RU2188431C2 - Method for determining liver disorders in leprosy patients - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves carrying out biochemical examination of leprosy patient blood serum. The amount of peroxidation-modified heparin-containing apo-B-containing lipoproteins is to be determined. The level of 3.45 mcmole/g of protein being found in the sample, liver disorder is to be diagnosed. EFFECT: high accuracy of diagnosis. 2 tbl

Description

 The invention relates to medicine, namely to leprology, and can be, in particular, used to determine the degree of liver damage in patients with leprosy.

 From the practice of medicine, a method for determining liver damage in patients with leprosy is known, which means that the content of bromine sulfalein is repeatedly determined in the blood serum by a biochemical method (Frank E., Lundin Jr., MD and Sister Hilary Ross, BS Liver disfunction in leprosy Results of the bromsulfalein retention test in 150 cases // International Journal of Leprosy, 1959.-Vol. 27.-NL- P.43-47). The disadvantages of this method are: the possibility of developing thrombophlebitis and an allergic reaction to the intravenous administration of bromosulfalene (dye), the scarcity and high cost of the dye, the great invasiveness of the technique, since during its implementation it is necessary to carry out 2 to 6 venipuncture, use up to 10 ml of serum for analysis, the need for a preliminary determination of the level of bilirubin in the blood, as well as a decrease in the diagnostic sensitivity and diagnostic effectiveness of the method for a number of pathological syndromes (leuko singing, cholestasis, water imbalance and protein metabolism), which does not allow to obtain a specific technical result - improving the accuracy of the method.

 There is also a method for determining liver damage in patients with leprosy, which consists in the fact that in the blood serum the biochemical method determines the indicators of protein, fat metabolism and enzymes (Venkatesan K., Bharadwaj VP Sequential biochemical investigations in lepromatous leprosy // Leprosy in India, 1978. - Vol.50.-N2.- P.166-172). The disadvantages of this method are: low diagnostic sensitivity and diagnostic efficiency, since the detected changes in the estimated biochemical parameters are characteristic of other diseases, and therefore may not be associated with liver damage, which does not allow to obtain a specific technical result - improving the accuracy of the method.

Closest to the proposed is a method for determining liver damage in patients with leprosy, which consists in the fact that the activity of gamma-glutamyl transpeptidase (B. Kumar, APS Narang and S. Kaur. Gamma-glutamil transpeptidase (GGT) in leprosy / / Indian Journal of Leprosy, 1985.-Vol. 57.-N4.-P.763-766). The similarity of this method with the proposed one lies in the fact that both of them relate to biochemical methods for determining liver damage. For analysis, blood serum obtained by the method of single venipuncture is used. The disadvantages of this method are:
- increased activity of the enzyme in the blood serum in acute and chronic pancreatitis, myocardial diseases;
- changes in enzyme activity as a result of exposure to ethanol and drugs (drugs, sedatives, microsomal oxidation inducers);
- changes in the activity of the enzyme in the blood serum characterize only two of the six main pathological syndromes of liver damage (cholestatic and toxic);
- low or normal levels of enzyme activity in serum in severe progressive forms of liver damage.

 Thus, the listed disadvantages indicate that the serum activity of gamma-glutamyltranspeptidase is not always associated with liver damage, which does not allow to determine organ damage. In case of liver damage, changes in the activity of the enzyme are multidirectional in nature, not fully reflecting the pathophysiological mechanisms of organ damage, which reduces the diagnostic efficiency and, therefore, the accuracy of the method when used to determine liver damage.

 The present invention solves the main problem - improving the accuracy of the method for determining liver damage in patients with leprosy, which allows us to assess the severity of the leprosy process and to dynamically monitor the functional state of the liver under the conditions of chemotherapy. The invention is expressed by a combination of essential features sufficient for the technical result provided by the invention.

 The problem is solved in that during a biochemical study of the blood serum of patients with leprosy, the amount of peroxide-modified heparin-deposited apo-B-containing lipoproteins is determined in it and at a level of 3.45 μmol / gram of protein in the sample and below, liver damage is judged.

 Improving the accuracy of determining liver damage in patients with leprosy makes it possible to carry out its early diagnosis against the background of long-term chemotherapy for leprosy, in the prevalence of low-symptom clinical forms of organ damage, regardless of the type, activity and duration of the leprosy process, which ensures high information content and reliability of the method.

 The problem of liver damage during leprosy remains one of the most relevant in modern leprology. Its most important aspect is the timely detection of liver damage, especially since the modern arsenal of biochemical methods is able to determine only 20-30% of such a pathology.

 Changes in the functional state of the liver during leprosy can be the result of exposure to the organ, both a specific process and modern anti-leprosy chemotherapeutic agents. Specific liver damage is observed already in the early stages of the disease and is clinically manifested as chronic persistent hepatitis, the outcome of which may be cirrhosis of the liver. The degree of liver damage is not always proportional to the duration of the leprosy process. The onset of its regression does not indicate liver intactness.

 In the context of therapeutic pathomorphism associated with the use of modern anti-leprosy drugs in the treatment of leprosy, severe manifestations of liver damage in patients with leprosy are quite rare. In a certain category of patients, latent malosymptomatic forms of such a pathology are possible. In this situation, the diagnostic information content of the already known methods for determining liver damage in patients with leprosy is reduced. At the same time, in the presence of functional insufficiency of the organ, the likelihood of adverse effects from the side of anti-leprosy chemotherapy, which patients receive for long periods, increases. Thus, the search for new methods for determining liver damage remains an urgent task of leprology.

 The liver is an organ that is actively involved in the metabolism, including lipid metabolism. Her defeat is accompanied by impaired lipoprotein metabolism. There is no doubt the role of activation of lipid peroxidation processes in its pathology. On the other hand, lipid peroxidation processes, accompanied by the accumulation of malondialdehyde (MDA), intensively occur precisely in circulating lipoproteins, and in human serum, these reactions occur exclusively in apo-B-containing lipoproteins, causing their peroxidation. Isolation of apo-B-containing lipoproteins does not affect the content of peroxidation products in them.

 The proposed method is as follows. The isolation of apo-B-containing lipoproteins was carried out by the chemical method according to M. Ledvina (1960), and the determination of the content of malondialdehyde in them by L.I. Andreeva et al. (1988). By the amount of malondialdehyde in heparin-deposited apo-B-containing lipoproteins, their peroxide modification is judged.

 For research, blood serum was used. Blood sampling in the amount of 1-1.5 ml is carried out in the morning on an empty stomach in 14-16 hours after the last meal in a dry clean test tube by puncture of the ulnar vein. To obtain serum, the blood is centrifuged at 900 g for 15 minutes.

In a test tube with 2 ml of a 0.025 M calcium chloride solution, 200 μl of serum is added, shaken and the initial absorbance (E ₁ ) is determined at a wavelength of 680 nm in a cuvette with a layer thickness of 1 cm against distilled water. After the measurement, the contents from the cuvette are transferred back to the same tube and 40 μl of a solution of heparin with a concentration of 1000 ME in 1 ml are added, rinsing the pipette several times with the contents of the tube. After 4 min, the optical density of the sample (E ₂ ) was re-measured at a wavelength of 680 nm against distilled water. After measurement, the contents from the cuvette are again transferred to the same tube. To better isolate heparin-deposited apo-B-containing lipoproteins, the sample is kept for 30 min at a temperature of + 4-6 ^o C. At the end of the incubation time, the tubes are centrifuged for 30 min at 1400 g at a temperature of + 4-6 ^o C. After that, carefully not to to disrupt the dense precipitate of heparin-deposited apo-B-containing lipoproteins, the supernatant is removed using a Pasteur pipette. In the resulting precipitate of heparin-deposited apo-B-containing lipoproteins, the content of MDA is determined.

For this, experimental and idle samples are prepared, which simultaneously pass through all subsequent stages. The test sample contains a precipitate of heparin-deposited apo-B-containing lipoproteins. A blank reagent sample was sampled, consisting of a solution of heptahydrate iron sulfate, phosphoric and thiobarbituric acids, taken in the same volumes and concentration as for the experimental sample. The precipitate of heparin-deposited apo-B-containing lipoproteins is dissolved in 100 μl of a freshly prepared solution of heptahydrate iron sulfate (28 mg per 10 ml of distilled water). The tubes are gently shaken, closed with ground stoppers and placed for 30 min in a thermostat at a temperature of +37 ^o C. At the end of the incubation time, the tubes are removed from the thermostat and 3 ml of a 1.0% phosphoric acid solution with pH = 2.0 and 1 are added to the samples ml of 0.6% freshly prepared solution of thiobarbituric acid. The tubes are closed with ground stoppers and placed in a boiling water bath (+100 ^o C) for one hour. Then the tubes are cooled in a stream of cold water, 3 ml of butanol are added to the samples, and they are thoroughly mixed by shaking for 2 min until a uniform white suspension with a pink hue is formed. To separate the phases, the samples are centrifuged for 15 min at 1400g. Immediately after centrifugation, the transparent upper butanol phase is carefully aspirated with a Pasteur pipette into a clean, dry tube and the optical density of the experimental (EOP) and blank (Ehol) samples is measured relative to butanol in a cuvette with a 1 cm layer at two wavelengths: 535 nm and 580 nm (E ₅₃₅ and E ₅₈₀ ).

Calculate the results. The amount of heparin-deposited apo-B-containing lipoproteins is calculated by the difference (ΔE) of the measured optical density of the sample before (E ₁ ) and after (E ₂ ) the heparin solution is introduced into the sample
ΔE = E ₂ -E ₁ .
To express the result in gram / liter of protein of heparin-deposited apo-B-containing lipoproteins (C ₁ ), the value of the difference in optical density (ΔE) is multiplied by a conversion factor equal to 2.44 (M. Ledvin, 1960).

С₂ - содержание перекисно-модифицированных гепариносажденных апо-В-содержащих липопротеидов в мкмоль/грамм белка в пробе;
С₁ - количество гепариносажденных апо-В-содержащих липопротеидов в грамм/литр белка;
Д_оп - оптическая плотность опытной пробы, определенная по разнице Е₅₃₅ - Е₅₈₀;
Д_хол - оптическая плотность холостой пробы, определенная по разнице Е₅₃₅-Е₅₈₀;
4 - объем бутанольной фазы в мл;
1,56•10 - коэффициент молярной экстинкции малонового диальдегида в моль^-1•см ^-1;
10⁶ - коэффициент перевода показателя C₂ в мкмоль/литр;
0,2 - объем сыворотки крови, из которого выделяли гепариносажденные апо-В-содержащие липопротеиды в мл.The content of peroxide-modified heparin-deposited apo-B-containing lipoproteins is calculated by the formula

C ₂ - the content of peroxide-modified heparin-deposited apo-B-containing lipoproteins in µmol / gram of protein in the sample;
C ₁ is the amount of heparin-deposited apo-B-containing lipoproteins in gram / liter of protein;
D _op - the optical density of the experimental sample, determined by the difference E ₅₃₅ - E ₅₈₀ ;
D _Hall - the optical density of the blank sample, determined by the difference E ₅₃₅ -E ₅₈₀ ;
4 - volume of the butanol phase in ml;
1.56 • 10 - coefficient of molar extinction of malondialdehyde in mol ^-1 • cm ^-1 ;
10 ⁶ - conversion coefficient of indicator C ₂ in µmol / liter;
0.2 - volume of blood serum from which heparin-deposited apo-B-containing lipoproteins in ml were isolated.

 All reagents used were of the “Clean For Analysis” (PSA) category. The work was carried out using a SPECORD spectrophotometer (Germany) and a RS-6 refrigerator centrifuge (Russia).

 Statistical processing of the results was carried out using MICROSOFT EXCEL spreadsheets with the calculation of the following statistical parameters: arithmetic mean (M), standard deviation (S) and student's criterion.

 The essence of the invention is illustrated by tables.

 From table 1 it is seen that the average value of the content of peroxide-modified heparin-deposited apo-B-containing lipoproteins (M) in leprosy patients with liver damage (first group) was 2.40 μmol / gram of protein in the sample. Given the standard deviation (S), the upper limit of the allowable interval was 3.45 μmol / gram protein in the sample. It was adopted as a criterion, with values equal to and below which, in patients with leprosy, liver damage was determined. In the group of patients who did not have liver damage (second group), the average value of the content of peroxide-modified heparin-deposited apo-B-containing lipoproteins was 5.45 μmol / gram of protein in the sample. Given the standard deviation, the lower limit of the confidence interval corresponded to 3.80 μmol / gram protein in the sample. The examined groups of patients did not have significant differences (P> 0.05) in terms of average serum gamma-glutamyl transpeptidase activity (prototype method), but significantly and significantly (P <0.05) differed in the content of peroxide-modified heparin-deposited apo-B -containing lipoproteins (the proposed method). Thus, the accuracy of the proposed method in comparison with the prototype method was higher, which makes it preferable for determining liver damage in patients with leprosy.

 The accuracy of determining liver damage in the general group of examined patients with leprosy (38 people) was evaluated taking into account the diagnostic sensitivity (DF) and diagnostic efficiency (DE) of the method, which were calculated according to Griner P.F. et al. (1981) (table 2).

 As can be seen from table 2, the sensitivity and diagnostic efficiency of the proposed method were 91.30% and 89.47%, respectively, which exceeds those indicators when using the prototype method (13.00% and 44.74%, respectively). Thus, the accuracy of determining liver damage in patients with leprosy of the proposed method was higher than when using the prototype method.

 The proposed method was successfully tested in the clinic of the Research Institute for the study of leprosy of the Ministry of Health of the Russian Federation for 38 patients with leprosy during 1999. Six patients suffered from an active form of the disease. In the remaining 32 people, the leprosy process was in a state of clinical remission. All patients were divided into two groups. The first group consisted of patients with persistent chronic hepatitis (23 people), who had a moderate liver enlargement at the time of the study. Most patients in this group had inconsistent dyspeptic and pain syndromes. The second group included 15 patients with leprosy, who had no history of liver damage in the history and at the time of the study.

 Below are the results of testing.

 Example 1

Patient L., born 1947, (medical history 3610). Sick leprosy for 44 years. From April 1993 to the present, there has been a relapse of leprosy. Diagnosis: leprosy, lepromatous type, LL _S. Chronic specific hepatitis. Clinically observed pain and dyspeptic syndromes, an increase in the liver by 2 cm from under the edge of the costal arch. Histology: infiltrate of lepromatous structure with the presence of single homogeneous and granular forms of M. leprae. The bacterioscopic index (BIN) is 1.17+. Liver scan: changes in the type of chronic hepatitis. To implement the proposed method, blood serum is used and liver damage is determined by the biochemical determination of peroxide-modified heparin-deposited apo-B-containing lipoproteins. Blood sampling was carried out in the morning on an empty stomach in 14-16 hours after the last meal in a dry clean test tube by puncture of the ulnar vein. To obtain serum, the blood is centrifuged.

In a test tube with 2 ml of a 0.025 M calcium chloride solution, 200 μl of serum is added, shaken and the initial absorbance (E ₁ ) is determined at a wavelength of 680 nm in a cuvette with a layer thickness of 1 cm against distilled water. After measurement, the contents from the cuvette are transferred back to the same tube and 40 μl of a solution of heparin with a concentration of 1000 ME in 1 ml are added. After 4 min, the optical density of the sample (E ₂ ) was re-measured at a wavelength of 680 nm against distilled water. The contents of the cuvette are again transferred to the same tube. The sample is held for 30 minutes at a temperature of + 4-6 ^{o C.} , after which the tubes are centrifuged for 30 minutes at 1400 g at a temperature of + 4-6 ^o C. Then the supernatant is removed using a Pasteur pipette. In the resulting precipitate of heparin-deposited apo-B-containing lipoproteins, the content of MDA is determined.

The test sample contains a precipitate of heparin-deposited apo-B-containing lipoproteins. A single sample consists of a solution of iron sulfate, phosphoric and thiobarbituric acids, taken in the same volumes and concentration as for the experimental sample. The precipitate of heparin-deposited apo-B-containing lipoproteins is dissolved in 100 μl of iron sulfate solution. The tubes are shaken, closed with stoppers and placed for 30 min in a thermostat at a temperature of +37 ^o C. At the end of the incubation time, 3 ml of a 1.0% phosphoric acid solution with pH = 2.0 and 1 ml of a 0.6% thiobarbituric solution are added to the samples acids. Test tubes are closed with stoppers and placed in a boiling water bath for one hour. Then the samples are cooled in a stream of cold water, 3 ml of butanol are added to them, mixed until a homogeneous white suspension with a pink tint is formed. Samples are centrifuged to separate phases. Immediately after centrifugation, the butanol phase is carefully transferred to a clean dry tube and the optical density of the experimental (EOP) and blank (Ehol) samples is measured relative to butanol in a cuvette with a layer thickness of 1 cm at two wavelengths: 535 nm and 580 nm (E ₅₃₅ and E ₅₈₀ )

 Calculate the results. The amount of heparin-deposited apo-B-containing lipoproteins is calculated. The result is expressed in grams / liter of protein heparin-deposited apo-B-containing lipoproteins, taking into account the conversion factor.

Концентрация перекисно-модифицированных гепариносажденных апо-В-содержащих липопротеидов составила 3,45 мкмоль/грамм белка в пробе. Таким образом, предложенный способ позволил достоверно определить поражение печени.The content of peroxide-modified heparin-deposited apo-B-containing lipoproteins is calculated by the formula

The concentration of peroxide-modified heparin-deposited apo-B-containing lipoproteins was 3.45 μmol / gram protein in the sample. Thus, the proposed method allowed to reliably determine liver damage.

 Example 2

 Patient K., born 1936, (medical history 3866). Sick leprosy for 18 years. Diagnosis: leprosy, lepromatous type, LLp, active stage with visceral manifestations. Clinically intermittent pain and dyspeptic syndromes. The liver is enlarged by 1.5-2 cm from under the edge of the costal arch. Histology: an infiltrate of lepromatous structure containing a large number of homogeneous and granular, often forming clusters such as "cigarette sticks" and "balls" of M. leprae. BIN = 2.5 +. Liver scan: marked changes in the type of chronic hepatitis. Determination of liver damage was carried out by the method described in example 1. The concentration of peroxide-modified heparin-deposited apo-B-containing lipoproteins was 2.46 μmol / gram of protein in the sample. Thus, the proposed method allowed to reliably determine liver damage.

 Example 3

 Patient M., born in 1936, (medical history 3866). Sick leprosy for 37 years. Diagnosis: leprosy, undifferentiated type, stage of regression. Chronic persistent hepatitis. Clinically intermittent dyspeptic syndrome. The liver is increased by 2 cm from under the edge of the costal arch. BIN = 0. Liver scan: pronounced changes in the type of chronic hepatitis. Determination of liver damage was carried out by the method described in example 1. The concentration of peroxide-modified heparin-deposited apo-B-containing lipoproteins was 1.58 per μmol / gram protein sample. Thus, the proposed method allowed to reliably determine liver damage.

 Example 4

Sick U-va, born in 1928, (medical history 3737). Sick with leprosy for 14 years. Diagnosis: leprosy, lepromatous type, LL _S , stage of regression. Clinically, there was no evidence of liver damage. BIN = 0. Liver scan: no signs of liver damage. Determination of liver damage was carried out by the method described in example 1. The concentration of peroxide-modified heparin-deposited apo-B-containing lipoproteins was 3.89 μmol / gram protein sample. Thus, the result indicates the absence of liver damage.

 Using the proposed method in comparison with the prototype method, a positive result is achieved, which consists in increasing the accuracy of determining liver damage in patients with leprosy on the background of the leprosy process and ongoing chemotherapy. The results of a clinical trial of the proposed method indicate its high diagnostic sensitivity and diagnostic efficiency, which allows for timely and accurate diagnosis of liver damage in patients with leprosy.

 Thus, the authors proposed a new, no one previously proposed method for determining liver damage in patients with leprosy.

 The application of the proposed method allows for accurate diagnosis of liver damage in patients with leprosy on the background of long-term chemotherapy. In the future, it is supposed to be used as a biochemical test to determine liver damage in patients with leprosy, and the method can be recommended for clinical use in anti-leprosy institutions.

Claims (1)

 A method for determining liver damage in patients with leprosy by biochemical examination of the blood serum of patients with leprosy, characterized in that the amount of peroxide-modified heparin-deposited apo-B-containing lipoproteins in the blood serum is determined and at a level of 3.45 μmol / g protein in the sample and below they are judged liver damage.

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