RU2498300C1 - Method for prediction of outcome of widespread purulent peritonitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: bioluminescent method is used to determine lactate dehydrogenase (LDG), malate dehydrogenase (MDG), NADP-dependent glutamate dehydrogenase (NADPGDG) and glutathione reductase (GR) activities in peripheral blood lymphocytes in patients with widespread purulent peritonitis. That is followed by calculating a metabolic coefficient (MC) representing the relation of a production of GR and NADPGDG activities to a production of LDG and MDG activities, i.e. MC=(GR×NADPGDG)/(LDG×MDG). If the MC is below 0.005, an unfavourable outcome of the disease is predicted, while the MC being 0.005 or higher, a favourable outcome of widespread purulent peritonitis is predicted.

EFFECT: using the declared method enables the higher early post-diagnosing prediction of the outcome of widespread purulent peritonitis and the higher level of prediction reliability.

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Description

The invention relates to medicine, namely to anesthesiology and resuscitation and can be used to determine the outcome of common purulent peritonitis (RGP).

A known method for predicting an unfavorable outcome of peritonitis widespread, consisting in a study in patients of the content of lactoferrin in peritoneal exudate on the first day of the postoperative period and with a value of less than 2500 ng / ml, an unfavorable outcome is predicted [2]. The disadvantage of this method is the need to study peritoneal exudate in the first day after surgery, which delays the onset of measures aimed at ensuring a favorable outcome of the disease.

There is a method for predicting the outcome of complex treatment of patients with RHP with abdominal administration of imosimase with determining the level of plasminogen, degradation products of fibrinogen and fibrin, as well as indicators of platelet aggregation and disaggregation upon admission and during treatment [3]. The disadvantage of this method is the duration of the research to form a prognosis of the outcome of the disease.

Closest to the proposed one is a method for predicting the outcome of a disease of patients with RHP with the determination of the concentration of total cholesterol (OXc), high density lipoprotein cholesterol (HDL-C), leuko-intoxication index (LII), leukocyte shift index (LIS) and concentration OXc is lower than 2.5 mmol / L, HDL-C is lower than 0.9, LII values above 4.0 and LIS above 6.0 predict an adverse treatment outcome [4]. The disadvantage of this method is the need to determine the claimed indicators only on the 2nd day of the disease, which delays the onset of measures aimed at ensuring a favorable outcome of RHP.

The objective of the proposed method is the early prediction (1 day after diagnosis) of the outcome of RGP.

The task is carried out due to the fact that using the bioluminescent method determine the activity of LDH, MDH, NADPHDH and GR in the peripheral blood lymphocytes of patients with RHP on the 1st day after diagnosis. Then calculate the metabolic coefficient (MK), which is the ratio of the products of the activity of GR and NADPHDH to the product of the activities of LDH and MDH, i.e.

MK = (GR × NADPHYDG) / (LDH × MDH).

With a value of MK below 0.005, an unfavorable outcome of the disease is predicted, with MK equal to or higher than 0.005, a favorable outcome of RHP is predicted.

The value of 0.005 was obtained empirically on the basis of a comparison of the calculated MK values and the data of subsequent observation of the clinical condition of patients with RHP. A MK value below 0.005 indicates a decrease in the activity of enzymatic reactions oxidizing NADPH, which realize antioxidant functions and carry out the transfer of intermediates from energy processes to amino acid metabolism reactions.

The method is as follows. In patients on the first day after the diagnosis of RHP, 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 method of A. Boyum (1968) [7]. Count the number of lymphocytes, 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 LDH, MDH, NADPHDG and GR lymphocytes using one of the known methods, for example, bioluminescent [5, 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 LDH, MDH, NADPHDH and GR. For this, in 50 μl of the incubation mixture containing the appropriate substrate and cofactor, add 50 μl of a suspension of destroyed lymphocytes. Specific values of the concentrations of substrates and coenzymes, as well as the pH of the medium for the determined enzymes are presented in table 1.

Table 1 The concentration of substrates and coenzymes and pH buffer for the determined enzymes Enzyme Substrate, mm Cofactor, mm buffer pH LDH Lactate - 2.0 OVER - 0.50 9.0 MDG Malat - 2.0 OVER - 2.50 9.8 NADFGDG Oxoglutarate - 100 NADPH - 0.0025 7.4 GR GSH - 0.5 NADPH - 0.0025 7.4 Note: media with pH 9.0 and 9.8 are prepared in Tris-HCl buffer (ICN Biomedicals Inc., USA); with a pH of 7.4 - on K ⁺ , Na ⁺ -phosphate buffer (buffer prepared from K ₂ HPO ₄ and NaH ₂ PO ₄ (Reakhim, Russia)).

It should be noted that in the incubation mixture to determine the activity of NADPHDH, NH ₄ CI is additionally added at a concentration of 5.0 mM.

After incubation of the test samples at 37 ° C for 30 minutes for LDH and MDH and 5 minutes for NADPHDG and GR, 50 μl of flavin mononucleotide (FMN) is added to 200 μl of the incubation mixture at a concentration of 1.5 × 10 ^-5 M, 50 μl 0. 0005% myristic aldehyde and 10 μl of the NADH: FMN oxidoreductase-luciferase enzyme system (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 indicators conditionally called the substrate background of enzymes are determined. Determined under the same conditions as for the above dehydrogenases, but instead of the appropriate 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, graphs of the dependence of the bioluminescence intensity on the concentration of NADH and NADPH are constructed (calibration graph). To do this, 200 μl of a standard solution of NADI or NADPH in the range of 10 ^-9 -10 ^-4 M is added to the bioluminometer cuvette containing FMN, myristic aldehyde and NAD (F) N: FMN oxidoreductase-luciferase in the concentrations indicated above, after which the intensity of bioluminescence is 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 are plotted for each pH buffer. The activity of enzymes is calculated by the formula:

A = Δ [C] × V / T

Where:

A - enzyme activity, E per 1 × 10 ⁴ lymphocytes (1E = 1 μmol / min [1]);

Δ [C] is the difference in the concentrations of NADH or NADPH in the samples "enzyme" and "background enzyme", mmol;

V is the sample volume, ml;

T is the incubation time, min

Then, according to the activity of LDH, MDH, NADPHYDH and GR, the MK value is calculated as the ratio of the products of the activity of GR and NADPHYDH to the product of the activities of LDH and MD. With a value of MK below 0.005, an unfavorable outcome of the disease is predicted, with MK equal to or higher than 0.005, a favorable outcome of RHP is predicted.

This method was tested on 31 patients with RPH.

table 2 Prediction of the outcome of RGP by the value of MK No. Prediction of RHP MK one Favorable outcome 0.179 2 Adverse outcome 0.002 3 Favorable outcome 0.006 four Favorable outcome 0.327 5 Adverse outcome 0.00003 6 Favorable outcome 0.183 7 Favorable outcome 0.394 8 Favorable outcome 7,589 9 Adverse outcome 0.001 10 Favorable outcome 0.212 eleven Favorable outcome 0,039 12 Adverse outcome 0.004 13 Favorable outcome 1.001 fourteen Adverse outcome 0,0008 fifteen Favorable outcome 0.324 16 Favorable outcome 0.008 17 Adverse outcome 0,0004 eighteen Favorable outcome 1,230 19 Adverse outcome 0.004 twenty Favorable outcome 0.056 21 Adverse outcome 0.001 22 Favorable outcome 0.532 23 Favorable outcome 0.946 24 Adverse outcome 0.003 25 Favorable outcome 0.290 26 Adverse outcome 0.002 27 Favorable outcome 0.0048 28 Favorable outcome 0.612 29th Favorable outcome 0,101 thirty Adverse outcome 0.001 31 Favorable outcome 0.273

According to the results of the determination of MK and the subsequent analysis of the outcome of the disease, it was found that 30 patients had a coincidence of prognosis (96.8% of matches). In one patient with a favorable outcome of the disease, MK was 0.0048. Therefore, the sensitivity of the method was 96.8%, specificity is 100%.

Clinical example 1.

Patient V., 33 years old (medical history No. 26230), was taken by ambulance to the MBUZ GKBSMP them. N.S. Karpovich, Krasnoyarsk with a clinic of peritonitis. Concomitant diagnosis: chronic pancreatitis. From the anamnesis it is known that it is sick for 2 days.

In the study of peripheral blood lymphocytes by the bioluminescent method with determination of the activity of LDH, MDH, NADPHDG and GR, it was found that the value of MK was 0.212 (a favorable outcome of RPG is predicted).

The patient was operated on an emergency basis. The operation revealed perforated gastric ulcer, common purulent peritonitis. Perforated ulcer suturing, debridement, abdominal drainage were performed. After 48 hours, a programmed sanitation of the abdominal cavity was performed.

In the postoperative period, for 7 days in the ICU conditions, infusion, antibacterial, symptomatic therapy, and dressings were performed. Then, to stabilize the condition, he was transferred to the surgical department, where conservative therapy was continued.

The postoperative period without complications. The patient was discharged on the 21st day in satisfactory condition for outpatient treatment by a surgeon at the place of residence.

Clinical example 2.

Patient G., 44 years old (medical history No. 16323), was delivered by ambulance to the MBUZ GKBSMP them. N.S. Karpovich, Krasnoyarsk with a clinic of peritonitis. From the anamnesis it is known that it is sick for 2 days, and a day ago abdominal pains took a diffuse character.

In the study of peripheral blood lymphocytes by a bioluminescent method with determination of the activity of LDH, MDH, NADPHDG and GR, it was found that the value of MK was 1.001 (a favorable outcome of RPG is predicted).

The patient was operated on an emergency basis. On the operation revealed gangrenous-perforated appendicitis, widespread purulent peritonitis. Appendectomy, rehabilitation, drainage of the abdominal cavity. On the 2nd and 4th day after the initial operation, programmed sanitation of the abdominal cavity was performed.

In the postoperative period, infusion, antibacterial, symptomatic therapy, and dressings were performed for 5 days in the ICU. Then conservative therapy is continued in the surgical department.

The postoperative period without complications. The patient was discharged on the 21st day in satisfactory condition for outpatient treatment by a surgeon at the place of residence.

Clinical example 3.

Patient G., 73 years old (medical history No. 11541), was taken by ambulance to the Moscow Children's Clinical Hospital. N.S. Karpovich, Krasnoyarsk with a clinic of strangulated ventral hernia, peritonitis. Concomitant diagnosis: IHD, hypertension. From the anamnesis it is known that it is sick for 40 hours.

In the study of peripheral blood lymphocytes by the bioluminescent method with determination of the activity of LDH, MDH, NADPHDG and GR, it was found that the value of MK was 0.004 (an unfavorable outcome of RPG is predicted).

The patient underwent an emergency herniolaparotomy. The operation revealed necrosis of the small intestine with perforation, widespread purulent peritonitis. The small intestine was resected with an anastomosis, sanation, and abdominal drainage. On the 2nd and 4th day after the initial operation, programmed sanitation of the abdominal cavity was performed.

In the postoperative period, in the conditions of ICU, infusion, antibacterial, symptomatic therapy, and dressings were performed. The postoperative period on the 13th day after the last program reorganization was complicated by eventration. Sewing event was performed. Despite the therapy, the patient's condition worsened, multiple organ failure increased. On the 34th day of hospitalization, death was ascertained.

Clinical example 4.

Patient S., 53 years old (medical history No. 14710), was taken by ambulance to the MBUZ GKBSMP them. N.S. Karpovich, Krasnoyarsk with a clinic of perforation of a hollow organ, peritonitis. From the anamnesis it is known that it is sick for 8 days.

In the study of peripheral blood lymphocytes by the bioluminescent method with the determination of the activity of LDH, MDH, NADPHDG and GR, it was found that the value of MK was 0.001 (an unfavorable outcome of RPG is predicted).

The patient was operated on in an emergency. The operation revealed perforation of the colon, common purulent peritonitis. A colostomy, debridement, and drainage of the abdominal cavity were performed. After 48 hours, a programmed sanitation of the abdominal cavity was performed.

In the postoperative period, in the conditions of ICU, infusion, antibacterial, symptomatic therapy, and dressings were performed. Despite the therapy, the patient's condition worsened, multiple organ failure increased with the development of septic shock. On the 3rd day of hospitalization, death was ascertained.

The advantages of the proposed method are:

1) the possibility of early prediction of the outcome of RGP - within 1 day after diagnosis;

2) a high level of reliability of the forecast - 96.8%. The proposed method can be recommended for use in clinical practice.

Sources used

1. Berezov T.T., Korovkin B.F. Biological chemistry. - M.: Medicine, 1998 .-- 704 p.

2. RF patent №2251700 C2, G01N 33/68, G01N 33/48, G01N 33/53, BIPM No. 13, 05/10/2005.

3. RF patent No. 2275632 C2, G01N 33/48, G01N 33/49, G01N 33/50, BIPM No. 12, 04/27/2006.

4. RF patent No. 2292048 C2, G01N 33/92, BIPM No. 2, 01.20.2007.

5. Savchenko A.A., Suntsova L.N. Highly sensitive determination of the activity of dehydrogenases in lymphocytes of human peripheral blood using the bioluminescent method // Laboratory. - 1989. - No. 11. - S.23-25.

6. Savchenko A.A. Bioluminescent determination of the activity of NAD- and NADP-dependent glutamate dehydrogenases of lymphocytes // Lab. a business. - 1991. - No. 11. - S.22-25.

7. Boyum A. Isolation of lymphocytes from blood and bone marroow // Scand. J. Clin. Lab. Invest. - 1968 .-- Vol.21 (Suppl. 97). - P.77-80.

Claims (1)

A method for predicting the outcome of widespread purulent peritonitis (RGP), including blood sampling and blood testing, characterized in that the activity of glutathione reductase - GR, NADP-dependent glutamate dehydrogenase - NADPHDH, lactate dehydrogenase - lactate dehydrogenase - is determined using peripheral blood lymphocytes using a bioluminescent method what is the metabolic coefficient calculated - MK, which is the ratio of the products of the activity of GR and NADPHDH to the product of the activities of LDH and MDH, and when the value of MK is not while 0.005 predict an unfavorable outcome of the disease, with MK equal to or higher than 0.005, a favorable outcome of RHP is predicted.