166
FARMACIA, 2008, Vol.LVI, 2
THE EFFECTS OF HEMODIALYSIS UPON
THE CONCENTRATION OF HOMOCYSTEINE
AND REACTIVE OXYGEN SPECIES
ANGELA ANTONESCU1, MARIANA MUREŞAN1*, OTILIA MICLE1,
LIANA MICLE1, LUCIANA DOBJANSCHI1, CAMELIA MRAZ1,
DOROFTEIU M.2
1
Faculty of Medicine and Pharmacy, Oradea, P-ta. 1 Decembrie 10, 410073
University of Medicine and Pharmacy “Iuliu Haţieganu” Cluj-Napoca
*
corresponding author: marianamur2002@yahoo.com
2
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 acid method, 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.
FARMACIA, 2008, Vol.LVI, 2
167
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 particularly
in 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.
168
FARMACIA, 2008, Vol.LVI, 2
RESULTS AND DISCUSSION
Before and after the hemodialysis, the homocysteine concentration
was significantly elevated in the blood of the studied patients (fig. 1).
homocysteine
mol/l
p<0.05
p<0.05
reference
before hemodyalisis
after hemodyalisis
21.93 + 5.66
14.12 + 1.7
7.97 + 1.57
Figure 1
The values of homocysteine in renal patients before
and after hemodyalisis, 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).
MDA
nmol/ml
p=1
reference
p>0.1
before hemodyalisis
after hemodyalisis
2.87 +0.35
3.15 + 0.45
1.91 + 0.33
Figure 2
The values of MDA in renal patients before
and after hemodyalisis, 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
169
FARMACIA, 2008, Vol.LVI, 2
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).
nmol/mg
proteins
p<0.001
p>0.1
p<0.001
4.72+ 0.26
reference
before
hemodyalisis
after
hemodyalisis
4.45 + 0.58
1.22 + 0.11
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).
ceruloplasmin
mg%
p<0.05
p>0.1
reference
before
hemodyalisis
after
hemodyalisis
32.11 + 1.62
25.80 + 1.23
28.11 + 1.55
Figure 4
The values of ceruloplasmin in renal patients before
and after hemodyalisis, compared to the control group
170
FARMACIA, 2008, Vol.LVI, 2
Table I
The values of biochemistry parameters in renal patients
Parameters
Creatinine
(mg%)
Urea
(mg%)
Uric acid
(mg%)
Total
proteins
(mg%)
Normal
values
0.6-1.1
mg%
15-45
mg%
3.5-7.2
mg%
6.6-8.7
mg%
May
June
months
July
August
September
8.52±2.64
9.14±2.1
9.36±1.87
8.81±2.07
8.47±2.25
158.73±36.42
152.15±35.21
165.38±57.78
145.68±28.97
146.87±36.90
6.77±1.11
6.87±1.03
7±0.69
6.9±0.83
6.6±0.91
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.
FARMACIA, 2008, Vol.LVI, 2
171
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.
172
FARMACIA, 2008, Vol.LVI, 2



A high level of homocysteine is associated with oxidative stress in
those patients.
To improve the metabolic profile, we suggest to use an antioxidant
treatment such as vitamin B6, B12, and folic acid.
Further investigations must establish the necessary doses for vitamin
B6, B12 and folic acid.
REFERENCES
1. Dou C, Xia D, Zhang L, Chen X, Flores P, Datta A, Yuan C.
Development of a novel enzymatic cycling assay for total
homocysteine, Cinical Chemistry 2005; 51, 1987-1989
2. Faure-Delanef L, Quere I, Chasse JF, Guerassimenko O, Lesaulnier M,
Bellet H, et.al. Methylenetetrahydrofolate reductase thermolabile
variant and human longevity, Am J Hum Genet 1997, 60, 999-1001
3. Friedman AN, Bostom AG, Selhub J, Levery AS, Rosenberg IH. The
kidney and homocysteine metabolism, J Am Soc Nephrol 2001, 12,
2181-35
4. Kalantar-Zadeh K, Block G, Humphreys MH, McAllister CJ, Kopple
JD: A low, rather than high, total plasma homocysteine is an indicator
of poor outcome in hemodialysis patients, J Am Soc Nephrol 2004, 15,
442-453
5. Mallamaci F, Zoccali C, Tripepi G, Fermo I, Benedetto FA, Cataliotti
A, Bellanuova I, Malatino LS, Soladrini A: Hyperhomocysteinemia
predicts cardiovascular outcomes in hemodialysis patients, Kidney Int
2002, 61, 609-614
6. McLean R, Jacques PF, Selhub J, Tucker KL, Samelson EJ, Broe KE,
et.al. Homocysteine as a predictive factor for hip fracture in older
persona, N Engl J Med 2004, 350, 2042-2049
7. Mezzano D, Pais EO, Aranda E, Panes O, Downey P, Ortiz M, Tagle R,
Gonzales F, Quiroga T, Caceres MS, Leighton F, Pereira J:
Inflammation, not hyperhomocysteinemie, is related to oxidative stress
and hemostatic and endothelial dysfunction in uremia, Kidney Int 2001,
60, 1844-1850
8. Moustapha A, Naso A, Nahlawi M, Gupta A, Arheart KL, Jacobsen
DW, et.al. Prospective study of hyperhomocysteinemia as an adverse
cardiovascular risk factor in end-stage renal disease, Circulation 1998,
97, 138-41
9. Obeid R, Kuhlmann M, Kohler H, Herrmann W. Response of
hyperhomocysteinemic dialysis patients, Clin Chem, 2005, 51, 196-201
FARMACIA, 2008, Vol.LVI, 2
173
10. Pastore A, De Angelis S, Kasciani S, Ruggia R, Di Giovamberardino G,
Noce A, Splendiani G, Cortese C, Federici G, Dessi M: Effects of folic
acid before and after vitamine B12 on plasma homocysteine
concentrations in hemodialysis patients with known MTHFR
genotypes, Clinical Chemistry, 2006, 52, 1:145-148
11. Robinson K, Gupta A, Dennis V, Arheart K, Chaudhary D, Green R,
et.al. Hyperhomocysteinemia confers an independent increased risk of
atherosclerosis in end-stage renal disease and is closely linked to
plasma folate and pyridoxine concentrations, Circulation 1996, 94,
2743-8
12. Sirrs S, Duncan L, Djurdjev O, Nussbaumer G, Ganz G, Frohlich J,
Levin A: Homocysteine and vascular access complications in
haemodialysis patients: Insights into a complex metabolic relationship,
Nephrol Dial Transplant, 1999; 14, 738-743
13. Stam F, van Guldener C, Ter Wee PM, Jakobs C, De Meer K,
Stehouwer CDA: Effect of folic acid on methionine and homocysteine
metabolism in end-stage renal disease, Kidney Int. 2005, 67:259-264
14. Suliman ME, Barany P, Zadeh KK, Lindholm B, Stenvinkel P.
Homocysteine in uraemia – a puzzling and conflicting story, Nephr.
Dialys. Transplant, 2005, 20(1):16-21
15. Suliman ME, Qureshi AR, Barany P, Stenvinkel P, Filho JC,
Anderstam B, Heimburger O, Lindholm B, Bergstrom J:
Hyperhomocysteinemia, nutritional status, and cardiovascular disease in
hemodialysis patients, Kidney Int, 2000, 57:1721-1735
16. Suliman ME, Stenvinkel P., Heimburger O et.al. Plasma sulfur amino
acid in relation to cardiovascular disease, nutritional status, and diabetes
mellitus in patients with chronic renal failure at start on therapy, Am. J.
Kidney Dis. 2002; 40:480-488
17. Wrone EM, Zehnder JL, Hornberger JM, McCann LM, Coplon NS,
Fortmann SP: An MTHFR variant, homocysteine, and cardiovascular
comorbidity in renal disease, Kideny Int. 2001, 60:1106-1113.