The object of the study was the blood plasma of 39 patients not previously exposed to anti-tumor treatment: 17 males (47 - 74 years) and 22 females (34 - 67 years) with cancer of kidney, bladder, prostate, ovaries, larynx, colon, uterus, pancreas, and gallbladder. Analysis of distribution of patients in stages and the diagnosis showed that the I^st stage was with 19% of patients, the II^nd stage was with 19%, the III^rd stage with 42%, the IV^th stage was 20%; cancer of: kidney was detected in 8 patients (27%), bladder in 3 (6%), prostate in 3 (6%), ovarian in 2 (5%), larynx in 2 (5%), intestine in 14 (37%), uterus in 3 (6%), pancreas in 1 (4%), gall bladder in 1 (4%).

The blood plasma of 21 patients was also examined with recurrent cancer of epithelial tissues before treatment and after the first course of chemotherapy. The first group included patients with recurrent colorectal cancer: 5 men and 6 women aged 57 to 62 years. All these patients had the IV^th stage of disease with distant metastases. The second group consisted of patients with recurrent bladder cancer: 8 men and 2 women aged 48 to 64 years. The patients of this group had the III^rd stage of disease without metastasis.

The control blood plasma was taken from 14 actually healthy people without harmful habits: 6 men (24 - 74 years) and 8 women (25 - 65 years).

Treatment of recurrent colon cancer included 8 courses of chemotherapy by cytostatics in conventional doses according to Protocol of International Clinical Recommendations. In case of relapse of bladder cancer the treatment included 6 courses of chemotherapy by carboplatin, methotrexate, vinblastin. The dose of carboplatin was calculated by AUC-6 index.

Patients in both groups were observed throughout the year and depending on the long-term results. Two above-mentioned groups were divided into two subgroups: those with the achieved objective response (stabilization or partial response) or with progression of the disease. The results of treatment were evaluated by computer tomography using the RESIST criteria; the computer tomography was carried out before treatment and every 8 weeks during therapy.

Free radical activity (FRA) was estimated by the method of induced biochemiluminescence [9] on BKhL-07 biochemiluminometer conjugated with IBM computer. BKhL-07 is designed to register light fluxes that occur in biological samples as a result of the flow of enzymatic and chemical processes. The method is based on the fact that in the present system the catalytic decomposition of hydrogen peroxide by metal ions with variable valence-ferrous iron occurs: ROOH + Fe 2 + → RO • + OH − + Fe 3 + (Fenton reaction). The resulting RO • and OH − react with activation of free radical oxidation in a biological substrate which leads to the formation of an unstable tetroxide which disintegrates with the release of a quantum of light recorded at BHL-07. The intensity of the luminescence process is affected by the full complex of compounds with both oxidative and prooxidant effects.

0.1 ml of blood plasma, 0.4 ml of phosphate buffer (pH = 7.5), 0.4 ml of ferrous sulfate solution (0.05 mMol) were added to the measuring cuvette. The cuvette was placed in BHL-07, and then 0.2 ml of 2% hydrogen peroxide solution was added. Measurement of free radical activity occurs automatically.

An informative indicator (I_max) is the maximum intensity of the glow of the sample under investigation measured in pulses/sec. I_max reflects the free radical activity of the sample.

The oxidative modification of proteins (OMP) was determined by the level of carbonyl derivatives found in reaction with 2,4-dinitrophenylhydrazine [10] . The method is based on the reaction of interaction between the oxidized amino acid protein residues with 2,4-dinitrophenylhydrazine (2,4-DNPH) with formation of 2,4-dinitrophenylhydrazones which are spectrophotometrically detected.

Control and experimental samples contained 0.05 ml of serum and 0.95 ml of phosphate buffer (pH 7.4), 1 ml of solution of 2,4-DNPH was poured into the experimental samples, and 1 ml of 2N HCl was added to the control samples. To precipitate proteins, 1 ml of 1N HClO₄ was added to each sample. Samples were incubated at room temperature for one hour, and then centrifuged at 3000 rpm for 15 min. The precipitate was washed by adding 3 ml of a mixture of ethyl acetate-ethanol (1:1), centrifuged at 3000 rpm for 15 min. When this occurred, the extraction of lipids and 2,4-DNPH, which did not react with the carbonyl groups of oxidized proteins, took place. The obtained precipitate was dissolved in 8 M urea solution. The carbonyl derivatives obtained as a result of the oxidative modification of the proteins interacted with 2,4-DNPH to form 2.4 dinitrophenyl hydrazones.

The family of DNPH-derivatives includes aldehyde (ADNPH) and ketone derivatives.

230 nm―aliphatic aldehyde dinitrophenylhydrazones of neutral nature (ADNPH)

270 nm―aliphatic aldehyde dinitrophenylhydrazones of basic nature

370 nm―aliphatic ketone dinitrophenylhydrazones of basic nature

430 nm―aliphatic aldehyde dinitrophenylhydrazones of basic nature

530 nm―aliphatic ketone dinitrophenylhydrazones of basic nature [7] [11] .

The obtained results were statistically processes using the Statplus software package, «AnalystSoft», 2018. While estimating the data set of biochemical parameters of patients with malignant tumors of epithelial tissues according to Shapiro-Wilk test, the normality of distribution was not confirmed; that is why for estimation of the significance in differences with the data of the control group the methods of nonparametric statistics were used: Mann Whitney U-test and Kolmogorov-Smirnov test. To analyze the correlation of the data of two samples, the Spearman correlation coefficient was used. Differences were considered to be statistically significant at p < 0.05.

Data from PS were aggregated and de-linked from personal information related to patients, medical institutions, and pharmacies: these are anonymous data. Therefore, no ethical issues are posed by the use of these data for this study.

Free radical activity of blood plasma increased at all stages of carcinogenesis as compared with healthy people (Figure 1).

At the III^rd and IV^th stages a higher statistically significant increase in free radical activity was observed which greatly exceeds that at the I^st, II^nd stages. The high level of I_max is likely due to the presence of distant metastases and this fact is reflected in the availability of reliable interconnection between these two parameters (r = 0.456). Thus, an increase is shown in free radical activity of blood plasma from the first stage of malignancies of epithelial tissues with further increase in subsequent stages which can be due to the detected activation of free radical oxidation at metastasis.

A significant activation of the total OMP was observed in malignant tumors of epithelial tissues, as evidenced by an increase in the content of carbonyl derivatives of proteins in the plasma, starting from the I^st stage of disease (by 71%, 146%, 259% and 82%, respectively) (Figure 2).

Special attention was paid to the activity of OMP at the initial and terminal stages of the disease, which demonstrate no great differences. But the analysis of the distribution of carbonyl derivatives of different wavelength showed a significant decrease of the OMP activity as compared to actually healthy people for the I^st stage of cancer (Figure 3).

A statistically significant reduction in absorption level is observed registered at 230 nm wavelength where aliphatic ADNPH of neutral nature are found. These compounds are the markers of protein fragmentation. On the contrary, the content of carbonyl derivatives at wavelengths of 270, 370 and 430 statistically significantly increases (Figure 3).

In the terminal (IV) stage of malignant neoplasms the level of phenylhydrazones, determined at 230, 370 and 530 nm, is significantly increased, that is, aliphatic ketone dinitrophenyl hydrazones are predominant which are the markers of protein aggregation characteristic of the later stages of oxidative stress (Figure 4).

With the development of the objective effect in patients after the 1^st course of treatment a statistically significant reduction of free radical activity of blood plasma is observed, in average by 20% (Figure 5). In the subsequent progression of tumor a tendency is shown to increase in free radical activity of blood plasma after the 1^st course of treatment.

With objective effect of polychemotherapy, the significant differences in the level of blood plasma OMP proteins with that before treatment were not found (Figure 6).

An increase in free radical activity of blood plasma in the absence of objective effect after polychemotherapy was accompanied by the activation of the total OMP―by 2.83 times in patients with recurrent colon cancer and by 2 times in patients with recurrent bladder cancer (Figure 6).

Analysis of phenylhydrazones distribution at different wavelengths, depending on the efficiency of cytostatic therapy, did not show significant changes in the case of disease progression (Figure 7, Figure 8).

Common to the cases of the objective effects of polychemotherapy has been a dramatic increase in the carbonyl derivatives recorded at 230 nm (Figure 9, Figure 10).