RU2284040C2 - Method for diagnosing metastases in lung root lymph node in pulmonary carcinoma cases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining lactate dehydrogenase activity and NADP-dependent glutamate dehydrogenase. Lactate dehydrogenase activity being equal to or greater than 12.50 mcU and NADP-dependent glutamate dehydrogenase activity being equal to or less than 0.25 mcU, no metastases in lung root lymph node in pulmonary carcinoma cases are thought to be available. Lactate dehydrogenase activity being less than 12.50 mcU and NADP-dependent glutamate dehydrogenase activity being greater than 0.25 mcU, the metastases are proved to be available.

EFFECT: high accuracy of diagnosis.

Description

The invention relates to medicine, more specifically to oncology. The method is intended for preoperative planning of treatment tactics.

A known laboratory method for the diagnosis of lung cancer metastases, based on the study of the erythrocyte sedimentation rate, color indicator, the average volume of red blood cells, the concentration of lymphocytes, sodium, the level of albumin and uric acid [Patent 1184125 SU, IPC ⁷ A 61 B 10/00. A method for the diagnosis of lung cancer metastases / 1st Moscow Medical Institute I.M.Sechenova, SU / Vapnik T.N., Shekhter A.I., Romanichev Yu.A., Ablitsov Yu.A.; claimed 1983.08.05; application 3632209/14; publ. 2002.11.20].

The known method is based on the analysis of blood parameters determined by various methods and having wide variations in normal and in various pathological conditions. In addition, with the exception of the concentration of lymphocytes in the blood, all of these parameters do not reflect the state of activity of the immune system, the lack of reactivity of which determines the appearance of metastases.

The known method is not accurate enough.

The objective of the invention is to improve the accuracy of diagnosis.

The problem is solved in that in the method for the diagnosis of metastases in the lymph nodes of the lung root in patients with lung cancer, including the study of biological material, according to the invention, the activity of lactate dehydrogenase (LDH) and the activity of NADP-dependent glutamate dehydrogenase (NADPHDG) in peripheral blood lymphocytes are determined, and the combination LDH activity above or equal to 12.50 μE and NADPHY activity equal to or lower than 0.25 μE indicates the absence of metastases, and a combination of LDH activity below 12.50 μE and NADPHDH activity in Chez MKE 0.25 indicates the presence of lymph node metastasis of lung root.

Lymphocytes equipped with receptors for all humoral regulatory substances respond to incoming impulses by a change in enzyme activity. The cell response depends on the intensity of the incoming signals, which is determined not only by the amount of regulatory substances circulating in the blood, but also by the sensitivity of immunocompetent cells to them - lymphocytes. With tumor growth, a complex of regulatory factors affects lymphocytes. On the one hand, activation signals stimulate the functional activity of immunocompetent cells, and on the other hand, the tumor itself synthesizes a number of immunosuppressive substances that inhibit the reactivity of immunocompetent cells. The most informative for the study of the metabolism of activated lymphocytes are redox enzymes. This is due to the fact that, being the main carriers of electrons in the cell, they carry out key reactions of cellular metabolism and coordinate conjugate metabolic pathways. LDH (EC 1.1.1.27) is the main regulator of the cytoplasmic level of NADH / NAD. In the case of an excess of NADH in the cytoplasm, LDH reduces pyruvate to lactate, which is then removed from the cell (anaerobic LDH reaction). At the same time, when aerobic processes are activated, LDH oxidizes lactate to pyruvate to form NADH (aerobic LDH reaction). NADPHDG (EC 1.4.1.4) performs NADP-dependent oxidative deamination of L-glutamic acid. Glutamate dehydrogenases are key in the system of intracellular nitrogen metabolism.

The method is as follows. From heparinized venous blood (25 IU / ml) of the examined patient with lung cancer, aimed at surgical treatment, lymphocytes are isolated. For this, heparinized blood is layered on ficoll-urographin (ρ = 1,077) in a ratio of 1.5: 5. Centrifugation is carried out at 400 g for 30 minutes. Cells located at the interface between the plasma and the ficoll-urographin gradient are harvested and washed three times in 199 medium. The number of selected lymphocytes in the Goryaev chamber is counted and the concentration of lymphocytes is adjusted to a concentration of 10 ⁶ cells / ml. Then, the suspension of isolated lymphocytes containing cells at a concentration of 1.0 million / ml, after a single freeze-thaw, is additionally destroyed by osmotic lysis with the addition of distilled water (1: 5 by volume). The activity of LDH and NADPHDG is determined by one of the known methods, for example, bioluminescent. The incubation mixture for determining LDH activity contains the following reagents: 50 μl of a suspension of destroyed lymphocytes, 50 μl of 2.0 mM lactate in 0.1 M Tris-HCl buffer pH 9.0, 100 μl 0.5 mM NAD ⁺ in 0.1 M Tris-HCl buffer pH 9.0. The incubation mixture for determining the activity of NADPHDG contains the following reagents: 50 μl of a suspension of destroyed lymphocytes, 50 μl of 0.5 mm glutamate in 0.1 M Tris-HCl buffer pH 9.8, 100 μl of 1.65 mm NADP ⁺ in 0.1 M Tris-HCl buffer pH 9.8. 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. The determination is carried out under the same conditions, but a buffer is added to the incubation mixture instead of the corresponding substrate. After incubation of the samples at 37 ° C for 30 minutes, 50 μl of flavin mononucleotide (FMN) at a concentration of 1.5 × 10 ^-5 M, 50 μl of 0.0005% myristic aldehyde and 10 μl of the NAD enzyme system are added to 200 μl of the incubation mixture ) N: FMN oxidoreductase-luciferase (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ -phosphate buffer with a pH of 7.0). After mixing the bioluminescent reagents and the incubation sample using a bioluminometer, for example "BLM-8803", measure the luminosity. Get the relative values of the activity of the studied enzymes. To obtain the absolute values of the activity, a graph of the dependence of the intensity of bioluminescence on the concentration of NAD (F) N (calibration graph) is plotted. For this, 200 μl of a standard solution of NAD (F) N in the range of 10 ^-9 -10 ^-4 M is introduced into a bioluminometer cuvette containing bioluminescent reagents at the concentrations indicated above, after which the intensity of bioluminescence is measured.

The activity of NAD (F) -dependent dehydrogenases is calculated by the formula:

,

,

Where:

A - dehydrogenase activity, μE (1 μE = 10 ^-6 E, 1 E = 1 μmol / min);

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

0.31 - sample volume, milliliters;

30 - incubation time, min.

The activities of LDH below 12.50 μE and NADPHDH above 0.25 μE indicate the presence of metastases in the lymph nodes of the root of the lung. LDH activity is higher or equal to 12.50 μE, and NADPHDH activity, equal to or lower than 0.25 μE, indicates the absence of metastases.

Based on the presence or absence of metastases, plan the tactics of combined treatment.

Example 1. Patient G., 75 years old. Case history No. 2259/189. He was hospitalized in the Department of Thoracic Surgery of the Krasnoyarsk Regional Oncology Center from 04/08/2003 to 04/28/2003 with a diagnosis of right lung cancer of the IA stage, T ₁ N ₀ M ₀ .

When studying the levels of dehydrogenase activity in blood lymphocytes, it was found that the activity of LDH is 26.89 μE, and the activity of NADPHDH is 0.12 μE.

Diagnosis - there are no metastases in the lymph nodes of the root of the lung. Based on the established diagnosis, preoperative radiation or chemotherapy is not recommended.

Operation 04.17.03., Performed a thoracotomy on the right. Extended lower lobectomy. A histological examination was performed No. 16200-201: squamous keratinizing cancer was determined in the preparation. In the lymph nodes, the picture of reactive hyperplasia, which confirms the result obtained by the proposed diagnostic method.

Example 2. Patient B., 62 years old. Case history No. 7374/662. He was hospitalized in the Department of Thoracic Surgery of the Krasnoyarsk Regional Oncology Center from 11.24.2003 to 12.21.2003 with a diagnosis of stage III left lung cancer, T ₃ N ₁ M ₀ .

When studying the levels of dehydrogenase activity in blood lymphocytes, it was found that the activity of LDH is 7.45 μE, and the activity of NADPHDH is 1.14 μE.

Diagnosis - metastases are present in the lymph nodes of the lung root. Based on the established diagnosis, preoperative radiation or chemotherapy may be recommended.

Operation 04.12.03., Performed thoracotomy on the left. Extended pulmonectomy. A histological examination was performed No. 50154-71: squamous cell carcinoma was detected in the preparation, metastases were detected in the bifurcation lymph nodes, which confirms the result obtained by the proposed diagnostic method.

The proposed method examined 175 people. The significance of differences was determined by the Mann-Whitney criterion.

Diagnosis of metastases in the lymph nodes of the root of the lung allows before surgery to more accurately determine the stage of the disease and the effectiveness of preoperative radiation or chemotherapy.

Claims (1)

A method for the diagnosis of metastases in the lymph nodes of the lung root in patients with lung cancer, including the study of biological material, characterized in that they determine the activity of lactate dehydrogenase (LDH) and the activity of NADP-dependent glutamate dehydrogenase (NADPHDH) in peripheral blood lymphocytes, while the combination of LDH activity is higher or equal 12.50 μE and NADPHDH activity equal to or lower than 0.25 μE indicates the absence of metastases, and a combination of LDH activity below 12.50 μE and NADPHDH activity above 0.25 μE indicates the presence of metastases.