Dehydroascorbic acid and oxidative stress in haemodialysis patients
Hidetsugu Nakayama,,
Shinichiro Akiyama,
Masahiro Inagaki1,
Yoshiaki Gotoh and
Katsuji Oguchi1
Department of Renal Medicine, Saiyu Soka Hospital, 1-21-37 Kitaya, Soka City, Saitama 340-0076 and
1 Department of Pharmacology, School of Medicine, Showa University, 1-5-8 Shinagawa-ku, Tokyo 142-8555, Japan
 |
Abstract
|
---|
Background. The amount of dehydroascorbic acid contained within total ascorbic acid (oxidized as well as non-oxidized forms) in plasma, hereafter referred to as the dehydroascorbic acid fraction, may be a measure of oxidative stress during haemodialysis. In the present study, we determined this fraction in chronic haemodialysis patients.
Methods. Using high performance liquid chromatography, dehydroascorbic acid and total ascorbic acid levels were measured in 80 maintenance haemodialysis patients for a period of >2 years as well as in 49 controls, to examine a possible association of these compounds with clinical parameters and/or drugs taken by the patients.
Results. Dialysis patients who had an increased plasma urate level (P<0.05) and had been taking allopurinol (P<0.05) or NSAID (non-steroid anti-inflammatory drugs) (P<0.01), and dialysis patients who were younger (
55 years), as compared with older dialysis patients (P<0.01), were found to have a lower dehydroascorbic acid fraction by multivariate analysis. Mean plasma dehydroascorbic acid levels and dehydroascorbic acid fractions were significantly lower in the younger haemodialysis patients (4.8±0.7 µmol/l and 28.4±3.9%) than in healthy younger controls (13.3±1.1 µmol/l and 41.1±1.8%) (P<0.0001 and P<0.01, respectively). Moreover, a correlation was found between plasma dehydroascorbic acid fraction and plasma lipid peroxide (r=0.66, P<0.01) in patients who had not been taking allopurinol and/or NSAID.
Conclusion. We found that dehydroascorbic acid fraction was related to patients' age, plasma urate level and to taking allopurinol or NSAID. Dehydroascorbic acid fraction may be another indirect index of oxidative stress.
Keywords: ascorbic acid; dehydroascorbic acid; haemodialysis
 |
Introduction
|
---|
Many reports shown that patients undergoing chronic haemodialysis have low serum ascorbic acid levels unless they take a supplement [1,2]. However, these studies included a number of patients whose ascorbic acid levels were comparable to healthy controls or were not correlated with any clinical factors.
Oxidative stress has been shown to be increased in haemodialysis patients [3]. Since ascorbic acid (reduced form) oxidizes to dehydroascorbic acid, it may have the potential to guard against these harmful factors. Dehydroascorbic acid may be one of the biomarkers of oxidative stress, but its clinical significance is not thoroughly understood [4,5]. Recently, there have been reports on dehydroascorbic acid levels in haemodialysis patients, however measurements in these reports used a spectrophotometer [6,7]. As spectrophotometry may have interference from non-specific substances [8], we used high performance liquid chromatography (HPLC) to measure plasma ascorbic acid and dehydroascorbic acid. This study aims to identify which factors are related to dehydroascorbic acid fraction, and to compare plasma dehydroascorbic acid levels in haemodialysis patients with those of healthy controls. Using multivariate analysis we identified certain clinical factors that seem to influence the dehydroascorbic acid fraction.
 |
Subjects and methods
|
---|
Patients
Eighty patients (37 male, 43 female) with chronic renal failure who had undergone maintenance haemodialysis for >2 years were studied. All were outpatients and underwent dialysis three times weekly for 34 h. None had any concurrent illness at the time of the study nor were they taking supplemental ascorbic acid. The patients were dialysed with Cuprophan (PC series, Asahi Medical Co. Tokyo, Japan, or SU series, TERUMO Co. Tokyo, Japan) or Polyacrylonitrile (PAN series, Asahi Medical Co. Tokyo, Japan) membrane with a bicarbonate dialysis solution. Patients over 75 years of age were dialysed for 3 h per session. The mean patient age was 55.7±1.2 years (range 2784 years). They had been on chronic haemodialysis for a mean period of 7.0±0.5 years (range 219 years). Patients were divided into two groups: a younger group (age
55 years) and an older group (age >55 years). The causes of chronic renal failure were chronic glomerulonephritis in 35 patients, diabetic nephropathy in 23, polycystic kidney in 10, nephrosclerosis in eight, and collagen disease in four. Eight patients had been taking non-steroid anti-inflammatory drugs (NSAID) and 26 patients had been taking allopurinol. Thirty-four healthy younger controls (16 male, 18 female) were selected from the hospital staff. The mean age of the younger controls was 46.5 years (range 2355 years). They were not taking any medication. Fifteen older healthy controls (seven male, eight female) were hypertensive outpatients who were taking only anti-hypertensive drugs. The mean age of the older controls was 68.9 years (range 6672 years).
Measurements
Before haemodialysis, blood samples from each patient were collected into tubes with lithium heparin, and the plasma was centrifuged at room temperature at 1500 g for 5 min. Then, 100 µl of plasma was mixed with 400 µl of 5% metaphosphoric acid containing 25 mM deferoxiamine. This solution was spun in the centrifuge at 4°C for 10 min at 3000 g. The supernatant was divided into units of 150 µl each for measurements of total ascorbic acid and reduced ascorbic acid. The dehydroascorbic acid level was calculated for that of total ascorbic acid minus that of reduced ascorbic acid. The ascorbic acid was analysed by HPLC with electrochemical detection, according to the method of Iriyama with some modifications [9]. Plasma lipid peroxide assay was performed by Determiner LPO kit (Kyowa Medex Co. Tokyo, Japan) according to Ohishi et al. [10].
Statistical analysis
The mean values for continuous variables were compared using the MannWhitney U-test. The two groups were compared using a two-by-two contingency table (Fisher's exact test). Differences of the means between groups were assessed using analysis of variance (ANOVA) (Fisher's test). Data that was not normally distributed was transformed to the logarithm of their respective values. Using Pearson's product moment correlation coefficients we assessed the relationships between various parameters (age, duration of haemodialysis, blood leukocyte count, haemoglobin, platelet count, total protein, total cholesterol, HDL cholesterol, triglycerides, urate, creatinine and iron levels) and total ascorbic acid levels or dehydroascorbic acid fractions. Multiple regression analysis was carried out to investigate independent variables including sex, age, duration of haemodialysis, urate level, medication of calcium antagonist, allopurinol and NSAID in the total ascorbic acid or the dehydroascorbic acid fraction. All significance tests were two-tailed, and the level of significance was set at 5%. All data are expressed as the mean±standard error. Statistical analysis was performed using a commercially available Macintosh personal computer, Stat-View 5.0 (Abacus Concepts, Inc., Berkeley, CA).
 |
Results
|
---|
Table 1
summarizes the relevant clinical characteristics of 80 patients. The younger group (
55 years) and the older group (>55 years) were well matched; there were no significant differences in the clinical parameters.
The mean plasma ascorbic acid level in haemodialysis patients was 29.4±4.7 µmol/l, which ranged from extremely low to high (1.2 µmol/l to 156.3 µmol/l). Table 2
shows the mean plasma total ascorbic acid level and dehydroascorbic acid level in the groups. The mean total ascorbic acid in the younger group was significantly higher than that in the older group (P<0.001). The mean dehydroascorbic acid fraction in the younger group was significantly lower than that in the order group (P<0.01). The mean dehydroascorbic acid fraction in the younger group was significantly lower than that in the younger controls (P<0.001).
By simple regression analysis, a weak correlation was found between the ascorbic acid level and the patient age (r=-0.380, P<0.01) (Figure 1
), and dehydroascorbic acid fraction and patient age (r=0.363, P<0.01) (Figure 2
). Other parameters showed no significant differences.
Multiple regression analysis found that patients with lower urate levels (P<0.05) and older patients (P<0.001) had lower total ascorbic acid levels. Patients with higher urate levels (P<0.05), those taking allopurinol (P<0.05) or NSAID (P<0.01), and younger patients (
55 years) (P<0.01) had lower dehydroascorbic acid fractions.
Since the use of allopurinol and NSAID affects dehydroascorbic acid fraction, the comparison of dehydroascorbic acid with lipid peroxide was made without those patients on allopurinol and NSAID. A significant correlation was found between lipid peroxide levels and dehydroascorbic acid fractions in patients who were not taking allopurinol and/or NSAID (r=0.66, P<0.01) (Figure 3
).
Table 3
shows comparisons of total ascorbic acid level, dehydroascorbic acid level and dehydroascorbic acid fraction in younger and older patients with variables of dialyser membrane, tobacco use, and use of allopurinol. There were no significant differences with variations of dialyser membrane and smoking. The mean dehydroascorbic acid levels in younger and older patients taking allopurinol were significantly lower than in those not taking allopurinol (P<0.05).
 |
Discussion
|
---|
Sullivan et al. observed that the serum ascorbic acid level decreased in patients undergoing maintenance haemodialysis [1]. Thus, ascorbic acid supplementation is considered necessary from this report [1]. However, Japanese studies showed normal ascorbic acid levels in patients undergoing haemodialysis [11,12]. Our findings were in accordance with the Japanese results. Another report documented that withdrawal of ascorbic acid supplementation was not associated with low plasma or leukocyte ascorbic acid levels [13]. These results suggest that there are some patients in whom plasma ascorbic acid levels are not low in uraemia. In our study, plasma ascorbic acid levels vary among patients. Our study included haemodialysis patients who were inactive, homebound, or who had a poor diet. Decreased dietary intake of ascorbic acid with increased age could be a major cause of low plasma ascorbic acid levels and thus provide another explanation of the increased utilization of ascorbic acid [14]. However, the mechanisms of this phenomenon are not known.
One report indicates that urate serves as a water-soluble potent antioxidant by means of radical scavenging and reducing activities, which are the strongest determinants of plasma antioxidative capacity [15]. It appeared that urate replaced some of the antioxidant functions of ascorbic acid, which in turn served to spare ascorbic acid [15]. Age was thought to be related to antioxidative capacity [15]. In some animal and human studies, the effect of allopurinol in the formation of reactive oxygen species is reported, particularly in ischaemic reperfusion injury of heart [16]. It has been hypothesized that calcium antagonists may exert antioxidant activity on certain tissue [17]. In this study, however, calcium antagonists did not alter dehydroascorbic acid fraction significantly in multivariate analysis. Finally some reports have found that NSAID have antioxidant activity by inhibiting the prostaglandin synthesize pathway [18]. The reported factors that concerned oxidative stress were related to dehydroascorbic acid fraction in this study.
Low dehydroascorbic acid level in dialysed patients corresponds to the report of Bakaev et al. [6]. The mechanisms of low dehydroascorbic acid are not known, however we have made some speculations. Lymphocytes have two distinct uptake mechanisms for ascorbic acid and dehydroascorbic acid [19]. The uptake rate of dehydroascorbic acid is 10 times higher than that for ascorbic acid [19], and intracellular dehydroascorbic acid is immediately reduced to ascorbic acid-dependent glutathione [20]. It may be that ascorbic acid as a scavenger is oxidized at or near the cell membranes, that it enters the cells as dehydroascorbic acid, reduces intracellulary to ascorbic acid and thus provokes a subsequent loss from plasma. Al-Ghamdi et al. reported that dehydroascorbic acid level in haemodialysis patients was not lower than in healthy controls [7]. The study of Al-Ghamdi et al. used whole blood instead of plasma to measure dehydroascorbic acid [7], which was different from our study. Intracellular ascorbic acid and dehydroascorbic acid levels were higher than plasma levels [2]. The uptake mechanisms of dehydroascorbic acid and the difference between intracellular and extracellular dehydroascorbic acid levels may explain this discrepancy.
Another possibility is that dehydroascorbic acid, which is oxidized to diketogulonic acid and subsequently metabolized to oxalate, is increased in haemodialysis patients. Bakaev et al. reported that plasma diketogulonic acid levels in uraemic patients were high [6]. A low level of dehydroascorbic acid may mean increased utilization or oxidation of dehydroascorbic acid to diketogulonic acid.
In conclusion, since the plasma dehydroascorbic acid was found to be correlated with the plasma lipid peroxide, since the dehydroascorbic acid fraction in the older haemodialysis group was higher than in the younger haemodialysis group, and since patients who were on allpopurinol had lower dehydroascorbic acid levels and dehydroascorbic acid fractions than those not taking allopurinol, we propose that the dehydroascorbic acid fraction may be considered as an indirect index of oxidative stress in such patients.
 |
Notes
|
---|
Present affiliation: H. Nakayama, Tsukuba Medical Center Hospital (Ibaraki Cancer Center Hospital) Division of Radiation Oncology, 1-3-1 Amakubo, Tsukuba City, Ibaraki 305-0005, Japan. 
Correspondence and offprint requests to: H. Nakayama, Tsukuba Medical Center Hospital (Ibaraki Cancer Center Hospital), 1-3-1 Amakubo, Tsukuba City, Ibaraki 305-0005, Japan. 
 |
References
|
---|
-
Sullivan JF, Eisenstein AB. Ascorbic acid depletion in patients undergoing chronic hemodialysis. Am J Clin Nutr1970; 23: 13391346[ISI][Medline]
-
DeBari VA, Frank O, Baker H, Needle MA. Water soluble vitamins in granulocytes, erythrocytes and plasma obtained from chronic hemodialysis patients. Am J Clin Nutr1984; 39: 410415[Abstract]
-
Gotoh M, Nagase K, Aoyagi K, Hirayama A, Takemura K, Ueda A, Tomida C, Kikuchi H, Koyama A. Thiobarbituric acid reactive substances are increased in the subcutaneous fat tissue of patients with end-stage renal disease. Nephrol Dial Transplant1997; 12: 713717[Abstract]
-
Lykkesfeldt J, Loft S, Nielsen JB, Poulsen HE. Ascorbic acid and dehydroascorbic acid as biomarkers of oxidative stress caused by smoking. Am J Clin Nutr1997; 65: 959963[Abstract]
-
Schorah CJ, Downing C, Piripitsi A, Gallivan L, Al-Hazaa AH, Sanderson MJ, Bodenhama A. Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma of critically ill patients. AM J Clin Nutr1996; 63: 760765[Abstract]
-
Bakaev VV, Efremov AV, Tityaev II. Low levels of dehydroascorbic acid in uremic serum and the partial correction of dehydroascorbic acid deficiency by haemodialysis. Nephrol Dial Transplant1999; 14: 14721474[Abstract]
-
Al-Ghamdi JM, Al-Jafari AA, Alhomida AS, Sobki SH, Al-Sulaiman MH, Al-Khader AA. Whole blood total, reduced and oxidized ascorbic acid levels in Saudi patients with chronic renal failure: influence of gender and chronic haemodialysis. Med Sci Res1998; 26: 343347[ISI]
-
Kishida E, Nishimoto Y, Kojo S. Specific determination of ascorbic acid with chemical derivatization and high-performance liquid chromatography. Anal Chem1992; 64: 15051507[ISI]
-
Iriyama K, Yoshiura M, Iwamoto Y, Ozaki Y. Simultaneous determination of uric and ascorbic acids in human serum by reversed phase high-performance liquid chromatography with electrochemical detection. Anal Biochem1984; 141: 238242[ISI][Medline]
-
Ohishi N, Ohkawa H, Miike A, Tatano T, Yagi K. A new assay method for lipid peroxides using a methylene blue derivative. Biochem Int1985; 10: 205211[ISI][Medline]
-
Marumo F, Kamata K, Okubo M. Deranged concentrations of water-soluble vitamins in the blood of undialysed and dialyzed patients with chronic renal failure. Int J Arttif Organs1986; 9: 1724
-
Ono K. The effect of vitamin C supplementation and with drawal on the mortality and morbidity of regular hemodialysis patients. Clin Nephrol1989; 31: 3134[ISI][Medline]
-
Ganguly R, Ramirez G, Fuller S, Curry A, Chamberlain P. Immunologic competence of hemodialysis patients following withdrawal of vitamin C supplement. Nephron1987; 47: 299304[ISI][Medline]
-
Burr ML, Sweetnam PM, Hurley RJ, Powell GH. Effects of age and intake on plasma-ascorbic-acid levels. Lancet1974; 2: 163164[Medline]
-
Ames BN, Cathcart R, Schwiers E, Hoschstein P. Uric acid peroxides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc Natl Acad Sci USA1981; 78: 68586868[Abstract]
-
Sisto T, Paajanen H, Mestsä-Ketelä T, Harmoinen A, Nordback I, Tarkka M. Pretreatment with antioxidants and allopurinol diminishes cardiac onset events in coronary artery bypass grafting. Ann Thorac Surg1995; 59: 15191523[Abstract/Free Full Text]
-
Henrry PD. Antiperoxidative actions of calcium antagonists and atherogenesis. J Cardiovasc Pharmacol1991; 18: S6S10
-
Kataoka M, Tonooka K, Imai K, Aimoto T. Hydroxyradical scavenging activity of nonsteroidal anti-inflammatory drugs. Free Radic Res1997; 4: 419427
-
Welch RW, Wang Y, Crossman A Jr, Park JB, Kirk KL, Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem1995; 270: 1258412592[Abstract/Free Full Text]
-
May JM, Mendiratta S, Hill KE, Bruk RF. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J Biol Chem1997; 272: 2260722610[Abstract/Free Full Text]
Received for publication: 7. 4.99
Revision received 16. 8.00.