Study participants
This study was conducted at the dialysis unit of the Department of Nephrology and Dialysis, University Clinical Hospital Centre, in Split. In total, a selected population of 45 adults (15 females and 30 males) on maintenance haemodialysis (HD) patients were included.
Eligible participants were enrolled if they met the following inclusion criteria: (i) aged 18 years or more, (ii) stable duration of maintenance HD of more than six months with a three times weekly dialysis program before study entry, (iii) continuity of HD regimen and (iv) those patients have never changed their modality of dialysis treatment (from peritoneal dialysis to HD). None of the patients received antibiotics, cytotoxic drugs, blood transfusions or corticosteroids during the 3 months prior to and during participation in the study. We also excluded those patients who had an implanted pacemaker or cardioverter defibrillator, stents or limb amputation; patients with liver cirrhosis and active underlying malignant disease or active infection at the beginning of the study; and patients who refused to participate in the study.
The causes of ESRD were arterial hypertension in 20 (44.5%) patients, diabetes mellitus in 7 (15.5%), chronic glomerulonephritis in 4 (8.9%), polycystic kidney disease in 5 (11.1%), and unknown in 9 (20%).
The fulfilment of these criteria was determined by interviewing both participants and their relatives, as well as by reviewing participants’ medical records.
The assessment of nutritional status, body mass composition, anthropometric measurements and blood sampling was performed in HD patients before the midweek HD session. Measurements were taken in July, October and January; altogether in 135 HD sessions. Blood was taken just prior to connecting the subject to the dialysis machine and before administering heparin. For determining the post HD blood urea concentration, a blood sample was obtained from the arterial line 2 min after the blood pump was reduced to 50 mL/min (slow-flow technique). All patients were receiving conventional 4 h HD, three times weekly, with bicarbonate dialysate at a flow rate of 500 mL/min and low molecular weight heparin as standard anticoagulation. The dialysis methods and pharmacological therapy have not changed (erythropoietin dose and dose of bone metabolism drugs were changed with dosage adjustment according to clinical guidelines) for the analysed months. High-flux polysulfone dialyzers (Fresenius Medical Care, Bad Homburg, Germany) were used mainly for HD.
All participants were informed of the purpose and nature of the study and provided written consent. The study protocol was accepted by the Hospital Ethics Committee of the University Hospital Centre Split (class 500-03/14-01/40, number: 2181-147-01/06/J.B.-14-2).
Biochemical and haematological analysis
Concentrations of total iron binding capacity (µmol/L), urea (mmol/L), creatinine (µmol/L), uric acid (µmol/L), serum albumin (g/L), phosphates (mmol/L) and C-reactive protein (CRP) (mg/L) were determined by standard laboratory methods.
The following parameters were determined by immunoturbidimetry: high sensitivity CRP (hsCRP) (mg/L) (Beckman Coulter, AU680, USA) and prealbumin (g/L) (Cobas Integra, Roche Diagnostics, Germany). A complete blood count was obtained with a hematology analyzer (Advia 120, Siemens, Erlangen, Germany).
Assessment of nutritional status
There is not a single measurement that provides complete assessment of the nutritional status of HD patients. To assess nutritional status in HD patients in this study, anthropometric measurements; the malnutrition inflammation score (MIS); the dialysis malnutrition score (DMS); body mass composition monitoring and laboratory parameters that reflect nutritional status such as serum albumin and prealbumin were used.
The following anthropometric parameters were collected for each study subject: height (cm), dry weight (kg), triceps skin fold (mm), waist circumference (WC), hip circumference (HC) and mid-upper arm circumference (MUAC). Additionally, waist-to-height ratio (WHtR) and BMI were calculated for each study subjects. The subjects stood upright, facing forward with their shoulders relaxed, and measures were taken with flexible, nonstretchable measuring tape.
The MIS assessment was performed according to the description by Kalantar-Zadeh et al. (28). The MIS is measured on a 4-point scale and is a quantitative nutrition screening tool consisting of four main parts: patients’ related medical history, physical examination, BMI and laboratory parameters. The sum of all components ranges from 0 to 30, and a higher score reflects a more severe degree of malnutrition and inflammation. The MIS was found to significantly correlate with hospitalization; mortality; and indices of nutrition, inflammation, and anaemia (29, 30).
Dialysis Malnutrition Score (DMS) consists of seven features; weight change, dietary intake, GI symptoms, functional capacity, co-morbidity, subcutaneous fat and signs of muscle wasting. Each component has a score from 1 (normal) to 5 (very severe). Thus the malnutrition score (sum of all seven components) is a number between 7 (normal) and 35 (severely malnourished). Lower score denotes tendency towards a normal nutritional status. A higher score is considered to be an indicator of the presence of malnutrition elements i.e. protein energy malnutrition (31).
The assessment of body mass composition was carried out using the Body Composition Monitor portable device (Fresenius Medical Care), which works on the principle of bioimpedance spectroscopy (32). The Body Composition Monitor is a valid method for assessing and monitoring hydration and nutritional status in haemodialysis patients (6). Measurements using the Body Composition Monitor (according to the manufacturer’s instructions) were made before the midweek dialysis session. Patients were in the supine position for approximately 5 min before the measurement. All Body Composition Monitor measurements were performed by the same trained operator with the study participant in the supine position on a nonconductive bed in resting conditions. Body Composition Monitor measurements included the following variables: overhydration volume (OH) (L), lean tissue mass (LTM) (kg), lean tissue index (LTI) (kg/m2), fat tissue mass (FTM), (kg), fat tissue index (FTI), (kg/m2), adipose tissue mass (ATM) (kg) and body cell mass (BCM) (kg) (33).
Oxidative stress
The blood levels of derivatives of reactive oxygen metabolites (d-ROMs) as indicator of protein oxidation and thiobarbituric reactive substances (TBARS) as indicator of lipid oxidation were measured for all study subjects. Total antioxidant capacity, as plasma protein reduced thiol content (THIOLS) and ferric reducing ability of plasma ( FRAP) were also measured.
The d-ROM assay is intended to measure the concentration of total hydroperoxides in serum or heparin plasma. The method was first described by Alberti et al. (34). It is based on the following principle. In an acidic buffered solution (pH = 4.8), iron ions are released from the serum (plasma) proteins and catalyze the in vitro transformation of hydroperoxides into alkoxyl and peroxyl radicals, which further react with the chromogen N,N-diethyl-p-phenylenediamine. The concentration of the colored complex is directly proportional to the concentration of the hydroperoxides that are present in the sample. The absorbance was measured at 505 nm, and the results are expressed in Caratelli units (CARR U). One CARR U corresponds to 0.08 mg/100 mL H2O2. The characteristics of the assay were evaluated and validated by Verde et al. (35), who reported that the assay is reliable even in patients with hyposideremic anemia.
The total antioxidant capacity, as the FRAP value, was measured. FRAP was measured by the ferric reducing/antioxidant and ascorbic acid (FRASC) assay (36). In this assay, the samples are treated with or without ascorbate oxidase, and antioxidants in the sample are evaluated as reductants of Fe3+ to Fe2+, which is chelated by tripyridyltriazine (TPTZ) to form an Fe2+–TPTZ complex absorbing at 593 nm. Absorbance was monitored with a UV–Vis spectrophotometer equipped with a six-cell holder and a thermostatically controlled bath. The results were compared with a standard curve prepared daily with different concentrations of vitamin C (ascorbic acid) and expressed as micromoles of vitamin C equivalents. The validity of this method for determination of vitamin C in heparinized human plasma has been shown previously (37).
The assay for plasma protein reduced thiol content (THIOLS) measures the major source of reducing equivalents (or antioxidant capacity) available in the plasma. Thiol groups were assayed according to the method of Ellman (38) as modified by Hu (39), as described in Himmelfab et al.(40). Briefly, 2 mL of buffer containing 0.1mol/L Tris,1 mmol/L EDTA, pH 8.2, and 100mL plasma was added to cuvettes, followed by 100uL 10 mmol/L 5dithio-bis (2-nitrobenzoic acid) (DTNB) in methanol. Blanks were run for each sample, prepared as above, with the exception that there was no DTNB in the methanol. Following incubation for 15 minutes at room temperature, sample absorbance was read at 412 nm. Results are expressed as umol/L of glutathione.
TBARS assay is based on reaction of malondialdehyde (MDA), one of end products of lipid peroxidation, with TBA (41). To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct (42). Absorbance was monitored by the above-mentioned UV-spectrophotometer. All measurements were carried out in triplicate. Results were compared with a standard curve prepared daily with different concentrations of MDA and expressed as lmol⁄ L MDA.
STATISTICAL METHODS
To discern the mean effect in the numeric variables of the three measurements,with the significance level of 0.05, power 0.95, and the effect size 0.25, the minimum sample size required was 44 subjects (calculated by G * Power 3.1.2, Franz Faul, University in Kiel, Germany). Categorical data are represented by absolute and relative frequencies. Numerical data were described by the median and the limits of the interquartile range. The normality of the distribution of numerical variables was tested by the Shapiro-Wilk test. Differences between numerical variables between measurements were tested by Friedman's test (Post hoc Conover). The correlation between numerical variables was evaluated by Spearman's correlation coefficient ρ (rho). Using multivariate regression analysis (stepwise), we determined the predictors that influence oxidative stress. All P values were two-sided. The significance level was set to Alpha = 0.05. MedCalc Statistical Software version 19.1.7 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2020) was used in the statistical analysis.