The study aims at conducting a comparative analysis of the vitamin status of urban and rural school children with levels of free radical reactions in their blood serum.

METHODS

Two experimental groups were created. The first group included urban school children living in the city of Orenburg (n = 250), while the second group consisted of school children living in villages located in Orenburg region (n = 200). Subgroups — 12–14 year olds (n = 150 and n = 100 among urban and rural school children respectively) and 15–17 year olds (n = 100 in both groups) — were marked out from each of the two experimental groups. The study included school children aged 12–17 years, who have no chronic diseases, have not been sick for a month before the study and residing in an area with the same anthropogenic load (within the group). The exclusion criterion here was unwillingness by the children or their parents to participate in the study. The Ethics Committee of Orenburg State Medical University approved the study. The parents of the school children gave a written consent for the participation of their children in the study. The samples for the study in the two groups were obtained simultaneously in three months after the children have commenced classes at the school.

Venous blood and urine were the biological material for the study. Blood was collected in the morning before the children could take their breakfast. 10 ml of blood was collected in Vacuette (BD, USA) vacuum tubes without an anticoagulant. It took 2 hours to deliver the blood samples to the lab. Transportation and storage was carried out at a temperature of 18–25 °C. Urine was collected in the morning after sleep or no earlier than after 2–3 hours after the previous urination. The urine samples (not less than 20 ml in volume) were collected in dry sterile vials. Transportation and storage took 3 hours. The biomaterial was transported and stored at a temperature of 18–25 °C.

Iron-initiated chemiluminescence was used to assess the level of free radical reactions in the blood serum. After collecting blood, it was held for 30 minutes and then centrifuged at 1500 rpm for 15 minutes. The resulting serum was diluted with phosphate buffer (2.72 g KH₂PO₄ and 7.82 g KCl in 1 liter of distilled water, pH 7.4). The resulting solution was titrated with saturated KOH solution until pH 7.45 was achieved. Chemiluminescence registration was performed according to Farkhutdinov technique [15] on Chemiluminometer-3 device (Lumex, Russia). The sensitivity of the device is about 200 photons/sec. The device was calibrated before commencement of work on reference light source (luminescent uranium glass JS-19). The maximal intensity of the fast signal, lightsum of the slow signal (S) and the tangent of the backside angle of the chemiluminescence signal were determined. Indicator values were given in relative units, having calculated their ratio for experimental and blank control samples.

The level of excretion of B vitamins and vitamin C in the urine and the content of vitamins A and E in the blood serum were examined.

Analysis of specimens for the presence of retinol and tocopherol was performed using bioliquid analyzer Fluorat 02-ABLF (Lumex, Russia). In preparing the samples, 1 ml of blood serum and 1 ml of ethanol were placed in one tube for centrifugation, while 1 ml of distilled water and 1 ml of ethanol (calibration pattern) were placed in the other tube. Both tubes were shaken in Vortex apparatus for 30 seconds after which 5 ml of hexane was added and again shaken for 1 minute. After shaking the tubes, they were centrifuged for 10 minutes at 1500 rpm. The separated hexane layer (extract) was used for fluorimetric analysis. Content of vitamin A or E (X, μg/ml) in the blood serum was calculated using the formula:

X = C_vit × V_E × Q / V_с,

where C_vit is vitamin concentration in the extract (measured in μg/ml); V_E is the extract volume (measured in ml); V_c is the blood serum volume taken for analysis (measured in ml); Q is the coefficient reflecting extract dilution.

Content of ascorbic acid and B vitamins was determined by the level of their excretion in urine. Visual titration with Tillman’s reagent (2,6-dichlorophenol sodium) was used for vitamin C. 10 ml of urine, 10 ml of distilled water and 1 ml of 10 % HCl solution were measured in two conical flasks. The contents of each flask was stirred and titrated with 0.001 N solution of Tileman’s reagent until pink color appeared and remained for 30 seconds. Vitamin C content (X, mg/h) was calculated by the formula:

X = 0,088 × А × C / B,

where 0.088 is the coefficient reflecting the amount of ascorbic acid, which is equivalent to 1 ml of 0.001 N solution of 2,6-dichlorophenol sodium (measured in mg); A is the arithmetic average of results of titration of 0.001 N solution of 2,6-dichlorophenol sodium of two urine samples (measured in ml); B is the amount of urine taken for titration (in ml); C is the average daily amount of urine (1500 ml for boys and 1200 ml for girls).

Thiamine excretion in the urine was determined by Wang & Harris technique. Riboflavin levels in urine were estimated by Maslennikova & Gvozdeva method, while those of pyridoxine were calculated by fluorimetric method [16].

The actual nutrition by the school children was estimated based on the meal taken by them in the last 24 hours [17]. This was done using a questionnaire. After that, the biological values of the diets were estimated using tables of chemical composition of food products proposed by Skurikhin & Tutelian [18]. The data obtained were compared with physiological norms for each age group [19].

The sample size was calculated using Sanetliyev’s formula (1968) where n is the number of observations, t is the confidence coefficient, p is the prevalence indicator, q = 100 % – p, ∆ is the confidence interval. Considering that in medical research, 95% is the minimum confidence level, which corresponds to a confidence factor t = 1.96, we took p equal to q, i. e. 50 %, in order to maximize the product of p and q, while 100 % was taken as the confidence interval. The amount of minimum sample calculated in this way (ensuring representativeness) was n = 100. Student’s test was applied with subsequent calculation of significance (p). This was to identify statistically significant differences in the experimental groups. The data were statistically processed using software package Statistica 5.0, which automatically calculated the mean values, standard deviation and standard error of the mean.