RU2651030C1 - Method for predicting the outcome of acute destructive pancreatitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is intended to determine the outcome of acute destructive pancreatitis (ADP). In patients with ADP, in the first 24 hours after the diagnosis the activities of NADP- and NADPH-dependent glutamate dehydrogenase are determined in the neutrophils of the venous blood of patients using the bioluminescent method, after which the ratio of dehydrogenase activities is calculated. If the ratio is below 0.035, an adverse outcome of the disease is predicted, with an equal 0.035 or higher - a benign outcome of ADP.

EFFECT: method allows to determine the risk of an adverse outcome in the early period of the disease.

1 cl, 2 tbl, 4 ex

Description

The invention relates to medicine, namely to anesthesiology and intensive care, and can be used to determine the outcome of acute destructive pancreatitis (ODP).

At present, ARP remains one of the problem areas of emergency abdominal surgery. Despite the use of modern detoxification technologies in the treatment of ODP, the development of new methods of surgical interventions, and the improvement of drug therapy, this pathology is characterized by high mortality [1, 10].

A known method for predicting the occurrence, clinical course and outcome of acute alcoholic pancreatitis is that DNA is isolated from the blood of patients with acute alcoholic pancreatitis and the GSTM1 and GSTT1 mutations are investigated by polymerase chain reaction [5]. If a “zero” GSTM1 genotype or a “zero” GSTT1 genotype is detected, the development of a destructive form of alcoholic pancreatitis is predicted, and when a combination of “zero” genotypes by the GSTM1 and GSTT1 genes is detected, the development of total pancreatic lesion is predicted. This method predicts only for patients with acute alcoholic pancreatitis and with a low degree of accuracy (80%).

A known method for predicting the outcome of destructive pancreatitis is that a patient is prepared from a patient's blood serum for infrared spectral analysis, an infrared spectroscopy of the sample is carried out with absorption spectra recorded in the region of 1200-1000 cm ^-1 and based on the obtained values of the indicators according to the logistic regression model equation determine the likelihood of an adverse outcome of acute destructive pancreatitis [4].

The disadvantage of this method is the difficulty in execution.

The closest in technical essence to the proposed method is a method for predicting the likelihood of an unfavorable outcome of pancreatic necrosis in the postoperative period, namely, in patients with pancreatic necrosis in the pre- and postoperative periods, the parameters of red blood cell peroxide resistance are evaluated and the decrease in erythrocyte chemiluminescence intensity to 61.3 9.4 or less, against the background of an increase in the light sum to 814.6 ± 53.8 mV⋅s or more, an unfavorable outcome is predicted by the third day of the postoperative period [3].

The disadvantage of this method is that for the prognosis of patients with pancreatic necrosis in the pre- and postoperative period, blood is drawn from a vein several times. In addition, the forecast is carried out using laboratory data obtained within three days, which, due to the rapid course of the disease, is too long.

The objective of the invention is the early prediction (on the first day after diagnosis) of the outcome of acute destructive pancreatitis.

The task is carried out due to the fact that during the first day after the diagnosis of NDP in the neutrophils of venous blood of patients using the bioluminescent method determine the activity of enzymes: NADP-dependent glutamate dehydrogenase and the activity of NADPH-dependent glutamate dehydrogenase, after which the ratio of dehydrogenase activity is calculated. If the value of the obtained indicator is less than 0.035, an unfavorable outcome of the NDC is predicted, and when the value of the indicator is equal to or greater than 0.035, the favorable outcome of the NDP is predicted.

The value of 0.035 was obtained experimentally based on a comparison of the calculated ratio of the activity of NADP-dependent glutamate dehydrogenase to the activity of NADPH-dependent glutamate dehydrogenase and data from subsequent monitoring of the clinical condition of patients with NDP. The value of the ratio of the activity of NADP-dependent glutamate dehydrogenase to the activity of NADPH-dependent glutamate dehydrogenase below 0.035 indicates the prevalence of the outflow of substrates from energy processes to amino acid metabolism reactions, which, accordingly, will lead to a decrease in the intensity of bioenergetic processes in cells and their functional reactivity.

Neutrophils are a highly reactive link in the immune system. They are the first to mobilize into the focus of inflammation, the effectiveness of antimicrobial protection of the body largely depends on their phagocytic activity [7, 8, 9]. Perceiving numerous signals about the destabilization of the internal environment, neutrophils modulate their functions aimed at its restoration. Activated neutrophils themselves become powerful effectors of triggering and regulatory mechanisms of cascade reactions that ensure the development of inflammation. This is due to the fact that neutrophils can not only produce cytotoxic molecules as effectors, but also synthesize a wide range of different cytokines as regulatory cells [7, 8]. It has been proven that the functional activity of neutrophils is largely determined by the level of activity of their metabolic processes [7, 6]. NADP- and NADPH-dependent glutamate dehydrogenase (NADPHDG and NADPH-GDH, respectively, EC 1.4.1.4) performs oxidative deamination of L-glutamic acid. The reaction includes an anaerobic phase of dehydrogenation of glutamic acid with the formation of an intermediate product - iminoglutaric acid, after which spontaneous hydrolysis occurs with the formation of ammonia and α-ketoglutaric acid. The enzymatic reactions of glutamate dehydrogenases are reversible, respectively, ammonia in the presence of NADPH and α-ketoglutaric acid can participate in the synthesis of glutamate. Glutamate dehydrogenase is one of the most studied oligomeric nitrogen metabolism enzymes with a molecular weight of 312,000, which consists of 6 subunits. The enzyme shows its activity only in multimeric form. This feature characterizes the allosteric mechanism of glutamate dehydrogenase regulation and defines this enzyme as regulatory in the amino acid metabolism system [2].

The method is as follows.

In patients, on the first day after the diagnosis of NDP, venous blood in the amount of 2 ml is taken from the ulnar vein by free flow into heparin tubes. Neutrophils are isolated from whole heparinized blood by centrifugation in a double density gradient of ficoll-urographin: ρ = 1,077 g / cm ³ - for the separation of lymphocytes, ρ = 1,119 g / cm ³ - for the selection of neutrophils. Count the number of neutrophils, for example, in the Goryaev’s chamber. When controlling the morphological composition of leukocyte suspensions determine the purity of neutrophil output, which is at least 97%. 1 million isolated cells are used to determine the activity of NADPHDG and NADPH-GDH neutrophils by one of the known methods, for example, bioluminescent [6]. For this, a suspension of isolated neutrophils, containing cells in an amount 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 dithiotreitol. Then directly determine the activity of NADPHDG and NADPH-GDH. For this, in 50 μl of the incubation mixture containing the appropriate substrate and cofactor, add 50 μl of a suspension of destroyed neutrophils. 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.

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

After incubation of the test samples at 37 ° C (within 30 minutes for NADPHDG and 5 minutes for NADPH-GDH), 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 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-3607", 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 absolute activity values, graphs of the dependence of the intensity of bioluminescence on the concentration of NADPH are constructed (calibration graph). For this, 200 μl of a standard NADPH solution 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 at 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 is the enzyme activity, E per 1 × 10 ⁴ neutrophils (1E = 1 μmol / min [2]);

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

V is the sample volume, ml;

T - incubation time, min

Then calculate the ratio of the activity of NADP-dependent glutamate dehydrogenase to the activity of NADPH-glutamate dehydrogenase. If the ratio is below 0.035, an unfavorable outcome of the disease is predicted, with a value of 0.035 or higher, a favorable outcome of NDP is predicted.

This method has been tested on 25 patients with ODP.

According to the results of the examination of patients with ADP according to the claimed method, it was found that out of 25 patients with ADP in 21 (84%), a favorable outcome of the disease was predicted. In these patients, the ratio of the activities of NADPHDH and NADPH-GDH was 0.036-20.126 (above 0.035). An unfavorable outcome of the disease was predicted in 4 (16%) patients with ADP, the ratio of the activities of NADPHDH and NADPH-GDH was 0.0008-0.034 (below 0.035). According to the results of further clinical observation, the coincidence of the prognosis in 100% of cases was established.

Clinical example 1.

Patient D., 34 years old (medical history No. 24957), was taken by ambulance to the MBUZ GKBSMP them. N.S. Karpovich, Krasnoyarsk with a clinic of acute pancreatitis. From the anamnesis it is known that it is sick for 2 days. In the analyzes, leukocytosis is 15.6 × 10 ⁹ / L, urea is 12.3 mmol / L, blood amylase is 2410 U / L (normal up to 100 U / L), urine amylase 16340 U / L (normal up to 500 U / L). Ultrasound scan of the abdominal cavity shows signs of ACP.

A study of the claimed method. The activity of NADP-dependent glutamate dehydrogenase and NAFDN-glutamate dehydrogenase was determined by the bioluminescent method on the first day after the diagnosis of NDP in neutrophils of the patient’s peripheral blood. The ratio of dehydrogenase activities was 0.238 (above 0.035). Forecast: favorable outcome.

In the conditions of the ICU of the surgical department, conservative therapy was carried out: infusion, antispasmodics, secretolytics, NSAIDs, antibiotics. After 48 hours of therapy, the condition was not clinically improved, peritoneal symptoms appeared.

The patient underwent emergency laparoscopy. Laparoscopy revealed signs of fatty pancreatic necrosis, common enzymatic peritonitis. Remediation, drainage of the omental bursa and abdominal cavity were performed.

In the postoperative period, infusion, antibacterial, symptomatic therapy, secretolitics were carried out for 7 days in the ICU. Then, to stabilize the condition, the patient 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., 54 years old (medical history No. 18333), was taken by ambulance to the Moscow Children's Clinical Hospital. N.S. Karpovich, Krasnoyarsk with the clinic of the ODP. From the anamnesis it is known that it is sick for 5 days. The diagnosis is confirmed by laboratory, ultrasound. Clinically, there is peritoneal symptomatology.

A study of the claimed method. The activity of NADP-dependent glutamate dehydrogenase and NAFDN-glutamate dehydrogenase was determined by the bioluminescent method on the first day after the diagnosis of NDP in neutrophils of the patient’s peripheral blood. The ratio of dehydrogenase activities was 0.036 (above 0.035). Forecast: favorable outcome.

The patient underwent emergency laparoscopy. When laparoscopy revealed mixed pancreatic necrosis, diffuse enzymatic peritonitis. Remediation, drainage of the omental bursa and abdominal cavity were performed. Despite the ongoing intensive care, the patient's condition without positive dynamics. On the 6th day of hospitalization according to clinical and laboratory data, the results of abdominal MSCT, the course of the disease was complicated by a right retroperitoneal phlegmon with signs of infection.

The patient was operated on an emergency basis. The operation revealed hemorrhagic pancreatic necrosis of the head and body of the pancreas, common enzymatic peritonitis. Produced cholecystostomy, omentobursostomy, debridement, drainage of the abdominal cavity, lumbotomy on the right, drainage of the retroperitoneal space.

In the postoperative period, infusion, antibacterial, symptomatic therapy, and dressings were performed for 14 days in the ICU. Then conservative therapy is continued in the surgical department. The postoperative period without complications. The patient was discharged on the 42nd day in satisfactory condition for outpatient treatment by a surgeon at the place of residence.

Clinical example 3.

Patient X., 40 years old (medical history No. 12673), was taken by ambulance to the Moscow Children's Clinical Hospital. N.S. Karpovich, Krasnoyarsk with the clinic of the ODP. From the anamnesis it is known that it is sick for 3 days. The diagnosis is confirmed by laboratory, ultrasound.

A study of the claimed method. The activity of NADP-dependent glutamate dehydrogenase and NAFDN-glutamate dehydrogenase was determined by the bioluminescent method on the first day after the diagnosis of NDP in neutrophils of the patient’s peripheral blood. The ratio of dehydrogenase activities was 0.003 (below 0.035). Forecast: adverse outcome.

In the conditions of the ICU of the surgical department, conservative therapy was carried out: infusion, antispasmodics, secretolytics, NSAIDs, antibiotics. After 7 days of therapy, the condition did not clinically improve; a clinic of infected pancreatic necrosis developed. According to the MSCT data of the abdominal cavity, an abscess of the omental bursa, bilateral retroperitoneal phlegmon is not excluded.

The patient underwent an emergency laparotomy. The operation revealed subtotal mixed pancreatic necrosis, an abscess of the omental bursa, bilateral retroperitoneal phlegmon. Cholecystostomy, necrosecestrectomy, omentobursostomy, debridement, drainage of the omental bursa, abdominal cavity, bilateral lumbotomy, drainage of the retroperitoneal space were performed.

In the postoperative period, in the conditions of ICU, infusion, antibacterial, symptomatic therapy, and dressings were performed. The postoperative period on the 14th day after the operation was complicated by bleeding from the stuffing bag. The stuffing bag was revised, hemostasis. Despite the therapy, the patient's condition progressively worsened, multiple organ failure increased. On the 27th day of hospitalization, death was ascertained.

Clinical example 4.

Patient N., 59 years old (medical history No. 11612), was taken by ambulance to the Moscow Children's Clinical Hospital. N.S. Karpovich, Krasnoyarsk with the clinic of the ODP. From the anamnesis it is known that it is sick for 6 days. The diagnosis is confirmed by laboratory, ultrasound.

A study of the claimed method. The activity of NADP-dependent glutamate dehydrogenase and NAFDN-glutamate dehydrogenase was determined by the bioluminescent method on the first day after the diagnosis of NDP in neutrophils of the patient’s peripheral blood. The ratio of dehydrogenase activities was 0.034 (below 0.035). Forecast: adverse outcome.

The patient underwent emergency laparoscopy. When laparoscopy revealed fatty pancreatic necrosis, diffuse enzymatic peritonitis. Remediation, drainage of the omental bursa and abdominal cavity were performed.

Despite the ongoing intensive care in the ICU, the patient's condition without positive dynamics. On the 8th day of hospitalization according to clinical, laboratory data, the results of abdominal MSCT, the patient developed infected pancreatic necrosis, left-side retroperitoneal phlegmon.

The patient was operated on in an emergency. The operation revealed necrotic paramezocolitis, diffuse purulent peritonitis. A necrosecvestrectomy, resection of the transverse colon with the removal of a transversostomy, rehabilitation, drainage of the abdominal cavity, lumbotomy on the left, drainage of the retroperitoneal space 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 progressively worsened, multiple organ failure increased. On the 19th day of hospitalization, death was ascertained.

The technical result of the proposed method:

- the possibility of an early prognosis of an adverse outcome of an NDP (within the first days after diagnosis);

- invasive intervention is limited to a single sampling of a small blood volume from a vein (2 ml);

- a high level of reliability of the forecast - 100%.

Thus, the method is informative, meets modern requirements for laboratory diagnostic methods, allows for timely prediction of the adverse outcome of NDC and can be recommended for use in clinical practice.

Information sources

1. Baychorov E.Kh., Greyasov V.I., Gagua A.K., Chuguevsky V.M., Evtikhov P.M. Acute destructive pancreatitis. Moscow: Litterra, 2015.200 s.

2. Biochemistry. Ed. E.S. Severin (2009), GEOTAR-Media, M., 768 p.

3. Patent 2398520 of the Russian Federation. A method for predicting the likelihood of an adverse outcome of pancreatic necrosis in the postoperative period / Cedrik N.I., Teplyakova O.V., Vinnik Yu.S., Filina N.G. Publ. 09/10/2010.

4. Patent No. 2277243 of the Russian Federation. A method for predicting the outcome of destructive pancreatitis / Petrov M.S., Gordetsov A.S. Publ. 05/27/2006.

5. Patent No. 2312348 of the Russian Federation. A method for predicting the risk of the onset, clinical course and outcome of acute alcoholic pancreatitis / Rukosueva M.A., Pervova O.V., Cherdantsev D.V., Vinnik Yu.S., Markova E.V., Zotova N.V., Snytko E.V. Publ. 12/10/2007.

6. Savchenko A.A. Determination of the activity of NAD (P) -dependent dehydrogenases in neutrophilic granulocytes by the bioluminescent method // Bulletin of Experimental Biology and Medicine. - 2015. - T. 159, No. 5. - S. 656-660.

7. Savchenko A.A., Zdzitovetsky D.E., Borisov A.G. Immunometabolic disorders with widespread purulent peritonitis. - Novosibirsk: Nauka, 2013 .-- 142 p.

8. Savchenko A.A., Zdzitovetsky D.E., Borisov A.G., Luzan N.A. Chemiluminescent activity of neutrophilic granulocytes and levels of cytokine concentration in patients with widespread purulent peritonitis // Cytokines and inflammation. - 2013. - T. 12, No. 1-2. - S. 115-119.

9. El-Benna J., Hurtado-Nedelec M., Marzaioli V., Marie J.C., Gougerot-Pocidalo M.A., Dang P.M. Priming of the neutrophil respiratory burst: role in host defense and inflammation // Immunol. Rev. - 2016. - Vol. 273, No. 1. - P. 180-193.

10. Vinokurov M.M., Saveliev V.V., Gogolev N.M., Yalynskya T.V. Immunomodulation and treatment of acute destructive pancreatitis in a multidisciplinary surgical hospital // Wiad. Lek. - 2015. - Vol. 68, No. 4. - P. 582-586.

Claims (1)

A method for predicting the outcome of acute destructive pancreatitis (NDP), including a blood test, characterized in that during the first days after the diagnosis of NDP in patients with venous blood neutrophils, the enzyme activity is determined using the bioluminescent method: NADP-dependent glutamate dehydrogenase and NADPH-dependent dehydrogen glutamine activity after which the ratio of dehydrogenase activities is calculated and, with a value of the obtained indicator less than 0.035, an unfavorable outcome of NDC is predicted, and when uu index equal to or greater than 0.035, predict favorable outcome MTO.