RU2395087C2 - Method of diagnosis of endogenous intoxication - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: drop of mixed saliva is dried and analysis of obtained facies structure is carried out with the microscope. Low degree of endogenous intoxication is detected at presence of dark pigmentation in the heart of facies central zone. An average degree of endogenous intoxication is detected at presence of dark pigmentation in the form of a ring at the edge of facies central zone. The high degree of endogenous intoxication is detected at presence of dark pigmentation across the facies central zone. The method is simple and available in implementation.

EFFECT: use of the method enables to determine degree of endogenous intoxication and conduct dynamic monitoring of intoxication level in course of treatment without invasive procedures.

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Description

The invention relates to medicine, namely to laboratory diagnostics, and can be used to diagnose intoxication and its severity.

State of the art

Endogenous intoxication is a syndrome characterized by the accumulation in tissues and biological fluids of the body of excess products of normal or perverse metabolism or cellular response - endogenous toxic substances. Several mechanisms of endogenous intoxication can be distinguished, including excessive production of endogenous toxic products, resorption of toxic substances, accumulation of products of lipid peroxidation and other inflammatory mediators, primarily tumor necrosis factor, translocation of the vital products of microorganisms and microbial bodies themselves, with subsequent autoimmune processes, and violation of the release of endogenous toxic products from the body by the natural detoxification organs [2, 4, 8, 9].

Endogenous intoxication is considered as one of the universal mechanisms of the pathogenesis of various diseases, which includes the release of toxic products into the blood from the pathological focus, their distribution throughout the body with blood flow, and effects on other organs and tissues. The development of pathological disorders in the body during intoxication depends on the balance of two opposite processes - the rate of formation and release of endotoxins into the blood, on the one hand, and the elimination (detoxification) of these substances, carried out by the body's protective systems, on the other. The development of a methodology for evidence-based biochemical assessment of the severity of the condition of patients in the development of endotoxemia is one of the urgent problems of modern medicine, clinical biochemistry. [3]

Intoxication develops in many diseases, in particular, with pathology of the kidneys and liver, and with oncological diseases [12, 13]. These diseases can occur both acutely and chronically with remissions and exacerbations, therefore, the problem arises of assessing the severity of intoxication, monitoring the condition of patients and evaluating the effectiveness of treatment. Known methods for biochemical studies of blood serum (determination of the concentration of medium-weight molecules (MSM), lipid peroxidation products, bilirubin, etc.) have a number of common disadvantages:

- it is necessary to take blood from a vein, that is, to perform invasive manipulation, which in addition can be difficult due to the peculiarities of the anatomical structure of the veins or the age of the subject;

- the need for a special biochemical laboratory equipped with sophisticated equipment and provided with reagents, which makes the tests expensive and not always available for medical institutions and for patients.

These shortcomings limit the number and frequency of these studies.

The known method of wedge-shaped dehydration [14], which does not require complicated expensive equipment and reagents. In the study of blood serum, the authors of the method found intoxication markers - wrinkles and toxic plaques that are observed in the serum facies, regardless of the cause of intoxication. Since no more than 0.1 ml of serum is needed for analysis, blood can be taken from the finger.

The method of wedge-shaped dehydration allows you to make visible the molecular organization of biological fluids by translating it at the macro level. After the droplet dries under standard conditions on a solid substrate, the amount of salts increases from the periphery to the center, and the amount of organic substances increases from the center to the periphery [14].

In terms of studying the degree of intoxication, saliva is interesting as a biological fluid, which can be taken from a patient with practically no limitation of quantity and frequency. Saliva is not associated with invasive procedures and can be carried out not only within the walls of a medical institution, but also at home. It is known that in addition to the digestive function, the salivary glands perform homeostatic and excretory functions. Bile acids and bilirubin, vitamins, antibiotics can be secreted with saliva, which indicates the excretory function of the salivary glands [17], Komarova LG and Alekseeva O.P. (2006) showed that the hematosalivation barrier is permeable to pyruvic acid, creatine, cholesterol, arachidonic acid, etc. [5]. GF Korotko (2006) believes that the hematosalivation barrier is not an obstacle for many toxic substances (heavy metals, alcohol, drugs, organochlorine compounds, and many others) [7].

In the work of S. N. Razumova, markers that appear in the facies of the oral fluid in various diseases of the oral cavity are described in detail [10]. S.N.Shatohina et al. [16] indicate that the presence of dark pigmentation along the edge of the central facies zone (CFF) of mixed saliva is a marker of intoxication. E.V. Agapova described the dark pigmentation of CZF crystals of mixed saliva with obstructive jaundice [1].

For the prototype of the present invention, a method for diagnosing endogenous intoxication and the degree of its severity according to the content of MSM in saliva was selected. For research take the supernatant obtained after sedimentation of mixed saliva collected in a test tube by spitting [8].

Definition: the supernatant is treated with a 15% trichloroacetic acid solution in a ratio of 1: 0.5. Thoroughly mix the contents of the tubes and after 5-7 minutes, centrifuge 30 minutes at 3000 rpm. After that, the supernatant is diluted with distilled water in a ratio of 1: 9. Then absorbance is measured on a spectrophotometer at wavelengths of 244, 254, 264, 274, 284 and 294 nm.

After protein precipitation, the supernatant retains molecules with a molecular mass below 10 kD, which are attributed the maximum toxic properties [86]. The presence of endogenous intoxication is assessed by determining the total level of medium-weight molecules (ΣMSM), their individual components in mixed saliva. In this case, a catabolic pool was distinguished in the general spectrum of MSM at wavelengths of 244-254 nm, and an anabolic pool of 264-294 nm.

The disadvantage of the prototype method is the need for special expensive equipment (spectrophotometer, centrifuge).

Disclosure of invention

The technical result of the invention is the cheapening, accessibility and ease of diagnosis of endogenous intoxication and its severity.

The technical result in the claimed method for the diagnosis of endogenous intoxication according to paragraph 1, is achieved by the fact that, as in the prototype method, the content of MSM of mixed saliva is analyzed. New in the method is that a drop of mixed saliva on a glass slide is pre-dried, the structure of the obtained facies is analyzed under a microscope, and endogenous intoxication is judged by the presence and location of varying degrees of pigmented agglomerate.

Moreover: the absence of endogenous intoxication is judged by the absence of dark pigmentation in the central zone of the facies; the low degree of severity of endogenous intoxication is judged by the presence in the very center of the central zone of the facies of pigmented agglomerate; the average severity of endogenous intoxication is judged by the presence of a pigmented ring along the edge of the central zone of the facies; a high degree of severity of endogenous intoxication is judged by the presence of dark pigmentation throughout the central zone of the facies. Necessary equipment:

1) glass slides,

2) pipette microdoser,

3) a light microscope.

The inventive method has the main advantage compared to the prototype - cheaper (does not require special expensive equipment and reagents) and the simplicity of the technical analysis.

Figure 1 - 4 presents the characteristic facies of mixed saliva of the gastroenterological patients examined. The morphological picture of mixed saliva has two zones: amorphous peripheral (A) and central, filled with salt crystals in the form of dendrites (B) (shown in figure 1).

A detailed analysis of the facies of gastroenterological patients showed that there were three variants of the distribution of dark pigmentation in the central saliva of saliva:

- dark pigmentation was concentrated only in the center of the CPF (A in figure 2),

- a dark ring along the edge of the CZF (A in figure 3),

- dark pigmentation was observed across the entire CZP (A in figure 4).

The inventive method is as follows. After thorough rinsing of the oral cavity, the mixed saliva is taken from the subjects on an empty stomach by spitting in a test tube, then it is defended in the refrigerator for a day. A drop of supernatant - mixed saliva is pre-dried on a glass slide and then the structure of the obtained facies is analyzed under a microscope, and endogenous intoxication is judged by the presence and location of varying degrees of pigmented agglomerate.

Moreover: the absence of endogenous intoxication is judged by the absence of dark pigmentation in the central zone of the facies; the low degree of severity of endogenous intoxication is judged by the presence in the very center of the central zone of the facies of pigmented agglomerate; the average severity of endogenous intoxication is judged by the presence of a pigmented ring along the edge of the central zone of the facies; a high degree of severity of endogenous intoxication is judged by the presence of dark pigmentation throughout the central zone of the facies.

Necessary equipment: slides, pipette microdoser, microscope with transmitted light. No reagents are required. The method is cheap and simple to execute, does not require special expensive equipment and a specially equipped laboratory. It can be used to detect endogenous intoxication, determine its degree and to dynamically monitor the level of intoxication during treatment.

To prove the reliability of the proposed method, 150 patients were examined on the basis of the gastroenterological department of the Nizhny Novgorod city clinical hospital No. 13, of which 62 men and 88 women. The age of the patients was from 30 to 84 years, the median was 54 years. All patients were diagnosed with a combined gastroenterological pathology (gastroesophageal reflux disease, chronic gatroduodenitis, sphincter of Oddi dysfunction, chronic constipation). As concomitant diseases, 53 people (35.3%) were diagnosed with cardiovascular diseases (coronary heart disease, arterial hypertension, chronic cerebrovascular accident), and 20 people (13.3%) had liver diseases (cirrhosis, fatty hepatosis), in 7 people (4.7%) - chronic bronchopulmonary diseases (bronchial asthma, chronic bronchitis), in 6 people (4%) - non-insulin-dependent diabetes mellitus. Thus, in most of the examined patients, diseases were observed that could be the causes of the development of endogenous intoxication.

Upon admission to the hospital and before discharge, they took blood from a vein and mixed saliva. Methods of saliva collection: in the morning on an empty stomach before brushing your teeth, rinse thoroughly 2-3 times with distilled water, then spit 2-3 times into the sink, then spit into a graduated tube for 10 minutes. While collecting saliva, you need to breathe through your nose and not talk. After that, the collected liquid was defended in the refrigerator for a day, as a result, the cell elements settled. For the study, supernatant was taken.

Blood serum and oral fluid were examined by the method of wedge-shaped dehydration, and the content of medium-weight molecules (MSM) was determined in them. In addition, the concentration of total bilirubin in blood serum was determined using a colorimetric photometric test on an OLIMPUS analyzer ().

Statistical processing of the results was carried out using Microsoft Excel 2000 and Statistica 6.0. Since the majority of distributions in the samples differed from the normal one, the number of objects of study (n), the minimum and maximum values of the characteristic, and the median (the value to the right and left of which equal numbers of the characteristic values of the given sample are located on the axis of the characteristic values) were indicated to describe the samples. To clarify the differences between the samples, the nonparametric Wilcoxon test for pairwise comparisons and the Mann-Whitney U test were used. To analyze the differences between several samples (more than two), the ANOVA method was used. The critical level of significance (p) when testing statistical hypotheses was assumed to be 0.05. In all tables, the significance level was shown in bold, provided that p <0.05. To study the relationship between the two signs, the non-parametric Spearman method was used. It was believed that a linear relationship between the signs exists with a correlation coefficient r≥0.4, significance level p <0.05 [11].

The morphological picture of mixed saliva in all examined patients had two zones: amorphous peripheral (A) and central, filled with salt crystals in the form of dendrites (B) (shown in figure 1). Dark pigmentation in the PCF of saliva before treatment was detected in 93 patients. A detailed analysis of the distribution of dark pigmentation in the CHF saliva of gastroenterological patients showed that there were three options:

- in 25 people (27%), dark pigmentation was concentrated only in the very center of the CPF (A in figure 2),

- in 50 people (54%) in the facies of the oral fluid, a dark ring was observed along the edge of the CPF (A in figure 3),

- in 18 people (19%), dark pigmentation was observed across the entire CZP (A in figure 4).

After treatment, dark pigmentation was observed much less frequently, located along the entire center of the center and along the edge of the center, and the frequency of saliva samples, in which dark pigmentation was located only in the center of the center or was absent, increased statistically significantly (Table 1).

Table 1
The frequency of occurrence of dark pigmentation in the central zone of the facies of mixed saliva before and after treatment Dark pigmentation in the Central Park of the Russian Federation Before treatment After treatment Across the FZF * 12% 2% Along the edge of the Central Park * 33% 10.5% In the center of the Central Park * 17% 37% No dark pigmentation * 38% 50.5% * - p <0.05 - critical level of significance

These data suggest that the different arrangement of dark pigmentation in the central focal zone of the Russian Federation depends on the severity of intoxication, and the following notation was introduced: 0 - no dark pigmentation, 1 - dark pigmentation is concentrated in the very center of the central phase, 2 - dark pigmentation in the form of a ring along the edge of the central central phase , 3 - dark pigmentation throughout the entire cenospheric center.

As can be seen from table 2, the extinction values of blood serum in the range from 244 to 274 nm were higher than normal, which indicates the presence of endogenous intoxication in the examined patients. Especially strong were the levels of MSM of the catabolic pool. It includes products of the catabolism of protein molecules and such low molecular weight metabolites as urea, creatine, creatinine, uric acid, products of purine metabolism, etc., as well as nucleotides and their derivatives - metabolites of nucleoprotein metabolism [8]. Such a shift of the main maximum towards the catabolic component of the MSM spectrum is characteristic of the fourth variant of the metabolic response of the body to endogenous intoxication according to the classification of T. V. Kopytova [6]. The variant may be referred to as a “nucleoprotein type catabolic response”.

Correlation analysis showed the presence of a statistically significant positive correlation of the spectrograms of blood serum and mixed saliva over the entire studied spectrum of MSM except 244 nm. In addition, extinction values at wavelengths of 264 and 274 nm did not statistically significantly differ in blood serum and mixed saliva.

table 2
The content of MSM in blood plasma and mixed saliva before treatment and their correlation MSM, conv. units 244 nm 254 nm 264 nm 274 nm 284 nm 294 nm Blood serum 0.1-0.81, median 0.64 * 0.26-0.68, median 0.34 * 0.2-0.63, median 0.3 * 0.23-0.67, median 0.34 * 0.23-0.63, median 0.35 0.14-0.42, median 0.22 Mixed saliva 0.18-2.2, median 1.02 0.07-0.96, median 0.49 0.06-0.73, median 0.32 0.05-0.67, median 0.31 0.05-0.57, median 0.26 0.04-0.41, median 0.2 Differences by Wilcoxon criterion p = 0,0004 p = 0,0006 p = 0.057 p = 0.08 p = 0.001 p = 0.01 Correlation r = -0.03, p = 0.8 r = 0.45, p = 0.04 r = 0.69, p = 0.002 r = 0.69, p = 0.0005 r = 0.7, p = 0.0005 r = 0.71, p = 0.0003 * - median values are higher than normal.

From these data, it can be concluded that MSM are excreted by the salivary glands. The difference in extinction values at wavelengths of 244 and 254 nm (catabolic pool of MSM) can be explained by pathological processes occurring in the oral cavity.

Correlation analysis of the relationship revealed statistically significant positive relationships between the degree of severity of dark pigmentation in the CZF (from 0 to 3) with all types of MSM in mixed saliva: 244 nm - r = 0.63, 254 nm - r = 0.56, 264 nm - r = 0.53, 274 nm - r = 0.46, 284 nm - r = 0.46, 294 nm - r = 0.46. The highest correlation coefficients were observed for the catabolic pool of MSM. In all cases, p <0.05.

Comparison of extinction values at different wavelengths for different morphological patterns of mixed saliva demonstrated statistically significant differences in most cases (Table 3).

The analysis of the tables showed that for the first time positive correlations of dark pigmentation in CZP with the content of MSM in mixed saliva are shown, which allows non-invasive diagnosis of endogenous intoxication by the proposed method.

Table 3
The content of MSM in the oral fluid with a different morphological picture of its facies MSM, conv. units 244 nm 254 nm 264 nm 274 nm 284 nm 294 nm No TP (0) 0.08-0.67, median 0.26 0.07-0.32, median 0.15 0.06-0.22, median 0.12 0.05-0.17, median 0.11 0.05-0.14, median 0.09 0.04-0.1, median 0.075 TP in the very center of the Central Park (1) 0.66-1.35, median 0.94 0.33-0.84, median 0.44 0.17-0.53, median 0.28 0.13-0.44, median 0.26 0.09-0.36, median 0.24 0.05-0.28, median 0.16 TP along the edge of the Central Park (2) 0.79-1.27, median 1.02 0.39-0.82, median 0.5 0.2-0.58, median 0.31 0.15-0.57, median 0.3 0.09-0.54, median 0.27 0.07-0.4, median 0.2 TP across the entire Central Park (3) 0.78-2.2, median 1.31 0.39-0.96, median 0.74 0.22-0.73, median 0.53 0.15-0.67, median 0.38 0.11-0.57, median 0.31 0.08-0.42, median 0.22 ANOVA comparison p = 0,0002 p = 0.015 p = 0.046 p = 0.005 p = 0.005 p = 0.26 TP - dark pigmentation

As an illustration of the health of the proposed method for non-invasive diagnosis of endogenous intoxication, we give examples that confirm the practical application of this method.

Example 1. Patient S., 65 years old, was admitted to the gastroenterological department with complaints of pain and heaviness in the epigastrium and right hypochondrium of a constant nature, heartburn, belching, air, bitterness in the mouth, nausea, bloating, constipation (stool 2 times a week) .

Over the course of 10 years, he was repeatedly treated for chronic pancreatitis, 5 years ago liver cirrhosis of toxic origin was first diagnosed. Deterioration within a month after another abuse of alcohol. Chronic constipation - for 15 years.

Upon receipt, the state of moderate severity. Consciousness is clear. Icterus of the skin and sclera, palmar erythema. There are scattered dry rales in the lungs, harsh breathing. Heart sounds are rhythmic, muffled, heart rate 96 beats / min, blood pressure 150/95 mm Hg The abdomen is somewhat tense and painful on palpation in the upper half. The liver protrudes from the hypochondrium by 4-5 cm, dense, tuberous. The enlarged spleen is palpated.

Examination results: General blood test indicators: hemoglobin - 108 g / l, red blood cells - 3.6 × 10 ¹² / l, white blood cells 9.2 × 10 ⁹ / l, ESR-26 mm / h, platelets - 160 × 10 ⁹ / l Biochemical blood test: ALT - 5N (5 times higher than normal), ACT-8N, bilirubin - 102 mmol / l, alkaline phosphatase - 4N, total protein - 63 g / l.

FGDS: Erosive reflux esophagitis. Varicose veins of the esophagus II degree, portal gastropathy.

Ultrasound: The liver is enlarged (anteroposterior size 14.8 cm), the structure is coarse-grained, echogenicity is reduced. The diameter of the portal vein is 14 mm. Choledoch 4 mm. Gall bladder 8.4 × 3.2 cm, the wall is sealed. The pancreas with uneven contours, echogenicity is increased, the size of the head is 3.4 cm. The spleen is 13 × 5 cm.

Sigmoidoscopy: Internal hemorrhoids without exacerbation.

Irrigoscopy: no pathology detected.

The study of the morphological picture of the oral fluid: a narrow peripheral zone with a network of three-beam cracks and a large number of crystalline inclusions in the form of grains of different sizes, the central zone with a large number of small dendrites, partially destroyed, with black pigmentation throughout the zone. Conclusion: high endogenous intoxication.

Clinical diagnosis: Liver cirrhosis of toxic etiology, Childe-Pugh class A with symptoms of portal hypertension (varicose veins of the esophagus, splenomegaly) and initial manifestations of liver failure. Oral gastropathy. Chronic recurrent pancreatitis in the stage of moderate exacerbation. GERD: erosive reflux esophagitis. Chronic functional constipation. Arterial hypertension II, risk 2.

The treatment: inside omeprazole 20 mg / day, mezim 10,000 units × 3 times a day, dicetel 50 mg × 3 times a day, dufalac 30 ml / day; injections of platifillin, cefataxime, essentiale, reamberin, B vitamins.

After 3 weeks of treatment, pain in the upper abdomen and dyspeptic syndrome were eliminated, jaundice decreased, and stool returned to normal. Blood biochemical parameters improved: ALT-2N, ACT-2N, bilirubin - 46 mmol / l, alkaline phosphatase - 3N, total protein - 64 g / l.

According to FGDS, erosion in the esophagus was epithelialized.

Study of the morphological picture of the oral fluid: a narrow peripheral zone with a small amount of crystalline inclusions in the form of small grains, a central zone with a large number of small dendrites with black pigmentation along the edge of the zone. Conclusion: moderate endogenous intoxication.

Example 2. Patient I., 51 years old, was admitted to the gastroenterological department with complaints of heartburn occurring an hour after eating, air belching, painful passage of food through the esophagus, a feeling of heaviness in the epigastrium after eating, constipation.

Sick for 10 years. Periodically took antacids and enveloping drugs with a good positive effect. During the last six months, heartburn became stubborn, pain appeared along the esophagus during meals.

Upon receipt, the condition is satisfactory. Normal nutrition. In the lungs, vesicular breathing. Rhythmic heart sounds, heart rate-76 beats. per minute. AD-110/70 mm Hg The tongue is moist, coated at the root with a white coating. The abdomen is soft, painful in the epigastrium and right hypochondrium. The liver is painless along the edge of the costal arch.

Examination: General and biochemical blood tests - without deviation from normal values.

When x-ray examination: the esophagus is freely passable for liquid barium, the elasticity of its walls is preserved. The cardia does not completely close. Hernia of the esophagus. Gastroesophageal reflux. Signs of chronic gastroduodenitis. Hypermotor dyskinesia of the duodenal bulb.

FGDS: The mucous membrane of the esophagus is light pink, swollen in the lower third, whitish, friable, with single scattered whitish spots (foci of glycogenic acanthosis) and a smooth vascular pattern. In the lower third in front of the Z-line (the transition of the esophageal epithelium into the gastric) on the mucosa there are multiple defects of the mucosa, connecting with each other, extending to 2 or more folds, occupying less than 75% of the esophagus circumference. The cardia yawns. The diaphragmatic pulp is displaced distal to the Z-line by 2 cm. The gastric mucosa is moderately hyperemic, swollen. The duodenal bulb is of the usual form. The mucous is pale pink. Conclusion: Erosive reflux esophagitis, degree C. Diaphragmatic hernia. Chronic diffuse antral gastritis with spread to the body of the stomach. The pyloric Helicobacter test is positive.

Ultrasound of the abdominal cavity: echoes of chronic stoneless cholecystitis. Diffuse structural changes in the liver and pancreas by type of fatty degeneration.

FCC: chronic hemorrhoids without exacerbation.

The study of the morphological picture of the oral fluid: a narrow peripheral zone without cracks and crystalline inclusions, the central zone with a large number of long thin dendrites, with dark pigmentation along the edge of the zone. Conclusion: moderate endogenous intoxication.

Clinical diagnosis: Gastroesophageal reflux disease: erosive reflux esophagitis. Hernia of the esophagus. Chronic gastroduodenitis associated with pyloric helicobacter. Chronic stoneless cholecystitis. Liver steatosis. Chronic functional constipation.

The treatment was carried out: inside omeprazole 40 mg / day, dicetel 150 mg / day, maalox 1 tbsp. spoon 3 times a day, flemoxin 2.0 g / day 10 days, Fromilide 1.0 g / day 10 days; injections of cerucal, vitamins of group B.

Within 10 days of treatment, the patient's condition improved: heartburn, belching, a feeling of heaviness in the epigastrium were eliminated, stool normalized.

During the control endoscopic examination, erosion in the esophagus was epithelized.

Investigation of the morphological picture of the oral fluid: a wide peripheral zone without cracks, with a small number of crystalline inclusions in the form of small grains, a central zone with a large number of long thin dendrites and a pigmented agglomerate in the very center of the zone. Conclusion: endogenous intoxication of low degree.

The claimed invention as having simplicity, cheapness, accessibility, as well as non-invasiveness can be widely used in medicine, in laboratory diagnostics and for the diagnosis of intoxication and its severity. It can be used to detect endogenous intoxication, determine its degree and to dynamically monitor the level of intoxication during treatment.

Information sources

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Claims (1)

A method for diagnosing the severity of endogenous intoxication, characterized in that the drop of mixed saliva is dried, the structure of the obtained facies is analyzed under a microscope, while the low severity of endogenous intoxication is judged by the presence of dark pigmentation in the very center of the central zone of the facies, and the average severity of endogenous intoxication judged by the presence of dark pigmentation in the form of a ring along the edge of the central zone of the facies, a high degree of severity of endogenous intoxication is judged by the presence of dark th pigmentation throughout the central zone of facies.

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