Effects of methionine loading on plasma and erythrocyte sulphur amino acids and sulph-hydryls before and after co-factor supplementation in haemodialysis patients

Mohamed E. Suliman1, José C. Divino Filho1,2, Peter Bárány1, Björn Anderstam1, Bengt Lindholm1 and Jonas Bergström,1

1 Divisions of Baxter Novum and Renal Medicine, Department of Clinical Science, Huddinge University Hospital 2 Sophiahemmet Dialysis Unit, Karolinska Institutet, Stockholm, Sweden



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hyperhomocysteinaemia, which is potentially atherogenic, is common in chronic haemodialysis (HD) patients but the reason for this is not yet known. The methionine (Met) loading test (MLT) is used to test the capacity of homocysteine (Hcy) disposal by the trans-sulphuration pathway and thus may provide information on the metabolism of sulphur amino acids. The availability of vitamin B6 and folic acid, as co-factors for Hcy metabolism may affect the response to MLT. In the present study, we compared the effect of Met loading on plasma and erythrocyte (RBC) sulphur amino acids and sulph-hydryls before and after co-factor supplementation in healthy subjects and HD patients.

Methods. In 10 HD patients and 10 healthy subjects the effect of Met loading, 0.1 g/kg BW, on plasma and RBC methionine metabolites was studied over 7 h, before and after 4 weeks supplementation with high daily doses of vitamin B6 (200 mg) and folic acid (15 mg).

Results. MLT before vitamin supplementation in HD patients, compared to the healthy subjects, caused significantly greater increases in plasma Hcy levels (43±12 vs 15±5 µmol/l), cysteinesulphinic acid (CSA) (1.34 vs 0.36 µmol/l) and {gamma}-glutamylcysteine (0.98±0.83 vs –01±0.42 µmol/l) and no decline in plasma cysteine (Cys) (0.5±33.9 vs -31±26 µmol/l), but no significant differences in plasma taurine, cysteinylglycine, and glutathione concentrations. In RBCs there was a small increase in Hcy levels and a more marked increase in Tau levels, with no difference between the healthy subjects and HD patients. Vitamin supplementation in pharmacological doses failed to correct the abnormal responses to MLT in the HD patients.

Conclusions. Oral methionine loading in HD patients leads to higher accumulation of Hcy and other Met metabolites in plasma and RBCs than in healthy subjects, indicating impaired metabolism of sulphur amino acids via the trans-sulphuration pathway. Supplementation with high doses of vitamin B6 and folic acid does not correct this impairment, suggesting that it most probably is not due to lack of these co-factors.

Keywords: erythrocyte; folic acid; haemodialysis; homocysteine; methionine loading test; sulphur amino acids; vitamin B6



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In hyperhomocysteinaemia, a characteristic feature of patients treated with maintenance haemodialysis (HD) [1,2], both plasma and erythrocyte (RBC) total homocysteine (Hcy) concentrations are elevated [2,3]. Hyperhomocysteinaemia is considered to be an independent risk factor for atherosclerosis in the general population [4] and there is evidence, although conflicting, that it may predispose to cardiovascular disease in patients with chronic renal failure [57]. Dialysis patients have also been reported to have low plasma and muscle concentrations of taurine (Tau) [8] in the presence of elevated concentrations of cysteinesulphinic acid (CSA) [9], suggesting an inhibition of CSA decarboxylase, which is a rate-limiting enzyme in the synthesis of Tau from CSA. The cause of this metabolic effect and its clinical implications are not known.

In mild hyperhomocysteinaemia it is generally assumed that high basal Hcy concentrations reflect impairment of the re-methylation pathway [10]. Such impairment may be due to low folate or vitamin B12 levels, or due to defects in the gene encoding for 5,10-methylenetetrahydrofolate reductase (MTHFR). The product of this enzymatic reaction, 5-methyltetrahydrofolate (MTHF), acts as a methyl donor in re-methylation of Hcy [11]. A second route of disposal of Hcy is via the trans-sulphuration pathway. Impairment of trans-sulphuration, which may be caused by defects in the cystathionine ß-synthase (CBS) gene or a vitamin B6 deficiency, may be diagnosed by an abnormal increase in plasma Hcy 4–8 h after the oral methionine (Met) loading test (MLT) [12]. This abnormality can be attenuated in B6-deficient subjects by treatment with vitamin B6 [13]. The concentration of intracellular Hcy is normally low. When Hcy production increases or its metabolism is inhibited, Hcy is exported from the cells into the extracellular medium as an alternative route of Hcy disposal, thus markedly increasing the extracellular Hcy concentration [14]. Figure 1Go summarizes the sulphur amino acid (sAA) metabolic pathways.



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Fig. 1. The methionine metabolic pathway. BHMTF, betaine-homocysteine methyl transferase; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; CSA, cysteinesulphinic acid; CSAD, cysteinesulphinic acid decarboxylase.

 
In a previous study we examined in detail the fasting plasma and RBC concentrations of sAA in HD patients and healthy subjects, and the effects of high doses of folic acid and vitamin B6 supplementation [2]. In the present study of the same population, the effects of oral methionine loading on plasma and RBC methionine metabolites in HD patients and healthy subjects were studied over 7 h. In addition, the effects of supplementation with high doses of vitamin B6 and folic acid on MLT were evaluated.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Ten HD patients (six men) with a mean age of 66 years (range 44–82) and 10 healthy subjects (five men) with a mean age of 45 years (range 32–65) participated in the study. Body mass index (BMI, mean±SD) was within normal range for the HD patients (22±3) and healthy subjects (22±2). The biochemical characteristics of both groups are shown in Table 1Go. Additional clinical data for the same population have been reported elsewhere [2]. The HD patients were dialysed three times weekly, using hollow-fibre dialysers, glucose-containing dialysate with bicarbonate as the buffer, blood flow between 300 and 350 ml/min, and a dialysate flow of 500 ml/min. The protein equivalent of nitrogen appearance normalized to body weight (nPNA) and the dialysis dose of the HD patients, expressed as Kt/Vurea, were calculated, based on urea kinetic modelling [15]. All patients had a nPNA >1.0 g protein/kg body weight/day and the mean Kt/Vurea was 1.64±0.25.


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Table 1. Baseline biochemical characteristics of haemodialysis patients (HD) and healthy subjects (HS) (mean±SD)

 
Routine medication included daily supplementation with water-soluble vitamins, including vitamin B6 (10 mg daily), sodium bicarbonate, phosphate binders, and diuretics. No patient was on folic acid or B12 supplementation during the 3-month period before starting the study. The nature, purpose, and potential risks of the study were carefully explained to all participants before they agreed to take part. The local Ethics Committee of Karolinska Institutet at Huddinge University Hospital approved the study protocol.

At 07.00 hours, the MLT was done on a mid-week dialysis day, after an overnight fast, by giving L-methionine orally in a dose of 0.1 g/kg body weight, in about 200 ml of fruit juice. Blood samples were collected immediately before (fasting) and at 2, 4, 6 and 7 h after the methionine intake. Patients then underwent HD and a blood sample was collected from each patient after the dialysis session and again before the next session (48 h). All participants received the same type of breakfast between 07.00 and 08.00 hours and the same type of lunch after the 6-h sample. The breakfast consisted of two slices of bread with butter and cheese and the lunch was a low-protein meal.

When the MLT was completed, the healthy subjects and HD patients were instructed to take vitamin tablets containing 15 mg folic acid and 200 mg vitamin B6 daily for 4 weeks. Both groups were encouraged to continue their usual diet during the study. The HD patients took their usual medication and the dialysis prescriptions were not changed throughout the study. The MLT was repeated 4 weeks later and blood samples were taken under the same condition as before.

Blood samples were collected in cooled EDTA tubes, centrifuged immediately at 4°C and then the plasma and RBCs were separated and treated, as previously described [2]. The plasma and RBCs sulph-hydryls (Hcy, cysteine (Cys), cysteinylglycine (Cys-Gly), {gamma}-glutamylcysteine ({gamma}-Glu-Cys) and glutathione (GSH)) were determined by high-performance liquid chromatography (HPLC) using fluorescence detection, according to the method of Ubbink et al [16], with only a minor modification, the HPLC analysis time being extended from 6 to 10 min. The plasma and RBC free AA concentrations were determined by HPLC, as previously described [9]. Concentrations of pyridoxal-5'-phosphate (PLP) in plasma and RBCs were determined with HPLC, according to Kimura et al [17]. Folate and vitamin B12 concentrations were determined with the Dualcount SPNB (solid phase no boil) radioimmunoassay kit from DPC (Diagnostic Product Corporation, Los Angeles, CA, USA).

Statistical analysis
Values are expressed as mean±SD. The concentration of the area under the curve over 7 h was calculated with the trapezoidal rule method. The data from the HD patients and healthy subjects before and after vitamin supplementation were compared with the Mann–Whitney U test. Within-group comparisons were evaluated with Wilcoxon's signed-rank test. Correlations were tested with Spearman's rank correlation test. A probability value of <0.05 was considered significant.



   Results
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 Subjects and methods
 Results
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The basal concentrations of Met metabolites differed significantly between the HD patients and the healthy subjects. We therefore used the maximum changes in metabolite concentrations, calculated as the difference between the basal concentration and the concentration at the time after Met loading when the mean difference was maximal, for comparisons between the groups. Tables 2Go and Table 3Go show for each metabolite the basal concentration, maximum change in concentration after Met loading, and the difference between the maximum change and basal concentrations ({Delta}conc.) in plasma and RBC concentrations respectively, before and after vitamin B6 and folic acid supplementation in the HD patients and the healthy subjects.


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Table 2. Basal, maximum and change ({Delta}conc.) in the plasma concentrations (µmol/l) of various sAA and sulph-hydryls in response to methionine loading before and after vitamin supplementation in healthy subjects and haemodialysis patients

 

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Table 3. Basal, maximum and change ({Delta}conc.) in the erythrocyte concentrations (µmol/l ICW) of various sAA and sulph-hydryls in response to methionine loading before and after vitamin supplementation in healthy subjects and haemodialysis patients

 
Calculating the maximum change may introduce a bias into the data since the value depends on only one-point determination after MLT [13]. Therefore, to determine the effect of MLT on Met metabolites more exactly, the effect of methionine supplementation was expressed as an area under the concentration curve over 7 h. Since the response was superimposed on the basal area, we calculated the incremental area (total area minus basal area under the curve). Only a few unimportant discrepancies were observed between the maximum changes in metabolite levels, as shown in Tables 2Go and 3Go, and changes expressed as the incremental areas (data not shown) after Met loading.

In the healthy subjects, MLT caused significant increases in the plasma concentrations of Hcy, CSA and Cys-Gly, a fall in plasma concentration of Cys by more than 10% and a slight fall in plasma GSH, compared to the basal values (Table 2Go, Figure 3Go). The Hcy concentration increased slightly in the RBCs (Table 3Go). The plasma Tau concentration did not change, while the RBC Tau concentration increased by more than 100% compared to the basal level (Tables 2Go and 3Go). The responses to MLT showed no significant change after supplementation with vitamins for 4 weeks, except that plasma Met increased significantly more and RBC Met significantly less compared to values before vitamin supplementation (Tables 2Go and 3Go, Figure 2Go).



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Fig. 3. The effect of the oral methionine loading test (MLT) on the plasma and erythrocyte (RBC) homocysteine concentrations during 7 h • before and {circ} after supplementation with 200 mg B6 and 15 mg folic acid daily for 4 weeks in healthy subjects (HS) and haemodialysis (HD) patients. The HD patient values in plasma and RBCs show the effects of HD treatment and the concentrations 48 h after MLT. EHD, end of haemodialysis; ICW, intracellular water.

 


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Fig. 2. The effect of the oral methionine loading test (MLT) on the plasma and erythrocyte (RBC) methionine concentrations during 7 h, • before and {circ} after supplementation with 200 mg B6 and 15 mg folic acid daily for 4 weeks in healthy subjects (HS) and haemodialysis (HD) patients. The HD patient values in plasma and RBCs show the effects of the HD treatment and the concentrations 48 h after MLT. EHD, end of haemodialysis; ICW, intracellular water.

 
In the HD patients, MLT caused significantly higher increases in the plasma Hcy concentration than in the healthy subjects, whereas the increase of Hcy in RBCs did not differ (Tables 2Go and 3Go, Figure 3Go). Plasma Cys was unchanged (no reduction after MLT as in healthy subjects), plasma CSA and {gamma}-Glu-Cys increased, while plasma Cys-Gly and GSH decreased (Table 2Go). Vitamin supplementation resulted in a lower maximum plasma concentration of Hcy after Met loading than the levels before vitamin supplementation, and a slightly (but not significantly) lower increase in Hcy from the basal level. After methionine loading, the plasma CSA increased significantly more in the HD patients than in the healthy subjects, while plasma Cys-Gly and GSH decreased in both groups (Tables 2Go and 3Go, Figures 2Go and 3Go). As in the healthy subjects, the plasma Tau concentration did not change, while the RBC Tau concentration increased markedly after MLT (Tables 2Go and 3Go). The increase in RBC Tau concentration was not significantly different before and after vitamin supplementation.

The basal concentrations of plasma folate, PLP and vitamin B12 were within the normal range; while the RBC concentrations of folate and PLP in HD patients were significantly higher than in the healthy subjects. Of these vitamins only plasma folate correlated significantly with the maximal postload Hcy level in the healthy subjects (P=0.03) but not in the patients.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Selhub and Miller [11] advanced a unified biochemical hypothesis to explain the pathogenesis of hyperhomocysteinaemia, according to which impairment of one Hcy metabolic pathway necessarily leads to impairment of the other one, resulting in hyperhomocysteinaemia. Essential to this hypothesis is the co-ordinating role of S-adenosylmethionine (SAM), which is both an allosteric inhibitor of MTHFR and an activator of CBS. Impaired re-methylation of Hcy decreases de novo Met synthesis, which greatly reduces the cellular SAM concentration and Hcy accumulation because of unstimulated CBS. Impairment of trans-sulphuration initially accelerates de novo synthesis of Met by re-methylation until SAM accumulates to such an extent that it is impossible to dispose of excessive Hcy by re-methylation.

The reason why basal plasma Hcy is elevated in CRF patients is not yet known. It has been shown that after Hcy loading the plasma clearance of Hcy is reduced in patients with CRF, indicating that the disposal of Hcy is impaired [18]. There is also a net uptake of Hcy by the rat kidney in vivo [19], suggesting that reduced renal elimination might play a role. On the other hand, no significant renal extraction of Hcy occurs in normal man during fasting [20], thus contradicting the suggestion that reduced renal elimination might be a cause of hyperhomocysteinaemia. However, it is still possible that the kidney may be involved in the elimination of Hcy in the non-fasting condition or after MLT. Homocysteine is generated from Met via SAM and S-adenosylhomocysteine (SAH). In HD patients, plasma levels of both these metabolites are reportedly elevated. SAH, but not SAM, accumulates in RBCs, and the SAM:SAH ratio is low in both plasma and RBCs [3,21]. SAH is a potent inhibitor of enzymatic methylation reactions, and consequently the SAM-dependent co-ordinated regulation of the re-methylation and trans-sulphuration pathways can be disturbed [11]; but it is not yet clear to what extent an impaired co-ordination of these pathways is involved in hyperhomocysteinaemia of patients with renal failure.

Supplementation with pharmacological oral doses of folic acid reduces plasma Hcy levels in HD patients, but does not normalize them [2,22,23]. The use of MTHF orally appears to be more efficient, resulting in normalization of plasma Hcy levels in about one-third of the patients, concomitantly with increases in RBC levels of SAM and Met, indicating an improvement in the re-methylation [24]. Recently, it has been shown in HD patients that treatment with 50 mg parenteral folinic acid (5-formyl tetrahydrofolate) and 250 mg pyridoxine once a week normalizes fasting plasma Hcy levels in 78% of patients, presumably by restoring the impaired capacity of re-methylation of Hcy to Met [25]. These results suggest that the primary cause of hyperhomocysteinaemia in HD patients is impaired re-methylation, presumably caused by resistance to folate action due to abnormal metabolism of folic acid into functionally active MTHF [26]. The results also suggest that impairments in folate metabolism can be circumvented by intravenous administration of folinic acid, which is interconvertible with 5,10-methylenetetrahydrofolate, from which MTHF is synthesized. However, it should be emphasized that until now no controlled study has been published, which compares the effect of folic acid with folinic acid or MTHF administration in equivalent doses and with the same period and mode of administration.

We reported previously [2] that the total plasma concentrations of Hcy, Cys, GSH, Cys-Gly, {gamma}-Glu-Cys and CSA and the RBC concentrations of Hcy and Cys-Gly were significantly higher in HD patients than in healthy subjects and that supplementation with high doses of B6 and folic acid lowered the plasma concentration of Hcy, although not to normal levels in the HD patients. The present study, which involves the same population, provides for the first time data describing changes after Met loading not only in plasma but also in RBCs in healthy subjects and HD patients. Moreover, we studied the effect not only on Hcy but also on several other sulphur compounds (Tables 2Go and 3Go). The MLT was repeated after 4 weeks supplementation with 15 mg folic acid and 200 mg B6, i.e. pharmacological doses of both vitamins.

We observed that Met loading before vitamin supplementation resulted in an increase in the plasma Hcy concentration, which was about three times higher in the HD patients than in healthy subjects. However, in RBCs, in which the basal Hcy levels were much lower than in plasma, the concentration increased much less, with no difference between the groups, indicating that the intracellular environment is protected from excessive accumulation of Hcy, presumably by active export from the cells. After 4-weeks treatment with high doses of folic acid and B6, the fasting and maximum plasma Hcy levels after Met loading decreased in both groups. However, the post-load increase was only slightly but not significantly lower in the HD patients after vitamin supplementation, indicating that pharmacological doses of folic acid and B6 over 4 weeks did not influence Hcy trans-sulphuration to any appreciable extent. Our patients were evidently not B6 depleted prior to the study, presumably because they took multivitamin tablets daily, including 10 mg B6.

There are few previous studies of Hcy using the MLT in patients with CRF. Hultberg et al [27] has reported slightly higher post Met load levels of plasma Hcy in a small mixed group of CRF patients, in whom the vitamin status was not well defined. In stable renal transplant patients with slight renal insufficiency (s-creatinine 133 µmol/l), Bostom et al [28] found higher fasting plasma Hcy levels and larger increases in plasma Hcy 2 h after Met loading than in controls, suggesting impairment of the trans-sulphuration. One reason for this might have been B6 depletion, since there was an inverse relation between post-load Hcy and B6 levels. Van Guldener et al [29] reported in HD patients a large increase in the plasma Hcy levels 6 h after Met loading, which was significantly smaller after 12 and 52 weeks of supplementation with 5 mg folic acid daily, with or without betaine. They speculated that the elevated post-load Hcy levels are not necessarily caused by trans-sulphuration impairment alone or, alternatively, that folic acid may have indirect effects on trans-sulphuration, e.g. via changes in intracellular regulatory factors such as SAM and SAH. Our results in the HD patients are largely in agreement with those of van Guldener et al [29], except that the post-load increase in plasma Hcy was not significantly lower after vitamin supplementation than before, possibly due to the small number of subjects in our study.

Taken together, these findings suggest that trans-sulphuration is impaired in chronic HD patients. This might be secondary to a disturbance in re-methylation, as outlined by Selhub and Miller [11], or due to a direct effect of uraemic intoxication, although it cannot be excluded that impaired renal function might play a role in the reduced disposal of Hcy after Met loading. A recent study by van Guldener et al [30], using stable isotopes, showed that Hcy re-methylation was decreased in HD patients, whereas trans-sulphuration did not differ from that in healthy persons. However, their study was performed after an overnight fast, preceded by 3 days on a fixed protein intake, i.e. under such steady-state conditions that the flux through the trans-sulphuration pathway should balance the food intake of Met and the net release of Met from body proteins, which should be approximately equal in HD patients and controls. Hence, it is conceivable that, after an acute Met load, an impairment of the trans-sulphuration in HD patients might be revealed, which is not seen in steady-state conditions.

After Met loading, the marked increase in plasma Met in the HD patients was similar to that seen in the healthy subjects and the magnitude and time-course of the increase in RBC Met was also essentially similar in the HD patients and healthy subjects (Figure 2Go). The parallel increase of Met to similar levels in plasma and RBCs indicates that the RBC membrane is readily permeable to Met. Erythrocytes have been shown to play an important role in the inter-organ transport of various amino acids [31]. Our results suggest that the rapid disappearance of Met from plasma after an oral load may be due to rapid cellular uptake and efficient inter-organ distribution and intracellular metabolism of Met.

In agreement with a previous study [32] the hyperhomocysteinaemia induced after Met loading in the healthy subjects was associated with a transient reduction in plasma total Cys, which was attributed to the displacement of Cys from its protein binding sites by Hcy [32]. This may cause an increase in free Cys, which is then easily metabolized. However, we observed in the HD patients that the total Cys concentration after Met loading did not change. An explanation might be that in patients with no renal function, a surplus of free Cys, generated by displacement from its protein binding site by Hcy, is not adequately disposed of due to impaired metabolism, and is consequently retained in the body.

As earlier reported, the plasma CSA concentration was much higher in the HD patients than in healthy subjects [9]. Likewise, after Met loading the increase in CSA was higher in the HD patients and the responses were not significantly affected by vitamin supplementation. These findings are in keeping with a reduced activity in uraemia of cysteinesulphinic acid decarboxylase [9], which is the key enzyme for the endogenous production of Tau from Cys.

The plasma Tau levels did not change significantly after MLT; however, the interindividual variations were large. On the other hand, RBC Tau levels increased markedly in both groups, indicating that Tau, generated from Met, was taken up in the RBCs and presumably disposed of by transport to and storage in various cells and tissues. Evidently, the defect in CSA metabolism in renal failure does not respond to pharmacological doses of folate and vitamin B6.

GSH, which is considered to be an important antioxidant in blood, is present in high concentration in RBCs while the concentration in plasmas is low, although higher in HD patients than in healthy subject [2]. The Met loading had no significant effect on GSH levels in RBC (except for a small increase in the healthy subjects before vitamin supplementation) and small decreases in plasma GSH were observed both in the healthy subjects (before vitamin supplementation) and in the HD patients (before and after supplementation). These observations suggest that GSH is but little affected by Met loading. In agreement with this conclusion is the lack of significant effects on the levels in RBC of {gamma}-Glu-Cys, a precursor of GSH, and Glu-Gly, a product of GSH metabolism.

To summarize, the findings in this study indicate that oral methionine loading in HD patients causes accumulation of Hcy and some other methionine metabolites in plasma and to a lesser extent in RBCs and that metabolism of sAA via the trans-sulphuration pathway is impaired. Supplementation with high doses of pyridoxine and folic acid does not correct the impairment in trans-sulphuration, indicating that this abnormality is not due to deficiency of these co-factors.



   Acknowledgments
 
This study was supported by the Karolinska Institutet, Stockholm, Sweden and by grants from the Baxter Healthcare Corporation, McGaw Park, Illinois, USA. The authors thank the entire staff of the Sophiahemmet Dialysis Unit, Stockholm.



   Notes
 
Professor Jonas Bergström, Division of Baxter Novum and Renal Medicine, K-56, Huddinge University Hospital, Karolinska Institutet, S-14186 Stockholm, Sweden. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 15. 9.99
Revision received 4. 9.00.