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FARMACIA, 2008, Vol.LVI, 2

The effects of hemodialysis upon the concentrationof homocysteine and reactive oxygen species

ANGELAANTONESCU1, MARIANAMUREŞAN1*, OTILIAMICLE1, LIANAMICLE1,LUCIANA DOBJANSCHI1,CAMELIA MRAZ1, DOROFTEIU M.2

1Faculty of Medicine and Pharmacy, Oradea, P-ta.1 Decembrie 10, 410073

2University of Medicine and Pharmacy “Iuliu Haţieganu” Cluj-Napoca

*corresponding author:

Abstract

Numerous recent studies have revealed a high incidence of hyperhomocysteinemia in renal patients.

The aim of this paper was to point out the associations between the level of homocysteine and the oxidative stress in patients submitted to hemodialysis.

We investigated 30 chronic patients during 5 months from May till September 2007, before and after the periodic hemodialysis.

In order to prove the oxidative stress, we evaluated the level of serum malondyaldehide (MDA) using the tiobarbituric acidmethod, the concentration of carbonylated proteins being determined in the presence of guanidine hydrochloride and the level of circulating ceruloplasmin measured with the Ravin method.

The concentration of homocysteine was determined using an enzymatic method. We also measured a few biochemical parameters such as urea, uric acid, creatinine, total proteins. In the control group we made the same investigations. The results pointed out an increased level of homocysteine before dialysis, also a high concentration of MDA and carbonylated proteins and a low level of ceruloplasmin. After hemodialysis, the MDA and carbonylated proteins concentration are almost the same. The antioxidant response of the organism was absent, which was also demonstrated by the unchanged level of ceruloplasmin.

After hemodialysis, the level of homocysteine was lower. Regarding the biochemical parameters,urea and creatinine presented high level but the rest of them were in normal range.

Increased plasma concentration of homocysteine and oxidative stress markers are found in patients submitted to hemodialysis.

Rezumat

Numeroase studii recente au demonstrat o incidenţă crescută a hiperhomocisteinemiei la pacienţii cu afecţiuni renale.

Scopul lucrării a fost acela de a sublinia relaţia dintre concentraţia homocisteinei şi stresul oxidativ la bolnavii hemodializaţi.

Am investigat un grup de 30 de pacienţi hemodializaţi cronic pe o perioadă de 5 luni (din mai până în septembrie 2007). Pentru a dovedi prezenţa unui stres oxidativ am determinat concentraţia serică a malondialdehidei (MDA) prin metoda cu acid tiobarbituric, a proteinelor carbonilate (metoda cu guanidină hidroclorică) şi ceruloplasmina prin metoda Ravin. Nivelul plasmatic al homocisteinei a fost determinat printr-o metodă enzimatică.

S-au analizat, de asemenea, o serie de parametri biochimici precum: ureea, creatinina, acidul uric, proteine totale.

Înainte de hemodializă, rezultatele au evidenţiat o hiperhomocisteinemie, un nivel seric crescut de MDA şi proteine carbonilate şi valori scăzute ale ceruloplasminei. După hemodializă, concentraţia MDA şi a proteinelor carbonilate se menţine aproximativ la aceleaşi valori. Nivelul plasmatic al ceruloplasminei rămâne nemodificat ceea ce demonstrează o capacitate antioxidantă scăzută a organismului.

După hemodializă, se reduce concentraţia homocisteinei. În ceea ce priveşte parametrii biochimici studiaţi,sunt în limite normale,cu excepţia creatininei şi ureei. Concentraţiile crescute de homocisteină şi de markeri ai stresului oxidativ sunt determinate de modificările funcţiei renale la pacienţii hemodializaţi.

  • reactive oxygen species (ROS)
  • homocysteine
  • hemodialysis
  • creatinine

INTRODUCTION

The kidney plays an important role in homocysteine metabolism [3]. Homocysteine is a non-protein sulfur containing aminoacid that is synthesized from methionine [10]. Increased total homocysteine is very common in renal patients [9,8]. Hyperhomocysteinemia is an additional factor that increases the risk of vascular diseases in general, and particularlyin renal patients [8,11]. In this paper we studied the concentration of total homocysteine in relation to antioxidant status and renal function in patient submited to hemodialysis.

MATERIALS AND METHODS

The study included 30 patients submitted to hemodialysis in the Clinical Hospital Oradea- Department of Nephrology, during five months, from May till September.

Before and after the hemodialysis, the concentration of homocysteine (total serum homocysteine was measured using the enzymatic homocysteine assay, cat no. FHER100, on Hitachi 912 instrument) of malondyaldehide(MDA) (with thiobarbituric acid) and carbonylated proteins (guanidin method) were assessed.

In the same time, the level of ceruloplasmin, the main antioxidant factor in plasma was measured (Ravin method). The results were compared to a control group.

We also estimated the level of creatinine and urea during those months.

RESULTS AND DISCUSSION

Before and after the hemodialysis, the homocysteine concentration was significantly elevated in the blood of the studied patients (fig. 1).

Figure 1

The values of homocysteine in renal patients before

and afterhemodyalisis,compared to the control group

The patients before hemodialysis had an increased level of MDA 2.87 ± 0.35 nmol/ml compared to the control group in which the concentration was 1.91 ± 0.33 nmol/ml. The difference is not significant (p>0.1). Hemodialysis changed slightly the level of MDA to 3.15 ± 0.45 nmol/ml (p=1) (fig. 2).

Figure 2

The values of MDA in renal patients before

and afterhemodyalisis,compared to the control group

The carbonylated proteins had also a high concentration in patients (4.72 ±0.26 nmol/mg proteins) compared to the control group in which the concentration was 1.22 ± 0.11 nmol/mg proteins. This difference is statistically significant (p< 0.001). After hemodialysis,it was not noticed a remarkable difference (p>0.1) (fig. 3).

Figure 3

The values of carbonylated proteins in renal patients before

and after hemodyalisis,compared to the control group

The concentration of ceruloplasmin in the serum of renal patients, before hemodialysis, was lower compared to the control group (from 32.11 ± 1.62 mg% in control group to 25.80 ± 1.23 mg% in patients.) (p< 0.05). After hemodialysis, the values were similar to those before the intervention (from 28.80 ± 1.23 mg % before intervention to 28.11 ± 1.55 mg% after intervention) (p> 0.1) (fig.4). We also assessed creatinine, urea and total protein (table I).

Figure 4

The values of ceruloplasmin in renal patients before

and after hemodyalisis,compared to the control group

Table I

The values of biochemistry parameters in renal patients

Parameters / Normal values / months
May / June / July / August / September
Creatinine (mg%) / 0.6-1.1 mg% / 8.52±2.64 / 9.14±2.1 / 9.36±1.87 / 8.81±2.07 / 8.47±2.25
Urea (mg%) / 15-45 mg% / 158.73±36.42 / 152.15±35.21 / 165.38±57.78 / 145.68±28.97 / 146.87±36.90
Uric acid (mg%) / 3.5-7.2 mg% / 6.77±1.11 / 6.87±1.03 / 7±0.69 / 6.9±0.83 / 6.6±0.91
Total proteins (mg%) / 6.6-8.7 mg% / 6.83±0.52 / 6.81±0.37 / 6.6±0.62 / 6.69±0.58 / 6.53±0.54

Hyperhomocysteinemia is present in the vast majority of maintenance dialysis patients according to almost all descriptive and prevalent studies [7,5,12,15,17].

About 80% of circulating homocysteine is protein bound by disulfide linkage [6]. The rest of unbound homocysteine combines by oxidation, either with itself to form the dimmer homocysteine, or with cysteine to form the mixed disulfide cysteine homocysteine. A small amount circulates as free homocysteine. Total homocysteine represents the sum of all forms of homocysteine including the oxidized and the free homocysteine [1].

Some researchers suggested that the markedly increased plasma homocysteine found in end-stage renal disease patients contributed independently to their very high incidence of fatal and nonfatal cardiovascular disease outcomes [2]. The increased concentration of homocysteine is mainly the result of impaired removal of homocysteine from the blood by the kidney and is independent of it`s vitamin concentration [1].

In our study, the renal patients before dialysis, presented a high level of homocysteine compared to the control group. The difference is statistically significant (p<0.05). The concentration of homocysteine in the patients serum,after dialysis, was significantly lower (p<0.05).

The pathophysiologic explanation for this phenomenon may be a deficiency in homocysteine clearance in the kidneys or an extrarenal cause.

Homocysteine oxidation generates reactive oxygen species. The serum level of MDA was high compared to the control group, but after hemodyalisis, the concentration slightly changed (p=1). The carbonylated proteins were also higher in patients compared to the reference group. The hemodialysis lowered unsignificantly (p>0.1) the level of serum carbonylated proteins.

The concentration of ceruloplasmin in the serum of patients before hemodyalisis and that of the control group were significantly lower (p<0.05). The level of oxidative stress persists after the hemodyalisis and the concentration of ceruloplasmin remains aproximatively constant (p>0,1).

Circulating total homocysteine exist mainly in protein bound form, albumin being the main homocysteine binding protein, but a poor correlation between plasma total homocysteine and serum albumin has been reported in end-stage renal disease patients [15]. In spite of this observation, a study in renal patients during dialysis treatment confirmed the correlation between the levels of total homocysteine and serum albumin [14].

In our study, the level of serum total protein during five months was almost the same and the concentration of homocysteine after dialysis decreased.

Other reports found a strong correlation between homocysteine and serum creatinine. This finding may reinforce the presence of a strong nutritional component of total homocysteine. The correlation between serum creatinine concentration and serum total homocysteine could also be the result of the metabolic association between them. The formation of creatine, the precursor of creatinine, depends on methyl donation by adenosylmethionine to become S-adenosyl-homocysteine leading to the formation of homocysteine. Recent studies have shown that patients with signs of malnutrition have a lower plasma total homocysteine level than patients with normal nutrition [16,15]. The concentration of creatinine in our investigations was significantly higher in comparison with the normal range. Also, urea had a high level due to uremic stage of the patients.

The blood possesses antioxidant factors like vitamin E, C, ceruloplasmin, bilirubin, transferrin, uric acid. Uric acid is a hydrosolubil antioxidant. Its efficiency like defence system is limited just to water solubile reactive oxygen species.

The concentration of uric acid in the group of renal patient studied was elevated. These results also demonstrate the presence of an oxidative stress.

Hyperhomocysteinemia may represent one of many factors in uraemia which contributes to an increased vascular risk. In end-stage renal disease patients, folic acid treatment lowers but does not normalize plasma homocysteine, whereas homocysteine remethylation to methionine increases in healthy controls. These findings indicate a persistent folate-independent defect in metabolic homocysteine clearance in end-stage of renal disease [13].

CONCLUSIONS

  • Concentrations of homocysteine in renal patients after hemodialysis are high.
  • A high level of homocysteine is associated with oxidative stress in those patients.
  • To improve the metabolic profile, we suggest to use anantioxidant treatment such as vitamin B6, B12, and folic acid.
  • Further investigations must establish the necessary doses for vitamin B6, B12 and folic acid.

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