286 Available online at www.annclinlabsci.org Correlation between Low Folate Levels and Hyperhomocysteinemia, but not with Vitamin B12 in Hypertensive Patients C. Scazzone 1, A. Bono 1, F. Tornese 2, R. Arsena 2, R. Schillaci 1, D. Butera 1, and S. Cottone 2 1 Department of Biopathology and Biomedical and Forensic Biotechnologies and 2 Department of Internal Medicine, Operative Unit of Nephrology and Hypertension, University of Palermo, Italy Abstract. Introduction. Hypertension is considered to be among the most important risk factors for cardiovascular and cerebrovascular diseases. In recent years, several investigators have reported that high plasma levels of total homocysteine (t-hcy) has a key role in the development of hypertension, and the deficiency of B complex vitamins could increase the risk of hypertension. The purpose of this study was to investigate the relationship between plasma homocysteine, folate and vitamin B12 in hypertensive patients. Materials and methods: In 116 patients with hypertension and 81 healthy subjects, total plasma homocysteine, vitamin B12 and folate levels were measured. Results and Discussion: Homocysteine was significantly higher in patients than in control subjects (22.9±3.5 versus 9.0±2.3 µmol/l respectively, p<0.001); the folate plasma concentrations in hypertensive patients were significantly lower than in control subjects (6.7±5.0 ng/ml and 9.0±4.4 ng/ml respectively, p<0.05). Moreover, no differences in vitamin B12 plasma levels were observed when comparing the levels of hypertensive patients and those of the controls (440±223 pg/ml vs 491±185 pg/ml respectively, p>0.05). Our results confirmed that, as previously observed, elevated t-hcy levels and low folate levels, but not vitamin B12 levels, are significantly associated with hypertension. Keywords: Folate, Homocysteine, Hypertension, Vitamin B12, Cobalamin, Vitamin B9 Introduction In developed countries, cardiovascular diseases are the leading cause of death. A number of important risk factors for cardiovascular disease are hyperlipidemia, smoking, obesity, diabetes, and hypertension. Several prospective studies demonstrated that hypertension is positively and independently associated with the incidence of cardiovascular and cerebrovascular diseases. Literature data have shown that among new risk factors, hyperhomocysteinemia is synergistic with other genetic and environmental factors in terms of its contribution to the development of hypertension [1,2,3,4]. Furthermore, hyperhomocysteinemia is a risk factor for many human diseases, including coronary atherosclerosis, myocardial infarction, stroke, peripheral vascular disease such Address correspondence to Dr. Concetta Scazzone, Department of Biopathology and Biomedical and Forensic Biotechnologies, University of Palermo, Italy; phone: +39 091 6553297; fax: +39 091 6553294; e mail: concetta.scazzone@unipa.it as venous and arterial thrombosis [5,6,7]. Homocysteine is a sulfur amino acid produced by the metabolism of the essential amino acid, methionine. Hyperhomocysteinemia may be caused by genetic defects located on genes coding for enzymes involved in its own metabolism, including a mutation in the cystathionine β-synthase gene, the C677T polymorphism of MTHFR, and methionine synthase A2756G variant [8,9]. Otherwise, hyperhomocysteinemia could be secondary to inadequate dietary supply of vitamin cofactors (such as folate and B-complex vitamins) required for cellular metabolism [10,11]. Several studies demonstrate that elevated levels of homocysteine are associated with low levels of folate, low levels of vitamin B12, and cardiovascular risk. The association between hyperhomocysteinemia and levels of B complex vitamin has been extensively analyzed, although previous studies have 0091-7370/14/0300-286. 2014 by the Association of Clinical Scientists, Inc.
Table 1. Demographic and clinical data of hypertensive patients and healthy controls. Characteristics Healthy controls Patients group P value (81) (58) Age(years) 60±12 54±14 <0.008 Body mass index (kg/m 2 ) 26±2 28.6±3 <0.01 Systolic blood pressure (mmhg) 113±10 140±19 <0.001 Diastolic blood pressure (mmhg) 73±4.4 84±11 <0.001 Serum creatinine ( mg/dl) 0.89±0.12 0.94±0.16 n.s. Serum glucose (mg dl-1) 89.5±9.8 90.6±0.6 >0.05 Plasma Homocysteine (µmol/l) 9.0 ±2.3 22.9±13.5 <0.001 Serum Vitamin B12 (pg/ml) 491±185 440±223 0.15 Serum Folate (ng/ml) 9.0±4.4 6.7±5.0 <0.01 Table 2. Biochemical parameters in hypertensive patients with/without chronic kidney disease. Characteristics Patients with Hypertension Patients with Hypertension and CKD (78) (38) Age (years) 53±13 58±16 Systolic blood pressure (mmhg) 140±20 137±19* Diastolic blood pressure (mmhg) 86±11 77±13* Serum creatinine (mg/dl) 0.9±0.2 1.5±0.3 Plasma Homocysteine (µmol/l) 18.5±11.8 26.8±14.5** Serum Vitamin B12 (pg/ml) 465±246 310±160* Serum Folate (ng/ml) 6.9±5.6 6.5±3.4* *p = ns **p<0.05 Traumatic neuroma of the anus 287 produced contrasting results. Some epidemiologic studies do not completely exclude that folate and other vitamins levels, regardless of the concentration of plasma homocysteine, could be associated with a risk of cardiovascular disease (CVD), although they are closely interconnected with the metabolism of homocysteine [12-16]. However, hyperhomocysteinemia could have a role in the development of hypertension. Homocysteine promotes atherogenic and thrombogenic events at the site of endothelial injury, and the auto-oxidation of homocysteine leads to an increased production of oxygen free radicals, resulting in reduction nitric oxide (NO) formation and vascular damage [17]. In the present study, we investigated the associations between biochemical parameters (such as homocysteine, vitamin B12, and folate plasma levels) and hypertension in Caucasian individuals. Materials and Methods Patients. From June 2012 to December 2012, a total of 116 hypertensive patients and 81 healthy subjects, all residents of Sicily, were recruited from the outpatient hypertension center at the University Hospital in Palermo. The group of patients with hypertension was composed of 84 males and 32 females, whereas the control group consisted of 81 subjects, 32 males and 49 females. The mean age (±SD) of patients and control individuals was 54±14 and 57±12 respectively. The patients were defined as hypertensive when the systolic/diastolic blood pressure (BP) was 140/90 mmhg or more, or when the patient was receiving antihypertensive therapy. The severity of hypertension was defined according to the European Society of Hypertension Guidelines [18]. Clinical BP was considered as the average of three consecutive measurements using a mercury sphygmomanometer after the subject had been sitting quietly for 5 minutes.
288 For each subject, plasma concentrations of homocysteine, vitamin B12, and folate were determined. In accordance with the manufacturer s instructions, a serum homocysteine concentration of 11 µmol/l was considered the threshold value for identifying high levels of homocysteine (Immulite; Diagnostic Products Corporation). The normal plasma levels of vitamin B12 and folate were 174-878 pg/ml and 3.0-17 ng/ml respectively (Immulite; Diagnostic Products Corporation). Exclusion criteria for the study included secondary or malignant hypertension, heart failure, a positive history or clinical signs of ischaemic heart disease, cerebrovascular disease, current use of vitamins, dyslipidaemia requiring pharmacological treatment, and known diabetes or fasting hyperglycaemia 126 mg dl. The local ethics committee approved the study protocol; written, informed consent was obtained from each patient. Biochemical analysis. Blood samples were collected in EDTA-treated Vacutainer tubes. Plasma and red blood cells were separated by centrifugation at 4000 rpm for 8 minutes, and the plasma was frozen at -20 C for subsequent analysis. The total plasma homocysteine concentration was determined using the Immulite system, an automated fluorescence polarization immunoassay based on the reduction of protein-bound mixed disulfide forms. Free homocysteine was enzymatically converted into S-adenosyl-L-homocysteine, which was then recognized by a monoclonal antibody. On the other hand, the levels of folate and vitamin B12 were determined in serum by using the kit Immulite. This assay consists of a solid-phase, competitive chemiluminescent enzyme immunoassay with preliminary heat. The solid phase, a polystyrene bead enclosed within an Immulite Test Unit, was coated with a monoclonal antibody specific for folate and vitamin B12 binding protein (Immulite; Diagnostic Products Corporation). Statistical Analysis. Data were expressed as means ± standard deviations (SD). Continuous variables were compared by student s t test. Correlation coefficients (r) were calculated to determine the relation between t-hcy, vitamin B12, and folate in the hypertensive patients group. A P value lower than <0.05 was considered statistically significant. Data were analyzed using the SPSS v. 13.0 statistics software. Results The purpose of our study was to assess the levels of homocysteine, vitamin B12, and folate in hypertensive patients in comparison with healthy control subjects, in order to determine the relationship between these biochemical parameters and hypertension. Table 1 presents the clinical characteristics of hypertensive patients and healthy controls. Our results suggest that hyperhomocysteinemia is constantly present in 77% of hypertensive patients. Significant differences in homocysteine levels were observed between cases and controls. In fact, the homocysteine was 22.9±13.5 µmol/l in the hypertensive group and 9.0±2.3 µmol/l in the control group (p<0.001). On the other hand, folate was significantly lower in the hypertensive group in comparison to the control group (6.7±5.0 vs 9.0±4.4 ng/ml; p<0.01). Finally, the plasma concentrations of vitamin B12 in both hypertensive patients and control subjects were within the normal range (440±223 pg/ml vs 491±184 pg/ml), and no statistically significant differences between the two groups were found. Mean concentrations of plasma homocysteine, serum folate, and vitamin B12 were evaluated in hypertensive patients disaggregated by sex. There were no significant differences in circulating levels of homocysteine between hypertensive women and men (26±13.5 vs 22±13.5 µmol/l respectively, p=0.22). Similarly, mean concentrations of folate (7.0±5.3 ng/ml vs 5.5±2.3 ng/ml p=0.15) and vitamin B12 (410±221 pg/ml vs 517±212 pg/ ml p=0.10) were not significantly different in hypertensive males as compared to that in females. There was no significant correlation between homocysteine and other parameters, including age and blood pressure (Systolic r=-0.15 p=ns, Diastolic r=-0.48 p=ns). In hypertensive patients, there was no statistically significant correlation between t-hcy and vitamin B12 (r=-0,04, p>0.73), but t-hcy was negatively correlated with folate (r=-0,41, p<0.001 (95% CI, -0641, -0217) (Figure 1). In fact 77% of hypertensive patients had high levels of t-hcy, and 78 out of 116 patients had a decreased concentration of plasma folate (<6 ng/ml). Since the hypertensive group included 38 patients with chronic kidney disease (CKD), we observed an increment of t-hcy significantly higher in the latter (26.8±14.5 vs 18.5±11.8), probably caused by a reduced homocysteine clearance from decreased glomerular filtration rate [19], as shown in Table 2.
Traumatic neuroma of the anus 289 Discussion In this study, we demonstrated that hypertensive patients had significantly higher homocysteine levels when compared to control subjects. We found no relationship between the level of homocysteine and the degree of hypertension. There was no correlation between homocysteine levels and systolic or diastolic blood pressure, despite increased homocysteine levels in hypertensive subjects as compared to that of controls. In addition, our study demonstrated that there is no statistically significant difference in the biochemical parameters (such as folate and vitamin B12) between hypertensive patients and hypertensive patients with CKD. Considerable research has reported that the deficiency of B complex vitamins may increase the risk of hyperhomocysteinemia because they are precursors of enzyme cofactors that are necessary for the conversion of homocysteine to methionine. Instead, our results suggest that elevated homocysteine levels are partly due to a deficit of folate. Some studies show that the intake of folic acid can lower homocysteine plasma levels, while vitamin B12 and B6 supplementation seems to have minor effects. Other studies show no correlation between low folate levels and hyperhomocysteinemia, even though folates are required for homocysteine metabolism [20,21,22]. In the literature, the data regarding the association between homocysteine and vitamin B12 is controversial. Some studies reported that levels of vitamin B12 are not correlated with hyperhomocysteinemia, while other authors showed that vitamin B12 levels can modulate those of homocysteine [23,24,25]. In our study there was no statistically significant difference in vitamin B12 plasma levels between the hypertensive and control groups (Table 1). Although vitamin B12 is an important cofactor for methionine synthase, our results do not support a role for vitamin B12 in modulating plasma homocysteine concentrations. Figure 1. Correlation between Homocysteine and folate levels in hypertensive patients However, our results support the important role of folate in remethylation of homocysteine to methionine, as folate is a cofactor in the modulation of methionine synthase and MTHFR enzymatic activity, affecting plasma levels of homocysteine [22,26]. Several studies have reported that folate supplementation can reduce the homocysteine levels in hypertensive patients. Numerous studies have demonstrated that folic acid reduces the concentration of plasma homocysteine by an average of 25%, and folic acid is a useful therapeutic agent to reduce the deleterious effects of homocysteine [27,28,29]. In fact, hyperhomocysteinemia enhances the formation of superoxide and reactive oxygen species, resulting in oxidative stress and damage to endothelium [30]. Many studies have shown that hyperhomocysteinemia can induce proliferation of smooth muscle cells, increased collagen synthesis, and platelet activation with increased platelet adhesion and aggregation [31,32,33]. In agreement with literature data, our research confirmed that hypertension is associated with increased plasma levels of homocysteine (OR:8,9; CI 95% 4.1-19). However, hyperhomocysteinemia is not an independent risk factor for hypertension. These diseases may result from synergism between hyperhomocysteinemia, low folate levels, and parameters of endothelial damage. Because of its toxic effects, hyperhomocysteinemia may enhance damage to arterial wall, leading to hypertension and contributing to cardiovascular pathology.
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