular history or vascular calcifications. Fisher's exact test was used for the comparison of percentages between the two groups. A logistic regression model was used to assess the influence of all variables to determine past cardiovascular events on the one hand and present vascular calcinosis on the other. Analysis of variance for repeated measurements was used to compare the variation of blood pressure, renin, cGMP and NOx after captopril stimulation [18].
Statistics were performed using the software SuperAnovaTM (Abacus Inc., Berkeley, CA), and for the logistic regression analysis, SOLO version 4 (by BMDP Statistical Software Inc., Los Angeles, CA).
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Results |
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According to the definition criteria, 17% of the patients were diabetic, 40% hypertensive and 25% smokers (Table 2). The relationships among the presence of cardiovascular events, traditional cardiovascular risk factors, homocyst(e)ine and haemostatic factors are shown in Table 2
. Lp(a) was higher in patients than in controls, and above the upper limit of normal (300 mg/l) in 33% of the patients. Total cholesterol was similar, whereas HDL cholesterol was lower in patients than in controls. Total cholesterol was >7 mmol/l in 10% of the patients. Fibrinogen and vWF concentrations were elevated in 95 and 62% of the patients, respectively. The prevalence of these factors was, however, not different in patients with or without a history of cardiovascular complications. After adjustment for all putative variables (continuous and nominal given in Tables 1 and 2
), past history of cardiovascular events was only independently associated with higher levels of homocyst(e)inaemia with an odds ratio (OR) of 1.06 [95% confidence interval (CI) 1.011.12; P<0.026]. When comparing patients with the lowest quartile of homocyst(e)ine values (tHcy<30 µmol/l, n=17) vs patients with the highest quartile of homocyst(e)ine values (tHcy>50 µmol/l, n=17), the OR for cardiovascular events between these two groups was 12.63 (95% CI 1.15144; P<0.05).
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Both aorta and hand vessel calcifications were insignificantly more prevalent among patients with a history of cardiovascular events than in patients without such complications (Table 2). By logistic regression, the presence of vascular calcifications (both hand vessels and aorta) was independently and significantly associated with age (OR 1.16; 95% CI 1.0011.34; P<0.05) and homocyst(e)inaemia only (OR 1.076; 95% CI 1.0021.16; P<0.05). When considered separately, aortic calcifications, were independently and significantly associated with age (OR 1.37; 95% CI 1.071.75; P<0.025), homocyst(e)inaemia (OR 1.14; 95% CI 1.021.27; P<0.05) and fibrinogen only (OR 9.74; 95% CI 1.2575.2; P<0.05). Interestingly, vascular calcifications were not associated with serum iPTH, calciumxphosphate product and oxalaemia.
Haemostatic factors and endothelial markers
Fibrinogen and vWF plasma concentrations and PAI-1 activity were significantly higher in haemodialysis patients than in controls, whereas ET-1 concentration was similar in patients and controls (Table 1). None of the three variables (fibrinogen, vWF and PAI-1) was related to homocyst(e)ine levels. Unlike fibrinogen, neither vWF nor PAI-1 was different in uraemic patients with or without cardiovascular history or vascular calcinosis.
The 63 patients were included in a 2 month trial of folate supplementation. Of these, only 53 patients (25 treated; 28 controls) completed the study; two patients were transplanted, two died and six patients dropped out because of non-compliance. Folate administration of 10 mg a day, orally, was remarkably well tolerated without any adverse effect. Plasma homocyst(e)ine decreased significantly in the folate-treated group (average decrease -23.4±38.4% in the folate-treated group vs +0.3±29.4% in the placebo group, P<0.001), yet without returning to normal (Table 3). In only one out of 25 treated patients did the homocyst(e)ine level decreased to the normal range. There was a highly significant positive correlation between the decrease in total plasma homocyst(e)ine and the pre-folate homocyst(e)ine level (r=0.945; P<0.0001), but not with the changes in plasma or erythrocyte folate. No significant change of endothelial markers or fibrinogen was observed despite the marked variation in plasma homocyst(e)ine, except for ET-1 which significantly decreased by 26% (Table 3
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Discussion |
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Folate or cobalamin deficiency is a major cause of acquired hyperhomocyst(e)inaemia [21] in the general population, where the concentration of homocyst(e)ine is negatively correlated with the intake and blood concentrations of folic acid, vitamin B12 and vitamin B6 [21]. Folate deficiency occasionally may occur in malnourished dialysis patients [12]. Biological markers of malnutrition such as plasma IGF-1 and albumin were only minimally depressed in our cohort. In contrast, there was definitely no association of high homocyst(e)ine with low folate levels; both plasma levels and intracellular stores of folates were normal in our patients. However, folate supplementation markedly reduced blood homocyst(e)ine levels in our uraemic patients, and the reduction was linearly related to pre-treatment levels. Yet, homocyst(e)ine levels did not return to normal despite a huge increase in plasma and intracellular folate levels. Recently, massive intracellular accumulation of S-adenosylhomocysteine was demonstrated in uraemic patients; this potent competitive inhibitor of methyltransferase reactions could impair re-methylation as well as numerous cellular functions [22]. The folate-dependent stimulation of the re-methylation in uraemic patients could be explained in terms of mass effect action and equilibrium shift [21].
Hyperhomocyst(e)inaemia is now recognized as an independent risk factor for cardiovascular complications [10]. The main biological risk factors independently associated with cardiovascular events in our cohort of haemodialysis patients were higher hyperhomocyst(e)inaemia and calciumxphosphate product; this association persisted after adjustment for other `traditional' risk factors. We previously have reported similar findings in a large series of uraemic patients not undergoing dialysis [11].
High homocyst(e)ine levels can induce oxidation of low-density lipoprotein [23] and increase incorporation of Lp(a) into fibrin [24]. Lipid disorders are encountered frequently in uraemic patients. Among the different lipid fractions, an increased level of apoprotein B, Lp(a), VLDL, chylomicrons, and a low level of HDL and apoprotein A-1 have been the best predictors of survival from cardiovascular diseases in uraemic patients [25]. In our study, Lp(a) was increased in one-third of all haemodialysis patients. There was no interaction between Lp(a), HDL cholesterol and homocyst(e)ine levels. Interestingly the highest levels of Lp(a) were found in patients without vascular lesion, suggesting a `survivor bias' effect [26].
Aortic calcifications are predictive of cardiovascular mortality. Indeed, aortic abdominal calcifications detected on radiography were found to be associated with a 6-fold higher risk of cardiovascular death in men aged 45 years independently of major cardiovascular risk factors [27]. Arterial calcification is composed of crystalline hydroxyapatite (Ca10[PO4]6[OH]2) and is closely linked to atherosclerosis in human arteries [28]. It has to be stressed, however, that media-calcinosis in dialysis patients is pathologically distinct from atherosclerotic lesions, which do not necessarily occur at the same time [3]. We found that the presence of calcifications of the small arteries or media-calcinosis of the aorta was associated essentially with patients' age, hyperhomocyst(e)inaemia and, for aortic calcifications, high fibrinogen concentrations. It is of note that, the arteriosclerotic lesions in children with homocystinuria due to defective cystathionine-ß-synthase are mainly of the fibrous or fibrocalcific type [7] and are reminiscent of those observed in dialysis patients [3].
Given experimental studies suggesting that hyperhomocyst(e)inaemia is responsible for endothelial lesions [29] and can change haemostatic conditions from antithrombotic to thrombogenic [3033], we were interested in looking for a correlation between homocyst(e)inaemia and some putative biological markers of endothelialhaemostatic dysfunction in vivo. Indeed, we found that fibrinogen, vWF and PAI-1 activity, but not ET-1, were elevated in our haemodialysis patients. The significant decrease in tHcy under folate supplementation was not associated with a similar reduction in plasma concentrations of markers of endothelial function, except for ET-1 which decreased significantly from initially normal values.
Fibrinogen is an inflammatory marker (hepatic acute phase reactant) as its synthesis is stimulated by cytokines, such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF). Fibrinogen is also involved in blood coagulation and in the progression of atherothrombotic disease by participating in platelet aggregation and in the formation of atheromatous plaques and parietal thrombi. Finally, fibrinogen is a major determinant of blood viscosity, an increase in which predisposes to thrombosis. Many recent studies indicate that fibrinogen is a potent, discriminant, and an independent risk factor of cardiovascular events [5,6,34]. The fibrinogen level was significantly increased in our haemodialysed patients, possibly due to the periodic cytokine-mediated inflammatory stimulation by the dialysis membrane, yet high fibrinogen levels have also been reported in uraemic non-dialysed patients [35]. In our study, high fibrinogen concentration was found to be associated independently with the presence of aortic vascular calcinosis but not with the past occurrence of cardiovascular events. However, in our patients, high fibrinogen was not concordant with hyperhomocyst(e)inaemia, in contrast to a previous report in non-renal patients with coronary artery disease and mildly elevated homocyst(e)ine and fibrinogen levels [36].
vWF is a glycoprotein originating from platelets, and even more from endothelial cells. It promotes platelet adhesion to damaged vessels and platelet aggregation in areas of high shear rates, thus contributing to thrombus formation. Increased levels of vWF in plasma are a marker of endothelial dysfunction or injury and are predictive of recurrent myocardial infarction [36]. vWF was increased in our dialysis population, an observation also reported recently [35,37,38].
PAI-1 is a 50 kDa protein synthesized and released by endothelial cells [39]. The activation of plasminogen at the fibrin surface or on cell membranes is the basis of fibrinolysis. During the process, plasminogen is transformed into plasmin by t-PA, whose activity is regulated by its specific inhibitor, PAI-1. High levels of PAI-1 result in low fibrinolytic activity and correlate with serum insulin, obesity and hypertriglyceridaemia [40]. High PAI-1 levels have been shown to predict the risk of recurrent myocardial infarction in younger non-uraemic men [41] and the occurrence of ischaemic accidents in atherosclerotic men [42]. Plasma PAI-1 forms complexes with and inactivates plasma t-PA, hence high plasma antigen levels of t-PA reflect high PAI-1 levels and low blood fibrinolytic activity [5]. PAI-1 production can be stimulated by oxidized LDL and cytokines such as TNF [43], both of which are elevated in haemodialysed patients, but also depends on a genetic polymorphism associated with myocardial infarction [44].
Eight weeks of oral folate supplementation significantly decreased homocyst(e)inaemia, yet reaching normal values in only one of 25 patients. Raising the question of the appropriate dose of folate, Bostom et al. have shown recently that supplementation with a multivitamin tablet containing 15 mg of folate, 100 mg of vitamin B6 and 1 mg of vitamin B12 could reduce tHcy to 15 µmol/l or less in one-third of dialysis patients having received 1 mg of folic acid daily previously [45]. Given the vast increase in plasma and intra-erythrocytic folate levels obtained in our patients, it is unlikely that higher doses of folate, e.g. 15 mg per day, could have led to a normalization of Hcy levels. Van Guldener et al. [46] did not find such a decrement after 15 mg of folate a day orally in their patients. On the other hand, Hong et al. found that 3 to 4-fold plasma folic acid concentrations did not suffice to reduce hyperhomocysteinaemia at all in dialysis patients receiving a daily dose of 5 mg [47].
We have not found any improvement of either the endothelialhaemostatic markers or endothelium-dependent vascular reactivity, as evaluated by the variations in serum cGMP and NO byproducts after stimulation by captopril. Our findings are in accordance with those of van Guldener et al. [46], who also found no change in endothelial function parameters, neither biologically, nor by means of flow-mediated vasodilatation studies, nor after ultrasound vessel wall analysis.
Based on epidemiological evidence [5,6,34,39], the elevated levels of fibrinogen, vWF and PAI-1 activity theoretically could account for the high cardiovascular morbidity and mortality observed in haemodialysis patients. However, and provided that measurement of plasma markers is sensitive enough to detect endothelial damage or haemostatic dysfunction, no firm functional link between hyperhomocyst(e)inaemia and these markers could be demonstrated in the present study.
In conclusion, haemodialysis patients exhibit a constellation of biological disturbances known as cardiovascular risk factors in non-uraemic subjects. The association of these abnormalities could account for the `accelerated atherosclerosis' observed in these patients. Among these, hyperhomocyst(e)inaemia emerges as an independent risk factor associated with a history of cardiovascular events. However, we could not demonstrate a functional link between hyperhomocyst(e)inaemia and markers of endothelial damage or dysfunction. While lowering hyperhomocyst(e)inaemia by folate supplementation was partially effective and may sound logical, further studies are needed to prove the effective of this approach on long-term cardiovascular morbidity and mortality in uraemic patients.
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Acknowledgments |
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References |
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