RU2412444C1 - Method of predicting exacerbation of chronic hepatitis c in teenage children - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: by means of bioluminescent method determined is activity of NADPH- and NADP-dependent reactions of glutamatdehydrogenases of peripheral blood lymphocytes of patients with chronic hepatitis C, and value of coefficient NADPH/NADP+ in enzymatic reactions of glutamatdehydrogenase is used to make conclusion about possibility to develop chronic hepatitis C exarcerbation. If coefficient value is ëÑ25, development of exarcerbation is predicted, if coefficient value is <25 - absence of exarcerbation. ^ EFFECT: application of method allows to make quick prediction of CHC exacerbation in children with clinical-biochemical remission at early stages. ^ 1 tbl, 4 ex

Description

The invention relates to medicine, namely to pediatrics, and can be used to determine the exacerbation of the course of chronic hepatitis C (CHC) in adolescent children.

It is known that lymphocytes that realize the basic functions of a specific immune response are the main cells in the immunological surveillance system and take an active part in the anti-infection protection system, including in case of viral hepatitis C (HCV) [2, 8]. It has been proven that the outcome of acute HCV and the nature of the course of chronic hepatitis C depend on the level of functional activity of lymphocytes [7]. At the same time, it was found that the functional activity of lymphocytes is largely determined by the level of their metabolic processes [6, 10]. Among the indicators that most objectively reflect the main parameters of intracellular metabolism, dehydrogenases can be attributed.

NADP-dependent glutamate dehydrogenase (NADPHDG, EC 1.4.1.4) provides oxidative deamination of L-glutamic acid using NADP ⁺ as a cofactor [1]. The enzymatic reaction of glutamate dehydrogenase is reversible, respectively, ammonia in the presence of NADPH and α-ketoglutaric acid can participate in the synthesis of glutamate. Glutamate dehydrogenase is an oligomeric enzyme with a molecular weight of 312,000, which consists of 6 subunits. The enzyme shows its activity only in multimeric form. When glutamate dehydrogenase is dissociated into subunits, which occurs in the presence of NADPH, GTP, and a number of steroid hormones, the enzyme loses its ability to oxidatively deamine glutamic acid, but acquires the ability to deamine a number of other amino acids. A similar feature characterizes the allosteric mechanism of glutamate dehydrogenase regulation and defines this enzyme as regulatory in the amino acid metabolism system.

A known method for predicting exacerbations of viral hepatitis, consisting in the study of bilirubin in blood serum, while at the same time at the peak of bilirubinemia and 7-10 days after it, the level of serum iron was additionally determined and when it increased compared to the initial level, an exacerbation was predicted [3]. The disadvantage of this method is the prediction of exacerbations only with a prolonged course of acute hepatitis.

A known method for predicting the rapid progression of the disease in patients with chronic hepatitis C is the study of body mass index, the quantitative blood count of lymphocytes expressing markers CD3 / CD25 ⁺ , CD3 / CD95 ⁺ and CD118 ⁺ lymphocytes, blood concentrations of insulin-like growth factor and transforming factor growth, HCV genotype and antigens of the HLA system. Based on the data obtained, a total indicator was calculated, based on the value of which the course of the disease was predicted (risk of liver cirrhosis) [5]. A significant disadvantage of this method is the difficulty of its implementation and the complexity of the calculations.

Closest to the proposed one is a method for predicting the course of chronic hepatitis C by determining microsomal and mitochondrial autoantibodies in blood serum and with an increase in their content by 25% or more, the course of chronic hepatitis C with exacerbations is predicted [4].

The disadvantage of this method is that it is implemented only in patients with biochemical and virological activity of the process and predicts only a prolonged course of chronic hepatitis C. However, the course of chronic hepatitis C consists of periods of exacerbations and remissions, and this method cannot be used to predict exacerbation of the disease in patients with clinical and biochemical remission.

In addition, in literary sources, no predictions of CHC in children were found, which determines the urgency of the problem.

The objective of the invention is the early prediction of exacerbation of chronic hepatitis C in adolescent children.

The task is carried out due to the fact that, using the bioluminescent method, the activities of NADPH and NADP ⁺ dependent reactions of glutamate dehydrogenases of peripheral blood lymphocytes of patients with chronic hepatitis C are determined, and the possibility of exacerbation is judged by the value of the NADPH / NADP ⁺ coefficient in the enzymatic reactions of glutamate dehydrogenase: ≥25 predict the development of exacerbation, with a coefficient value <25 - the absence of exacerbation.

The method is as follows. In adolescent children with chronic hepatitis C, venous blood is taken from the ulnar vein by free flow into heparin tubes. Lymphocytes are isolated. The density gradient of ficoll-verographin is centrifuged according to the standard procedure of A. Boyum (1968) [9]. The concentration of lymphocytes is calculated, for example, in the Goryaev chamber. When controlling the morphological composition of leukocyte suspensions determine the purity of the output of lymphocytes, which is at least 97%. 1 million selected cells are used to determine the activity of NADPH-GDH and NADP-GDH lymphocytes using one of the known methods, for example, bioluminescent [6]. For this, a suspension of isolated lymphocytes containing cells at a concentration of 1.0 million / ml is destroyed by osmotic lysis with the addition of distilled water (1: 5 by volume) and 1.0-2.0 mM dithiothreitol. Then directly determine the activity of NADPH-GDH and NADP-GDH. For this, 50 μl of a suspension of destroyed lymphocytes are added to 150 μl of the incubation mixture containing the appropriate substrate and cofactor (for the NADP-GDH enzyme, the substrate is glutamate - 0.5 mM, cofactor NADP ⁺ - 1.65 mm, buffer pH 9.8; for the NADPH-GDH enzyme, the substrate is 2-oxoglutarate - 100.0 mM, cofactor NADPH - 0.0025 mM, buffer pH 7.4).

It should be noted that in the incubation mixture to determine the activity of NADPH-GDH and NADH-GDH, NH ₄ Cl is additionally added at a concentration of 5.0 mM.

After incubation of the test samples at 37 ° C for 30 minutes for NADP-GDH or 5 minutes for NADPH-GDG, 50 μl of flavin mononucleotide (FMN) at a concentration of 1.5 × 10 ^-5 M, 50 μl 0, 0005% myristic aldehyde and 10 μl of the NADH: FMN enzyme system oxidoreductase-luciferase (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ phosphate buffer at pH 7.0). After mixing the bioluminescent reagents and the incubation sample using a biochemiluminometer, for example brand "BLM-8803", measure the glow. Given that the cells have a certain number of substrates for the course of various metabolic reactions, including those catalyzed by the studied enzymes, the parameters conventionally called "substrate background of enzymes" are determined. Determined under the same conditions as for the above dehydrogenases, but instead of the corresponding substrate, a buffer is added to the incubation mixture. As a result of measuring the glow on a bioluminometer, relative values of the activity of the studied enzymes are obtained. To obtain the absolute values of the activity, a graph of the dependence of the intensity of bioluminescence on the concentration of NADPH is constructed (calibration graph). For this, 200 μl of a standard NADPH solution in the range of 10 ^-9 -10 ^-4 M is added to bioluminometer cuvettes containing FMN, myristic aldehyde and NAD (F) N: FMN oxidoreductase-luciferase at the concentrations indicated above, after which the intensity of bioluminescence was measured. Due to the wide range of pH buffers used to determine dehydrogenase activity, as well as the pH dependence of the bioluminescence of the enzymatic system from luminous bacteria, calibration plots were plotted for each pH buffer. The activity of NAD (F) -dependent dehydrogenases is calculated by the formula

A = Δ [C] × V / T,

where A is the dehydrogenase activity, E per 1 × 10 ⁴ lymphocytes (1E = 1 μmol / min [1]);

Δ [C] is the difference in the concentrations of NAD (F) N in the samples "enzyme" and "background enzyme", mmol;

V is the sample volume, ml;

T is the incubation time, min

Then, by the ratio of activity levels of NADPH-GDH and NADP-GDH, the value of the NADPH / NADP ⁺ coefficient is found. The value of the coefficient, equal to and higher than 25, indicates the development of exacerbation of CHC, less than 25 - the absence of exacerbation of CHC. The value of 25 was obtained experimentally based on a comparison of the data of the bioluminescent analysis, the results of a medical examination and the subsequent exacerbation of chronic hepatitis C. The value of the NADPH / NADP ⁺ coefficient, equal to or greater than 25, indicates a pronounced prevalence of reductive amination over oxidative deamination, which reduces the number of substrates in energy processes and, accordingly, reduces the functional activity of lymphocytes.

This method was tested on 22 patients with chronic hepatitis C. All patients also underwent generally accepted laboratory examination methods upon admission to the hospital and in dynamics after 6 months: peripheral blood analysis, biochemical blood analysis with determination of the concentration of total bilirubin and its fractions, thymol test, serum aminotransferase (alanine aminotransferase (ALT) and aspartate aminotransferase ( ACT) Using an enzyme-linked immunosorbent assay (ELISA), antibodies were detected against the core protein and non-structural proteins of the virus (NS ₃ , NS ₄ , NS ₅ ). The method of polymerase chain reaction (PCR) was determined and HCV RNA. Prediction match was measured by ALT level.

Table 1 The prognosis of exacerbation of chronic hepatitis C by the value of the NADPH / NADF ⁺ coefficient No. Development / absence of exacerbation NADPH / NADF ⁺ one there will be no aggravation 0.254 2 there will be no aggravation 12,835 3 there will be no aggravation 8.547 four there will be no aggravation 0,120 5 there will be no aggravation 0.139 6 there will be no aggravation 1,203 7 there will be no aggravation 1,616 8 there will be no aggravation 12,269 9 there will be no aggravation 2,190 10 there will be no aggravation 7,853 eleven there will be no aggravation 4,996 12 there will be an aggravation 803.995 13 there will be an aggravation 281.703 fourteen there will be an aggravation 25,237 fifteen there will be no aggravation 12,369 16 there will be an aggravation 25,237 17 there will be no aggravation 0.678 eighteen there will be an aggravation 25,862 19 there will be no aggravation 0.139 twenty there will be no aggravation 6,416 21 there will be an aggravation 26,255 22 there will be an aggravation 89,635

The activity of NADPH-GDH and NADP-GDH in blood lymphocytes was determined using the bioluminescent method in children with chronic hepatitis C.

According to the results of a biochemical blood test, it was found that 15 children and adolescents did not have an exacerbation of CHC for 6 months (ALT and ACT activity were within normal limits), 7 - in the range of 3 weeks to 6 months, an exacerbation of CHC developed (upon repeated examination an increase in the activity of aminotransferases in blood serum was detected).

In all patients, a coincidence of the forecast made by determining the coefficient of NADPH / NADP ^{+ was found} .

Clinical example 1

Patient R., 14 years old (case history No. 135) was undergoing routine examination in the children's infectious ward of the Municipal Clinical Hospital No. 20 named after I.S.Berson of Krasnoyarsk with a diagnosis of Chronic viral hepatitis C, inactive. Sop. diagnosis: chronic gastritis, biliary dyskinesia. The child is registered in the dispensary for 2 years in the dispensary room of the hepatocenter of the Municipal Clinical Hospital No. 20 of Krasnoyarsk, when he was diagnosed with chronic viral hepatitis C.

From the anamnesis it is known that the child received a blood transfusion at the age of 8 months in connection with severe pneumonia.

Upon admission to the children's infectious diseases department of the Municipal Clinical Hospital №20 named after I.S.Berzon, Krasnoyarsk, the boy did not complain.

The results of the biochemical study are as follows: total bilirubin 14.6 μmol / L, ALT 0.6 mmol * h / L (within normal limits) ACT 0.24 (within normal limits) mmol * h / L.

In the study of peripheral blood lymphocytes by bioluminescence with the determination of the activity of NADPH and NADP-dependent glutamate dehydrogenase reactions, it was found that NADPH / NADP ⁺ amounted to 1.203, which indicates the absence of exacerbation.

When examined in the dispensary office of the hepatological center on the basis of the infectious diseases department of the Municipal Clinical Hospital No. 20 named after I.S.Berzon after 6 months, the activity of aminotransferases was also within normal limits.

Clinical example 2

Patient X., 17 years old (No. of medical history 562) was treated in the children's infectious ward of the Municipal Clinical Hospital № 20 named after I.S. Berzon of Krasnoyarsk with a diagnosis of Chronic viral hepatitis C, with a moderate degree of activity. Sop. diagnosis: Gilbert's syndrome.

From the anamnesis, it is known that the girl’s mother is also ill with chronic viral hepatitis C. The child has been undergoing dispensary registration for 9 years (since 1999) in the dispensary room of the Municipal Clinical Hospital No. 20 of Krasnoyarsk, when he was diagnosed with chronic viral hepatitis C. A routine examination in the dispensary office of the hepatocenter revealed hyperfermentemia and the child was sent to a hospital to clarify the nature of liver damage and resolve the issue of treatment tactics.

Upon admission to the hospital, the girl complained of abdominal pain, weakness, fatigue. During the examination, pronounced ictericity of the skin and sclera, a moderate enlargement of the liver (1 / 3-2 cm-2 cm) were revealed.

The results of the biochemical study are as follows: total bilirubin 104.7 μmol / L (normal up to 20 μmol / L), direct 35.9 indirect 71.5, ALT 2.15 mmol * h / l (3.2 times higher than normal) ACT 1,045 mmol * h / l (1.6 times higher than normal).

In the study of peripheral blood lymphocytes by bioluminescence with the determination of the activity of NADPH and NADP-dependent glutamate dehydrogenases, it was found that NADPH / NADP ⁺ was 8.547, which indicates the absence of exacerbation.

In the control biochemical analysis of blood, a decrease in the concentration of bilirubin and normalization of ALT and ACT were noted. On the 26th day of hospital stay, the patient was discharged.

When examined in the dispensary office of a hepatocenter on the basis of the infectious diseases department of the Municipal Clinical Hospital No. 20 after 6 months, the activity of aminotransferases remained at the level of normal indicators.

Clinical example 3

Patient S., 10 years old (case history No. 443), was on a routine examination in the children's infectious diseases unit of the Municipal Clinical Hospital No. 20 in Krasnoyarsk with a diagnosis of chronic viral hepatitis C, inactive.

From the anamnesis it is known that the child received a blood transfusion at the age of 3 years in connection with surgery. The patient is observed for 4 years in the dispensary room of the hepatocenter of the Municipal Clinical Hospital No. 20 of Krasnoyarsk with a diagnosis of chronic viral hepatitis C. During a routine examination in the dispensary office of the hepatocenter, hyperfermentemia was detected and the child was sent to a hospital to clarify the nature of liver damage and decide on tactics treatment.

The results of the biochemical study are as follows: total bilirubin 16.3 μmol / l, indirect reaction, ALT 0.6 mmol * h / l (within normal limits), ACT 0.25 mmol * h / l (within normal limits).

In the study of peripheral blood lymphocytes by bioluminescence with the determination of the activity of NADPH and NADP-dependent reactions of glutamate dehydrogenases, it was found that NADPH / NADP ⁺ amounted to 25.237, which indicates the development of exacerbation.

On the 10th day of hospital stay the patient was discharged. However, during an outpatient examination in the dispensary office of the hepatocenter on the basis of the infectious ward of the Municipal Clinical Hospital No. 20 named after I.S.Berzon after 6 months, a 1.3-fold increase in the activity of ALT and ACT was again noted.

Clinical example 4

Patient Ch., 16 years old (medical history No. 546), was examined and treated in the children's infectious diseases unit of the Municipal Clinical Hospital No. 20 of Krasnoyarsk with a diagnosis of Chronic Viral Hepatitis C, with a minimal degree of activity. Sop. diagnosis: chronic gastritis, dzhvp. From the anamnesis it is known that the child has been observed since 2008 in the dispensary room of the Municipal Clinical Hospital No. 20 of Krasnoyarsk with a diagnosis of Chronic Viral Hepatitis C. During a routine examination, an increase in ALT activity was recorded in the child and the boy was sent to a hospital.

The results of the biochemical study are as follows: total bilirubin 18.7 μmol / l, indirect reaction, ALT 1.05 mmol * h / l (1.5 times higher than normal), ACT 0.44 mmol * h / l (within normal limits) .

When studying peripheral blood lymphocytes by bioluminescence with the determination of the activity of NADPH and NADP-dependent glutamate dehydrogenase reactions, it was found that NADPH / NADP ⁺ amounted to 281,703, which indicates the development of exacerbation.

In the control biochemical analysis of blood, normalization of the activity of aminotransferases was noted.

On the 10th day of hospital stay the patient was discharged. However, during outpatient observation in the dispensary room after 6 months, an increase in ALT and ACT activity by 1.6 times was again noted.

The last two examples indicate the need for monthly monitoring of taking a biochemical blood test in patients with chronic hepatitis C and determining the activity of aminotransferases for earlier detection of exacerbation.

The advantages of the proposed method are:

1) the ability to predict exacerbation of chronic hepatitis C in children with clinical and biochemical remission;

2) a high level of coincidence of the forecast - 100%.

The proposed method can be recommended for use in clinical practice.

Sources used

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Claims (1)

A method for predicting exacerbation of chronic hepatitis C in adolescent children by blood testing, characterized in that, using the bioluminescent method, the activities of NADPH and NADP-dependent reactions of glutamate dehydrogenases of peripheral blood lymphocytes of patients with chronic hepatitis C are determined, and the possibility of an exacerbation of chronic hepatitis C is judged by magnitude ratio of NADPH / NADP ⁺ glutamate in enzymatic reactions: the value of the coefficient> 25 predict exacerbations development, When the value of the coefficient of <25 - the absence of an exacerbation.