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The influence of the hemodialysis treatment time under oxidative stress biomarkers in chronic renal failure patients
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Biomedicine & Pharmacotherapy 62 (2008) 378e382 www.elsevier.com/locate/biopha
Original article
The influence of the hemodialysis treatment time under oxidative stress biomarkers in chronic renal failure patients Juliana Valentini a, Denise Grotto a, Clo´vis Paniz a, Miguel Roehrs a, Geni Burg b, Solange C. Garcia a,* a
Department of Clinical and Toxicological Analysis, Federal University of Santa Maria, Campus Universita´rio, Caixa Postal 5061, CEP 97110-970, Santa Maria, Rio Grande do Sul , Brazil b Nurse of Unitate of Renal Clinical of Caridade and Casa of Sau´de Hospitals, Santa Maria, RS, Brazil Received 13 June 2007; accepted 24 October 2007 Available online 17 December 2007
Abstract Summary: Oxidative stress possibly helps to promote the progression and complication of chronic renal failure (CRF). Hemodialysis (HD) may aggravate oxidative stress. In addition long time of treatment may intensify the oxidative stress. Thus, the aim of this study was to evaluate the effect of prolonged HD treatment under parameters of the oxidative stress. Methods: Plasmatic thiobarbituric acid reactive substances (TBARS), plasmatic malondialdehyde (MDA), blood d-aminolevulinate dehydratase (ALA-D) activity, ALA-D reactivation index, and erythrocytic reduced glutathione (GSH) were measured into two different groups of HD patients: recent treatment (n ¼ 36; HD duration: 17.7 1.71 months), and long time of treatment (n ¼ 26; HD duration: 82.2 6.32 months), and in a control group (n ¼ 40). Results: Plasmatic TBARS and MDA levels were both elevated in HD patients. However, only MDA levels had positive correlation with time of HD treatment. Blood ALA-D activity was decreased in HD patients. The ALA-D reactivation index showed increase in HD patients, and it had correlation with the time of HD treatment. Erythrocytic GSH levels were increased in HD patients. Conclusions: Our results indicated that MDA levels and ALA-D reactivation index may be the better biomarkers to evaluate chronic oxidative stress in comparison with others markers analyzed in this study. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Hemodialysis treatment time; ALA-D activity; Oxidative stress; MDA; GSH
1. Introduction Inadequate antioxidant protection or excessive production of reactive oxygen species create a condition known as oxidative stress, which has an important role in the etiology of various disease and in aging [1,2]. This condition leads to structural and/or functional deterioration in cell components including DNA, proteins, carbohydrates and lipids [3]. In addition, this state is involved in many pathophysiological processes, particularly within the atherosclerosis context, inflammation and cancer
* Corresponding author. Tel.: þ55 55 3220 8941; fax: þ55 55 3220 8018. E-mail address: [email protected] (S.C. Garcia). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.10.017
[4e7], with higher prevalence in patients with chronic renal failure (CRF) compared with age-matched controls from the general population [3]. Indeed, CRF has emerged as a major example of disease-associated oxidative stress, because of the excessive production of oxidants accompanied by inadequate antioxidant defenses [8e10]. In CRF patients under hemodialysis (HD) treatment the formation of reactive oxygen species (ROS) is amplified, therefore beyond uremic toxins, the hemodialysis itself due to bio-incompatible dialysis water, non-sterile dialysate, poor quality of dialysis water, and back-leak of contaminants across the dialysis membrane. The other causes of enhanced oxidative stress are advanced age [11], high frequency of diabetes [12], chronic inflammatory state [13], excessive parenteral
J. Valentini et al. / Biomedicine & Pharmacotherapy 62 (2008) 378e382
iron administration [14], and time of hemodialysis treatment [15]. Previous studies have reported that longer time of HD treatment may amplify the oxidative stress, and it contributes to the increase of the morbidity and mortality in these patients, particularly in relation to cardiovascular disease [3,15,16]. The presence of ROS can cause damage in many molecules, such as lipids, leading to the production of malondialdehyde (MDA), an indicator of lipid peroxidation [17e19]. However, studies about influence of the time of HD treatment assessed the plasmatic levels of thiobarbituric acid reactive substances (TBARS), and its results were contradictors, with positive correlation between TBARS and the time of HD treatment [20], and not correlation [3]. These works did not report the MDA levels and their relation with time of HD treatment. d-Aminolevulinate dehydratase (ALA-D) is an essential enzyme for aerobics organisms, once it catalyses the synthesis of tetrapyrrolic compounds such as billins and hemes. ALA-D is a sulfhydryl enzyme which is dependent on Zn2þ ion binding to display full activity and it is sensitive to situations associated with oxidative stress [21,22]. Importantly, d-ALA-D activity is decreased in CRF, especially in HD treatment [23e26]. However, works relating d-ALA-D activity with duration of dialysis therapy not have. Based on the fact that the duration of dialysis treatment can amplify the oxidative stress, the present study was designated to evaluate the levels of plasmatic TBARS and MDA, blood ALA-D activity, ALA-D reactivation index, and erythrocytic reduced glutathione (GSH) levels in HD patients with different times of HD treatment compared with a control group. 2. Materials and methods
379
(HD time: 82.6 6.32 months), with 51 11.6 years of age (ranging between 29 and 75 years). Control group consisted of 40 healthy subjects (20 men, 20 women), with 43.15 7.1 years of age (ranging between 30 and 57 years), which did not have clinical history of renal diseases or another pathologies. All the volunteers did not receive vitamins, were non-smokers, and did not consume alcohol regularly. 2.3. Samples Blood venous samples (10 ml) were drawn from HD patients and controls subjects, and this were divided in heparinized tubes, EDTA-containing tubes, and tubes without anticoagulant. PlasmaeEDTA and serum were obtained by centrifugation at 1500g for 10 min at 4 C. 2.4. Hematological determinations Hemoglobin (Hb) and hematocrit (Hct) were determined in Cobas Micros system (Hematology Analyzer, Roche Diagnostics). 2.5. Lipid peroxidation Lipid peroxidation was estimated by the measurement of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA). TBARS assay were realized in plasmaeEDTA samples according to the method of Okhawa et al. [27]. The measurement of plasmatic MDAwas determined by high performance liquid chromatographic with visible detection (HPLCeVIS), according to the method of Grotto et al. [28].
2.1. Chemicals 2.6. d-ALA-D activity 50 -Aminolevulinic acid (ALA), 2-thiobarbituric acid (TBA), dithiothreitol (DTT), and reduced glutathione (GSH) were purchased from Sigma (St. Louis, USA). All other chemicals used in this study were of the highest purity available. 2.2. Subjects Sixty-two patients with diagnosis of CRF (35 men and 27 women) undergoing regular hemodialysis (HD) treatment at Caridade and Casa of Sau´de Hospitals, located in Santa Maria, RS, Brazil. A study protocol was approved by the Human Ethics Committee of the Health Science Center from the Federal University of Santa Maria (protocol no: 091/2003) and all the patients gave their informed consent prior to the inclusion in the study. Patients with alcoholism, smoking, diabetes, viral hepatitis and HIV, and use of any antioxidant vitamin within last 3 months were excluded. Patients HD included in the study were divided into two groups in accordance with the time of HD treatment: recent time (RT) of HD treatment group comprised 36 patients (HD time 17.7 1.71 months) with 53.6 12.8 years of age (ranging between 20 and 77 years). Long time (LT) HD treatment group comprised 26 patients
d-Aminolevulinate dehydratase activity was determined in the total blood according to the method of Sassa [29] with some modifications. The enzyme activity was determined by the rate of phorphobilinogen (PBG) formation in 1 h at 37 C, in the presence and absence of the reductor agent dithiothreitol (DTT e 2 mM final concentration). The enzyme reaction was initiated after 10 min of preincubation. The reaction was started by adding d-aminolevulinic acid (ALA) to a final concentration of 4 mM in phosphate buffered solution at pH 6.8, and incubation was carried out for 1 h a 37 C and the reaction product wasmeasured at 555 nm. The ALA-D reactivation index was estimated using the equation: A B=A 100; where A ¼ absorbance of ALA-D with DTT; B ¼ absorbance of ALA-D without DTT: 2.7. GSH assay Erythrocytes were deproteinized with an equal volume of trichloroacetic acid (TCA) 10% after hemolysis. GSH was measured in acid derivate with DTNB (5,50 -dithio-bis-2-nitrobenzoic acid) by high performance liquid chromatographic
J. Valentini et al. / Biomedicine & Pharmacotherapy 62 (2008) 378e382
380
(HPLC) with UV detector at 330 nm, using gradient elution at 39 C [30].
Table 2 Parameters analyzed in this study Controls, n ¼ 40
Parameters 1
2.8. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan’s test when appropriate. Results are expressed as means standard error (S.E.M). The association between analyzed parameters and characteristics of three study groups was evaluated by Pearson’s correlation for variables that had a normal distribution and by Spearman’s rank order correlation for variables that did not exhibit a normal distribution. Data were analyzed using the StatisticaÒ 6.0 software system (Statsoft Inc., 2001). 3. Results
TBARS (mM L ) MDA (mM L1) ALA-D (UI) ALA-D index of reactivation (%) GSH (mM/gHb)
RT, n ¼ 36
LT, n ¼ 26
7.14 0.65 4.53 0.15 20.10 0.88 18.90 2.05
a
11.72 0.61 6.36 0.20a 8.91 0.58a 77.30 5.80a
12.38 0.56a 7.52 0.68b 9.60 0.68 94.90 9.38b
5.31 0.21
7.78 0.30a
8.14 0.30a
Results expressed in mean standard error. a Significantly different from controls ( p < 0.05). b Significantly different from controls and from patients with recent time of HD treatment ( p < 0.05).
3.3. ALA-D activity
Information relating to each group studied including sex, age and time of HD treatment is shown in Table 1. Patients had sex ratios similar to healthy controls. The age was significantly higher in both groups of the HD patients (LT and RT) compared to the healthy subjects, being 51.0 11.6 and 53.6 12.8 vs 43.1 7.1 years old, respectively, ( p < 0.05). However, it did not observe the significant difference when comparing only the HD groups ( p > 0.05). Moreover, the age did not present correlation with the parameters of the oxidative stress analyzed in this study.
Blood d-ALA-D activity was significantly lower in HD patients (LT and RT) when compared to the healthy subjects (Table 2), being 9.60 0.68 and 8.91 0.58 vs 20.07 0.88 UI, respectively, ( p < 0.0001). In addition, d-ALA-D activity did not correlate with the time of the HD treatment. The involvement of SH groups in ALA-D inhibition was examined by testing the effect of dithiothreitol (DTT) on the enzyme. The addition of DTT (2 mM) to the assay mixture caused an increase of 77.3% and 94.9% in ALA-D activity in patients with long and recent time of HD treatment, respectively, (Fig. 2). In addition, ALA-D reactivation index correlated positively with the time of HD treatment (r ¼ 0.30; p < 0.05) (Fig. 3).
3.2. Lipid peroxidation
3.4. GSH assay
The results of the lipid peroxidation assessed by TBARS and MDA measurement are shown in Table 2. Plasmatic TBARS levels were significantly higher in HD patients (LT and RT) compared to the healthy subjects, being the TBARS levels of the 12.38 0.56 and 11.72 0.61 vs 7.14 0.65 mmol L1, respectively, ( p < 0.0001); and MDA levels 7.52 0.68 and 6.36 0.20 vs 4.53 0.15 mmol L1, respectively, ( p < 0.0001). In relation to the HD patients, the plasmatic MDA levels were higher in long time HD treatment when compared to recent time HD treatment ( p < 0.05). MDA levels correlated positively with the time of the HD treatment (r ¼ 0.29; p < 0.05) (Fig. 1), whereas TBARS levels did not correlate ( p > 0.05).
GSH levels were significantly higher in HD patients (LT and RT) than in healthy subjects (Table 2), being 8.14 0.30 and 7.78 0.30 vs 5.31 0.21 mmol/gHb, respectively, ( p < 0.0001). However, it didn’t present correlation with the time of HD treatment.
3.1. Characteristics of the studied population
Table 1 General characteristics of the population studied
Age (years) Time of HD treatment (months) Sex (men/women)
Long time HD treatment (LT), n ¼ 26
Recent time HD treatment (RT), n ¼ 36
Healthy subjects (controls), n ¼ 40
51 11.60a 82.60 6.32
53.6 12.80a 17.70 9.40
43.15 7.09 e
12/14
16/18
20/20
The values are expressed as mean standard error. a Significantly different from controls ( p < 0.05).
Fig. 1. Pearson’s positive correlation between MDA levels and time of HD treatment (r ¼ 0.29; p < 0.05).
J. Valentini et al. / Biomedicine & Pharmacotherapy 62 (2008) 378e382
Fig. 2. d-ALA-D activity in control, recent HD treatment (RT) and long HD treatment (LT) patients in the absence or presence of DTT 2 mM. Results are expressed as means SE; control, n ¼ 40; RT, n ¼ 36; and LT, n ¼ 26. *Significantly different from control. **Significantly different from control and from patients with RT.
4. Discussion This study showed clearly a relationship between oxidative stress parameters, and patients with chronic renal failure (CRF) on hemodialysis treatment, an extrarenal depurative technique, which promotes increase of oxidative stress. In these patients, oxidative stress is caused by several reasons, such as the use of low-biocompatible synthetic membranes and the lack of ultrapure dialysis water stand out [31]. Lipid peroxidation is hallmark of oxidative stress, which disrupts the structural integrity of cell membranes and can also lead to the formation of aldehydes, which in turn time damage lipids, proteins and DNA [32]. One of the most often used biomarker to investigate the oxidative damage in lipids is the measurement of thiobarbituric acid reactive substances (TBARS), which includes MDA as their major compound [33]. In our work, plasmatic TBARS and MDA levels were higher in HD patients, when compared to the healthy subjects. This is found in agreement with previous studies that reported
381
increase of the oxidative stress in HD patients [34e36]. However, it verified significant difference in the levels of MDA only when it was compared with the recent and long time HD treatment. In accordance with literature, these results suggest that the specificity of TBARS assay is low, and that measurement of MDA is more specific and sensitive for the evaluation of the lipid peroxidation status [33]. Blood ALA-D activity in HD patients was significantly lower than the healthy subjects, in agreement with previous studies [26,32]. However, basal blood ALA-D activity did not have significant difference when compared with recent and long time HD treatment. In addition, the reactivation index of enzyme activity was significantly increased in both groups of patients when compared with healthy controls, and it was significantly higher in patients with long time of HD treatment in comparison with patients with recent time of HD treatment. The increase in the reactivation index of the enzyme may be related to an overproduction of free radicals, which can be confirmed by an increase in MDA production. Free radicals can directly interact with thiol groups of proteins, oxidizing them to disulfides [37]. In line with these results, recent data from our and others laboratories indicate that situations associated with overproduction of free radicals can result in ALAD oxidation [22,26,38]. GSH is a tripeptidic thiol found in the inside of all animal cells and likely is the most important cellular antioxidant [31]. In this study the GSH levels were increased in HD patients, in agreement with previous studies [39,40]. This increase in the GSH levels could suggest an adaptive compensatory mechanism due to increased oxidative stress occurring in these patients [41]. In addition, we observed that GSH levels are not different between patients with recent and long time HD treatment. In conclusion, the results presented here indicate that MDA levels, one classical indicator of oxidative stress, suffer influence from time of HD treatment, and that their measurement may be more specific than the measurement of the TBARS levels in this situation. Besides, the increase in ALA-D reactivation index demonstrated that oxidative stress associated with time HD treatment can modify the activity of this enzyme, which is sensitive to oxidant agents or pro-oxidant situations. Thus, we could suggest that in chronic processes, as HD treatment with long time, the assessment of MDA levels and ALA-D reactivation index can be important such as biomarkers of oxidative damage in lipids and thiol proteins. Acknowledgements This study was supported by grant from Fapergs to Solange C. Garcia (Proade3) and S.C. Garcia is recipient of CNPq research fellowship. References
Fig. 3. Pearson’s positive correlation between ALA-D reactivation index and time of HD treatment (r ¼ 0.30; p < 0.05).
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[2] Kumaraguruparan R, Kabalimoothy J, Nagini S. Correlation of tissue lipid peroxidation and antioxidants with clinical stage and menopausal status in patients with adenocarcinoma of the breast. Clin Biochem 2005;38:154e8. [3] Ko¨ken T, Serteser M, Kahraman A, Go¨kc¸e C, Demir S. Changes in serum markers of oxidative stress with varying periods of haemodialysis. Nephrology 2004;9:77e82. [4] Dro¨ge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47e95. [5] Durak I, Kaomaz M, Cimen MY, Buyukkoc¸ak U, Ozturk HS. Blood oxidant/antioxidant status of atherosclerotic patients. Int J Cardiol 2001;77: 293e7. [6] Cerutti PA, Trump BF. Inflammation and oxidative stress in carcinogenesis. Cancer Cells 1991;3:1e7. [7] Bankson DD, Kestin M, Rifai N. Role of free radicals in cancer and atherosclerosis. Clin Lab Med 1993;13:463e80. [8] Mimic-Oka J, Simic T, Djukanovic L, Relji Z, Davicevi Z. Alteration in plasma antioxidant capacity in various degrees of renal failure. Clin Nephrol 1999;51:233e41. [9] Himmelfarb J, McMonagle E, McMenamin E. Plasma protein thiol and carbonyl formation in chronic renal failure. Kidney Int 2000;58:2571e8. [10] Ceballos-Picot I, Witko-Sarsat V, Merad-Boudia M, Nguyen AT, The´venin M, Jaudon MC, et al. Glutathione antioxidant system as a marker of oxidative stress in chronic renal failure. Free Radic Biol Med 1996;21:845e53. [11] Olivieri O, Stanzial AM, Girelli D, Trevisan MT, Guarini P, Terzi M. Selenium status, fatty acids, vitamins A and E, and aging: the Nove study. Am J Clin Nutr 1994;60:510e7. [12] Miyata T, Maeda K, Kurokawa K. Oxidation conspires with glycation to generate noxious advanced glycation end products in renal failure. Nephrol Dial Transplant 1998;12:255e8. [13] Zimmermann J, Herrlinger S, Pruy A, Metzeger T, Wanner C. Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int 1999;55:648e58. [14] Euchant E, Carbonnneau MA, Dubourg L, Thomas MJ, Perromat A, Clerc M. Lipoperoxidation in plasma and red blood cells of patients undergoing haemodialysis: vitamin A, E and iron status. Free Radic Biol Med 1994;16:339e46. [15] Nguyen-Khoa T, Massy ZA, De Bandt JP, Kebede M, Salama L, Lambrey G, et al. Oxidative stress and haemodialysis: role of inflammation and duration of dialysis treatment. Nephrol Dial Transplant 2001;16:335e40. [16] Drai J, Bannier E, Chazot C, Hurot JM, Goedert G, Jean G, et al. Oxidants and antioxidants in long-term haemodialysis patients. II Farmaco 2001;56:463e5. [17] Jansen YM, van Houten B, Borm PJ, Mossman BT. Cell and tissue responses to oxidative damage. Lab Invest 1993;69:261e74. [18] Stohs SJ. The role of free radicals in toxicity and disease. J Basic Clin Physiol Pharmacol 1995;6:205e28. [19] Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995;41:1819e28. [20] Weinstein T, Chagnac A, Korzets A. Haemolysis in haemodialysis patients: evidence for impaired defence mechanisms against oxidative stress. Nephrol Dial Transplant 2000;15:883e7. [21] Folmer V, Santos FW, Savegnago L, Britto VB, Nogueira CW, Rocha JBT. High sucrose consumption potentiates the sub-acute cadmium effect on Naþ/Kþ-ATPase but not on delta-aminolevulinate dehydratase in mice. Toxicol Lett 2004;153:333e41.
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Biomedicine & Pharmacotherapy 62 (2008) 378e382 www.elsevier.com/locate/biopha
Original article
The influence of the hemodialysis treatment time under oxidative stress biomarkers in chronic renal failure patients Juliana Valentini a, Denise Grotto a, Clo´vis Paniz a, Miguel Roehrs a, Geni Burg b, Solange C. Garcia a,* a
Department of Clinical and Toxicological Analysis, Federal University of Santa Maria, Campus Universita´rio, Caixa Postal 5061, CEP 97110-970, Santa Maria, Rio Grande do Sul , Brazil b Nurse of Unitate of Renal Clinical of Caridade and Casa of Sau´de Hospitals, Santa Maria, RS, Brazil Received 13 June 2007; accepted 24 October 2007 Available online 17 December 2007
Abstract Summary: Oxidative stress possibly helps to promote the progression and complication of chronic renal failure (CRF). Hemodialysis (HD) may aggravate oxidative stress. In addition long time of treatment may intensify the oxidative stress. Thus, the aim of this study was to evaluate the effect of prolonged HD treatment under parameters of the oxidative stress. Methods: Plasmatic thiobarbituric acid reactive substances (TBARS), plasmatic malondialdehyde (MDA), blood d-aminolevulinate dehydratase (ALA-D) activity, ALA-D reactivation index, and erythrocytic reduced glutathione (GSH) were measured into two different groups of HD patients: recent treatment (n ¼ 36; HD duration: 17.7 1.71 months), and long time of treatment (n ¼ 26; HD duration: 82.2 6.32 months), and in a control group (n ¼ 40). Results: Plasmatic TBARS and MDA levels were both elevated in HD patients. However, only MDA levels had positive correlation with time of HD treatment. Blood ALA-D activity was decreased in HD patients. The ALA-D reactivation index showed increase in HD patients, and it had correlation with the time of HD treatment. Erythrocytic GSH levels were increased in HD patients. Conclusions: Our results indicated that MDA levels and ALA-D reactivation index may be the better biomarkers to evaluate chronic oxidative stress in comparison with others markers analyzed in this study. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Hemodialysis treatment time; ALA-D activity; Oxidative stress; MDA; GSH
1. Introduction Inadequate antioxidant protection or excessive production of reactive oxygen species create a condition known as oxidative stress, which has an important role in the etiology of various disease and in aging [1,2]. This condition leads to structural and/or functional deterioration in cell components including DNA, proteins, carbohydrates and lipids [3]. In addition, this state is involved in many pathophysiological processes, particularly within the atherosclerosis context, inflammation and cancer
* Corresponding author. Tel.: þ55 55 3220 8941; fax: þ55 55 3220 8018. E-mail address: [email protected] (S.C. Garcia). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.10.017
[4e7], with higher prevalence in patients with chronic renal failure (CRF) compared with age-matched controls from the general population [3]. Indeed, CRF has emerged as a major example of disease-associated oxidative stress, because of the excessive production of oxidants accompanied by inadequate antioxidant defenses [8e10]. In CRF patients under hemodialysis (HD) treatment the formation of reactive oxygen species (ROS) is amplified, therefore beyond uremic toxins, the hemodialysis itself due to bio-incompatible dialysis water, non-sterile dialysate, poor quality of dialysis water, and back-leak of contaminants across the dialysis membrane. The other causes of enhanced oxidative stress are advanced age [11], high frequency of diabetes [12], chronic inflammatory state [13], excessive parenteral
J. Valentini et al. / Biomedicine & Pharmacotherapy 62 (2008) 378e382
iron administration [14], and time of hemodialysis treatment [15]. Previous studies have reported that longer time of HD treatment may amplify the oxidative stress, and it contributes to the increase of the morbidity and mortality in these patients, particularly in relation to cardiovascular disease [3,15,16]. The presence of ROS can cause damage in many molecules, such as lipids, leading to the production of malondialdehyde (MDA), an indicator of lipid peroxidation [17e19]. However, studies about influence of the time of HD treatment assessed the plasmatic levels of thiobarbituric acid reactive substances (TBARS), and its results were contradictors, with positive correlation between TBARS and the time of HD treatment [20], and not correlation [3]. These works did not report the MDA levels and their relation with time of HD treatment. d-Aminolevulinate dehydratase (ALA-D) is an essential enzyme for aerobics organisms, once it catalyses the synthesis of tetrapyrrolic compounds such as billins and hemes. ALA-D is a sulfhydryl enzyme which is dependent on Zn2þ ion binding to display full activity and it is sensitive to situations associated with oxidative stress [21,22]. Importantly, d-ALA-D activity is decreased in CRF, especially in HD treatment [23e26]. However, works relating d-ALA-D activity with duration of dialysis therapy not have. Based on the fact that the duration of dialysis treatment can amplify the oxidative stress, the present study was designated to evaluate the levels of plasmatic TBARS and MDA, blood ALA-D activity, ALA-D reactivation index, and erythrocytic reduced glutathione (GSH) levels in HD patients with different times of HD treatment compared with a control group. 2. Materials and methods
379
(HD time: 82.6 6.32 months), with 51 11.6 years of age (ranging between 29 and 75 years). Control group consisted of 40 healthy subjects (20 men, 20 women), with 43.15 7.1 years of age (ranging between 30 and 57 years), which did not have clinical history of renal diseases or another pathologies. All the volunteers did not receive vitamins, were non-smokers, and did not consume alcohol regularly. 2.3. Samples Blood venous samples (10 ml) were drawn from HD patients and controls subjects, and this were divided in heparinized tubes, EDTA-containing tubes, and tubes without anticoagulant. PlasmaeEDTA and serum were obtained by centrifugation at 1500g for 10 min at 4 C. 2.4. Hematological determinations Hemoglobin (Hb) and hematocrit (Hct) were determined in Cobas Micros system (Hematology Analyzer, Roche Diagnostics). 2.5. Lipid peroxidation Lipid peroxidation was estimated by the measurement of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA). TBARS assay were realized in plasmaeEDTA samples according to the method of Okhawa et al. [27]. The measurement of plasmatic MDAwas determined by high performance liquid chromatographic with visible detection (HPLCeVIS), according to the method of Grotto et al. [28].
2.1. Chemicals 2.6. d-ALA-D activity 50 -Aminolevulinic acid (ALA), 2-thiobarbituric acid (TBA), dithiothreitol (DTT), and reduced glutathione (GSH) were purchased from Sigma (St. Louis, USA). All other chemicals used in this study were of the highest purity available. 2.2. Subjects Sixty-two patients with diagnosis of CRF (35 men and 27 women) undergoing regular hemodialysis (HD) treatment at Caridade and Casa of Sau´de Hospitals, located in Santa Maria, RS, Brazil. A study protocol was approved by the Human Ethics Committee of the Health Science Center from the Federal University of Santa Maria (protocol no: 091/2003) and all the patients gave their informed consent prior to the inclusion in the study. Patients with alcoholism, smoking, diabetes, viral hepatitis and HIV, and use of any antioxidant vitamin within last 3 months were excluded. Patients HD included in the study were divided into two groups in accordance with the time of HD treatment: recent time (RT) of HD treatment group comprised 36 patients (HD time 17.7 1.71 months) with 53.6 12.8 years of age (ranging between 20 and 77 years). Long time (LT) HD treatment group comprised 26 patients
d-Aminolevulinate dehydratase activity was determined in the total blood according to the method of Sassa [29] with some modifications. The enzyme activity was determined by the rate of phorphobilinogen (PBG) formation in 1 h at 37 C, in the presence and absence of the reductor agent dithiothreitol (DTT e 2 mM final concentration). The enzyme reaction was initiated after 10 min of preincubation. The reaction was started by adding d-aminolevulinic acid (ALA) to a final concentration of 4 mM in phosphate buffered solution at pH 6.8, and incubation was carried out for 1 h a 37 C and the reaction product wasmeasured at 555 nm. The ALA-D reactivation index was estimated using the equation: A B=A 100; where A ¼ absorbance of ALA-D with DTT; B ¼ absorbance of ALA-D without DTT: 2.7. GSH assay Erythrocytes were deproteinized with an equal volume of trichloroacetic acid (TCA) 10% after hemolysis. GSH was measured in acid derivate with DTNB (5,50 -dithio-bis-2-nitrobenzoic acid) by high performance liquid chromatographic
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(HPLC) with UV detector at 330 nm, using gradient elution at 39 C [30].
Table 2 Parameters analyzed in this study Controls, n ¼ 40
Parameters 1
2.8. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan’s test when appropriate. Results are expressed as means standard error (S.E.M). The association between analyzed parameters and characteristics of three study groups was evaluated by Pearson’s correlation for variables that had a normal distribution and by Spearman’s rank order correlation for variables that did not exhibit a normal distribution. Data were analyzed using the StatisticaÒ 6.0 software system (Statsoft Inc., 2001). 3. Results
TBARS (mM L ) MDA (mM L1) ALA-D (UI) ALA-D index of reactivation (%) GSH (mM/gHb)
RT, n ¼ 36
LT, n ¼ 26
7.14 0.65 4.53 0.15 20.10 0.88 18.90 2.05
a
11.72 0.61 6.36 0.20a 8.91 0.58a 77.30 5.80a
12.38 0.56a 7.52 0.68b 9.60 0.68 94.90 9.38b
5.31 0.21
7.78 0.30a
8.14 0.30a
Results expressed in mean standard error. a Significantly different from controls ( p < 0.05). b Significantly different from controls and from patients with recent time of HD treatment ( p < 0.05).
3.3. ALA-D activity
Information relating to each group studied including sex, age and time of HD treatment is shown in Table 1. Patients had sex ratios similar to healthy controls. The age was significantly higher in both groups of the HD patients (LT and RT) compared to the healthy subjects, being 51.0 11.6 and 53.6 12.8 vs 43.1 7.1 years old, respectively, ( p < 0.05). However, it did not observe the significant difference when comparing only the HD groups ( p > 0.05). Moreover, the age did not present correlation with the parameters of the oxidative stress analyzed in this study.
Blood d-ALA-D activity was significantly lower in HD patients (LT and RT) when compared to the healthy subjects (Table 2), being 9.60 0.68 and 8.91 0.58 vs 20.07 0.88 UI, respectively, ( p < 0.0001). In addition, d-ALA-D activity did not correlate with the time of the HD treatment. The involvement of SH groups in ALA-D inhibition was examined by testing the effect of dithiothreitol (DTT) on the enzyme. The addition of DTT (2 mM) to the assay mixture caused an increase of 77.3% and 94.9% in ALA-D activity in patients with long and recent time of HD treatment, respectively, (Fig. 2). In addition, ALA-D reactivation index correlated positively with the time of HD treatment (r ¼ 0.30; p < 0.05) (Fig. 3).
3.2. Lipid peroxidation
3.4. GSH assay
The results of the lipid peroxidation assessed by TBARS and MDA measurement are shown in Table 2. Plasmatic TBARS levels were significantly higher in HD patients (LT and RT) compared to the healthy subjects, being the TBARS levels of the 12.38 0.56 and 11.72 0.61 vs 7.14 0.65 mmol L1, respectively, ( p < 0.0001); and MDA levels 7.52 0.68 and 6.36 0.20 vs 4.53 0.15 mmol L1, respectively, ( p < 0.0001). In relation to the HD patients, the plasmatic MDA levels were higher in long time HD treatment when compared to recent time HD treatment ( p < 0.05). MDA levels correlated positively with the time of the HD treatment (r ¼ 0.29; p < 0.05) (Fig. 1), whereas TBARS levels did not correlate ( p > 0.05).
GSH levels were significantly higher in HD patients (LT and RT) than in healthy subjects (Table 2), being 8.14 0.30 and 7.78 0.30 vs 5.31 0.21 mmol/gHb, respectively, ( p < 0.0001). However, it didn’t present correlation with the time of HD treatment.
3.1. Characteristics of the studied population
Table 1 General characteristics of the population studied
Age (years) Time of HD treatment (months) Sex (men/women)
Long time HD treatment (LT), n ¼ 26
Recent time HD treatment (RT), n ¼ 36
Healthy subjects (controls), n ¼ 40
51 11.60a 82.60 6.32
53.6 12.80a 17.70 9.40
43.15 7.09 e
12/14
16/18
20/20
The values are expressed as mean standard error. a Significantly different from controls ( p < 0.05).
Fig. 1. Pearson’s positive correlation between MDA levels and time of HD treatment (r ¼ 0.29; p < 0.05).
J. Valentini et al. / Biomedicine & Pharmacotherapy 62 (2008) 378e382
Fig. 2. d-ALA-D activity in control, recent HD treatment (RT) and long HD treatment (LT) patients in the absence or presence of DTT 2 mM. Results are expressed as means SE; control, n ¼ 40; RT, n ¼ 36; and LT, n ¼ 26. *Significantly different from control. **Significantly different from control and from patients with RT.
4. Discussion This study showed clearly a relationship between oxidative stress parameters, and patients with chronic renal failure (CRF) on hemodialysis treatment, an extrarenal depurative technique, which promotes increase of oxidative stress. In these patients, oxidative stress is caused by several reasons, such as the use of low-biocompatible synthetic membranes and the lack of ultrapure dialysis water stand out [31]. Lipid peroxidation is hallmark of oxidative stress, which disrupts the structural integrity of cell membranes and can also lead to the formation of aldehydes, which in turn time damage lipids, proteins and DNA [32]. One of the most often used biomarker to investigate the oxidative damage in lipids is the measurement of thiobarbituric acid reactive substances (TBARS), which includes MDA as their major compound [33]. In our work, plasmatic TBARS and MDA levels were higher in HD patients, when compared to the healthy subjects. This is found in agreement with previous studies that reported
381
increase of the oxidative stress in HD patients [34e36]. However, it verified significant difference in the levels of MDA only when it was compared with the recent and long time HD treatment. In accordance with literature, these results suggest that the specificity of TBARS assay is low, and that measurement of MDA is more specific and sensitive for the evaluation of the lipid peroxidation status [33]. Blood ALA-D activity in HD patients was significantly lower than the healthy subjects, in agreement with previous studies [26,32]. However, basal blood ALA-D activity did not have significant difference when compared with recent and long time HD treatment. In addition, the reactivation index of enzyme activity was significantly increased in both groups of patients when compared with healthy controls, and it was significantly higher in patients with long time of HD treatment in comparison with patients with recent time of HD treatment. The increase in the reactivation index of the enzyme may be related to an overproduction of free radicals, which can be confirmed by an increase in MDA production. Free radicals can directly interact with thiol groups of proteins, oxidizing them to disulfides [37]. In line with these results, recent data from our and others laboratories indicate that situations associated with overproduction of free radicals can result in ALAD oxidation [22,26,38]. GSH is a tripeptidic thiol found in the inside of all animal cells and likely is the most important cellular antioxidant [31]. In this study the GSH levels were increased in HD patients, in agreement with previous studies [39,40]. This increase in the GSH levels could suggest an adaptive compensatory mechanism due to increased oxidative stress occurring in these patients [41]. In addition, we observed that GSH levels are not different between patients with recent and long time HD treatment. In conclusion, the results presented here indicate that MDA levels, one classical indicator of oxidative stress, suffer influence from time of HD treatment, and that their measurement may be more specific than the measurement of the TBARS levels in this situation. Besides, the increase in ALA-D reactivation index demonstrated that oxidative stress associated with time HD treatment can modify the activity of this enzyme, which is sensitive to oxidant agents or pro-oxidant situations. Thus, we could suggest that in chronic processes, as HD treatment with long time, the assessment of MDA levels and ALA-D reactivation index can be important such as biomarkers of oxidative damage in lipids and thiol proteins. Acknowledgements This study was supported by grant from Fapergs to Solange C. Garcia (Proade3) and S.C. Garcia is recipient of CNPq research fellowship. References
Fig. 3. Pearson’s positive correlation between ALA-D reactivation index and time of HD treatment (r ¼ 0.30; p < 0.05).
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