Method for assay of effect of foodstuff on body

FIELD: medicine, immunology.

SUBSTANCE: method involves determination of thiol-disulfide ratio value (TDR) in blood sample after its incubation with a single dose of antigen in the specific foodstuff. Then in 30 min, 1 and 3 h after onset of incubation 2-, 4- and 8-fold dose of antigen is added repeatedly and the value TDR is determined in interval for 30 min, 1, 3 and 24 h of incubation. Then the curve for dependence of the TDR value from incubation time is plotted and the value AUC (areas under curve) for dependence of the TDR value of the incubation time is calculated. Invention provides the development of a method allowing carrying out assay of allergy, nutritional intolerance or usefulness of foodstuff. Invention can be used in assay of effect of foodstuff on the body.

EFFECT: improved assay method.

4 cl, 3 tbl, 2 ex

 

The invention relates to medicine, in particular to immunology, Allergology, nutrition, and can be used for the diagnosis of allergic diseases, intolerance or usefulness of food for any living organism (human or animal).

A known method for the diagnosis of allergic reactions of neutrophils in the blood (Vagrancy, 1985), based on the rate of damage of neutrophils.

There is also known a method for the diagnosis of food Allergy double-blind provocative test with placebo - DSPTAP (Bock S.A. et al. Double-blind, placebo-controlled food challenge (DBPCFC) as an office procedure: a manual //J.Allergy Clin. Immunol. - 1988. - Vol.82. - P.986), which is directed test suspicious allergies food when drinking a small amount of the test person again in ascending doses (ranging from doses of 125-500 mg followed by doubling the dose every 15-60 minutes) and determining the reaction of the organism. If the dose of product in 10 g of a patient moves without the development of the reaction, the test result is considered negative; if you have allergies after eating a specific food product in person to show symptoms (dyspepsia, dyspnea, tachycardia, hypotension, urticaria, diarrhea and other), the test is considered positive.

The disadvantage of these methods is nespecificnomu test with the social neutrophils blood and the lack of regulation for its use in determining food intolerances. In addition, testing with the use of suspect food allergies in vivo takes a long period of time (a day tested the reaction of one or a small number of food allergens) and may contribute to the development of adverse reactions or complications for the patient.

The closest to the essence of the claimed is a method of diagnosis of allergic diseases (see RF patent № 2098826, IPC 6 G 01 N 33/53, 33/48, publ. 10.12.1997, by setting the load test with antigen theophylline in vitro and simultaneous determination of blood parameters, change them diagnose, simultaneously with the production test with theophylline as antigen for determination of blood parameters are spontaneous reaction rosethorne with sheep erythrocytes and the number of rosette neutrophils equal to 1067 in 1 ml of blood, and teophelinresistent T-lymphocyte - 27% and below - diagnosed allergic disease.

The main disadvantage of this method is insufficient informative for the diagnosis of various forms of allergic diseases, as well as significant nespecificnomu method evaluation of immune disorders, which limits the scope of its practical application. While not developed conditions for the diagnosis of food Allergy and intolerance food is of aktov, there are no conditions for an objective evaluation of the potential beneficial effects of a test substance on the body.

The basis of the invention is the task of such improved method for the diagnosis of allergies, food intolerances and usefulness of food products, which account for use as antigen test substance and determining the quality of blood thiol-disulfide ratio (TDS) expands the scope of the diagnosis of various types of allergies, including food. Additionally, you receive the possibility to diagnose food intolerances or their beneficial influence on the studied organism. The proposed method allows for the simultaneous testing of a practically unlimited number of allergens in vitro that is absolutely safe for the patient.

This object is achieved in that in the method of determining the effects on the body of the food product by asking a load test with antigen in vitro and simultaneous determination of blood parameters, change them diagnose, according to the invention as an antigen using the test substance, as well as changes in blood parameters determine the thiol-disulfide ratio (TDS) compared to the control measurement is carried out by PEFC is the introduction into the blood of physiological solution, while TDS define many times during the incubation time sufficient to detect early and/or late reaction, but not less than 24 hours, and in the process of determining the TDS in the blood re-administered the test substance in the time interval of 0.5 hour, 1 hour, 3.0 hours, and each subsequent dose of the test substance increase in 2, 4 and 8 times, respectively; to determine allergic reaction or food intolerance test substance assess the value of reduction of area under curve (AUC - Area Under the Curve) according to TDS on the time of incubation; for determining the effect of a test substance to evaluate the magnitude of the increase in the area under the curve TDS on the time of incubation. The area under the curve was calculated using the trapezoid method (Karkishchenko N.N., Khoronko V.V., Sergeev S.A., Karkishchenko NR. Methods of calculating the area under the curve. In the book: The pharmacokinetics. - Phoenix, Rostov-on-don, 2000. - S-158).

The causal link between the proposed set of features and achievable medical results is as follows.

Every biological individual has a unique genotype, which is implemented individual spectrum of biochemical and enzymatic reactions, including to interact with food, food additives, drugs or other abused substances. Depending on the possible outcome of such interaction reaction to the organism can be evaluated as useful, harmful, or indifferent. To establish objective criteria such influence on biochemical processes helps it offer a way to assess the impact of exogenous substances tested in the flowing nature of the redox processes, the balance of Pro-antioxidant status, as well as the level of nonspecific resistance and adaptive capacity through assessment of thiol-disulfide exchange. In essence, the thiol-disulfide ratio in the blood at the level of the microorganism reflects the integral of the biochemical balance of the health-disease. Substances contributing to regenerative processes in the body, can potentially play a useful role for health factors and influence in the opposite direction, on the contrary, aggravate the failure of adaptive mechanisms or contribute to the deterioration of biochemical and physiological homeostasis and the development of pathological processes.

To select the diagnostic criteria conducted research in 37 individuals with allergic diseases, as well as in 78 patients with no allergies with the use of complex biochemical tests and methods allergological the in vitro diagnostics. To identify allergies to various allergens (pollen, household, medical, infectious, food) applied the scratch method, and a load test with the specific antigen in the blood in vitro. When setting the load test, the patient produced the blood from the cubital vein in a volume of 15 ml, mixed it with a 5% solution of sodium citrate. Then nitrate blood incubated with various allergens, and after 30 minutes, 1 hour and 3 hours after the start of incubation in a tube of blood was re-added sequentially increased in 2, 4 and 8 times, respectively, the dose of the specific allergen. At the same time before the start of incubation, and then after 30 minutes, 1 hour, 3 hours and 24 hours incubation of blood with the allergen content was determined thiol (-SH) and disulfide (-SS-) groups in the lysate of whole blood by the method of amperometric titrations with silver nitrate, and then calculated the thiol-disulfide ratio:

([-SH]/[-SS-]=TDS).

Thus, investigated TDS in the blood in the dynamics at intervals of 30 minutes, 1 hour, 3 hours, 24 hours incubation at escalation increase input in blood in vitro dose tested allergen, ranging from 1 dose (baseline), then 2 doses (added to the blood after 30 minutes incubation 1 dose)followed by 4 doses (added 30 minutes after 2 doses) and, finally, added 8 more doses of allergen (2 hours after por the previous introduction 4 doses of allergen); just a study in blood was injected 1+2+4+8=15 doses tested allergen. In the control instead of the allergen in the same time intervals were injected with saline (negative control) or a similar mode of increasing doses of histamine (positive control). Then build a plot of the values of TDS on the time of incubation and compared the area under this dependence compared with the control values (Δ AUC). Diagnosis of Allergy or food intolerance was performed on the basis of the decrease of the area under the curve TDS - incubation time compared with the negative control. At the same time take into account the degree of such reduction compared with the positive control (i.e. in the reaction with histamine). With a maximum reduction of the area under the curve up to the level of the positive control response to antigen was regarded as a manifestation of allergies, but less pronounced decrease as food intolerance. The positive influence of the antigen or food allergen on the redox balance and nonspecific resistance was evaluated by the increase in the area under the curve TDS - incubation time compared with the negative control.

Clinical-laboratory comparison revealed that in allergic effects in patients neb, who was udalos reduction of the area under the curve TDS - the incubation period compared with the negative control at 80-140% (Δ AUC in the range of -40%to+20%), and this figure was close to the positive control (reaction with histamine). In the absence of allergies, but with the presence of clinical signs of intolerance to food indicators Δ AUC ranged between 60-30%. The beneficial impact of food was seen when indicators Δ AUC is not less than 100%. In addition, reduction Δ AUC ranging from 99% to 60% did not have a material adverse reaction on the body and has been assessed by us as indifferent.

Carried out researches have shown (see table No. 3)that persons with allergies or food intolerance observed a significant reduction of the area under the curve TDS - incubation time compared with the figures of people who have no such violations. It is established that in patients with signs and symptoms of food intolerance, do not manifest clinical signs of Allergy (including in the absence of a positive scratch tests with allergens), had a different degree of reduction of the area under the curve TDS - incubation time compared with the negative control, but not reaching those indicators in positive control. This reflects, apparently, the different nature of the biochemical reaction is AI blood for a particular food product from a particular individual.

Presents the results of the dynamics of the mean values are supported by the data correlation analysis. Patients with allergies found a significant negative relationship according to the area under the curve TDS - incubation time (r - 0,89) compared with the control. Identified results provide a basis to diagnose in vitro as having food allergies, and to identify intolerance to specific foods in individuals with no positive reactions in allergic (scratch, immunological) tests with food allergens. Furthermore, the method allows to assess the positive impact of a specific food product on the status of redox processes and the level of nonspecific resistance of the organism.

Example 1.

Patient S., 49 years. Complains of itching and rashes on the skin of the head and chest, which first appeared about 1 year ago. The specific links between the disease and any allergen or food intolerance install failed. Objectively: General condition is satisfactory, the skin, the scalp and the sternum is determined papular rash, dry skin. A study of the impact on patient food by asking Allergy intradermal tests with sevime allergens, a positive reaction was not detected. Delivered clinical diagnosis: allergic dermatitis of the head and chest. Given the history, the absence of obvious causes allergic reactions in the skin, pilot testing of blood by way of setting up a load test antigens and test substances, which are used for food allergens were studied allergens 50 food (tables 1.1, 1.2, 1.3, 1.4 - attached). Revealed a significant decrease (compared with a negative control) area under the curve TDS - incubation time when a load test with cow's milk casein (decrease by 111%) and protein chicken eggs (decrease by 105%), which corresponded to the level of the positive control with histamine (decrease of 122%), i.e. essentially an allergic reaction. Was also less marked reduction Δ AUC (within 59-28% compared with a negative control) when testing 9 food (fish, carp, walleye Pollock, hake, pumpkin, oatmeal, barley, buckwheat, raspberry, green tea), which is interpreted as indicators of their intolerance. Among the 12 foods (the yolk of chicken eggs, rice and millet grits, rye flour, beet, Apple, banana, currant, plum, grapes, onions, coffee) revealed minimal reduction Δ AUC (within 99-60% compared to negative the nutrient control), which was regarded as indifferent action. And finally, when testing another 27 food was noted the increase Δ AUC above the negative control (taken as 100%), which assumed their beneficial effects on the body.

After exclusion of food allergenic foods (an elimination diet) and eating mostly foods with prognostically useful effect on the body (including neutral), signs of allergic dermatitis regressed within 4 weeks without any additional treatment.

Example 2.

Patient I., aged 47. She was complaining that after eating certain foods (fruit, meat) the feeling of heaviness in the epigastric region, bloating, frequent stools. Occasionally itching and rashes on the skin of the hands. These events usually last from several hours to days. A clear link between the intake of any food product and these complaints to identify failed. Gastroenterology examination of pathological changes in the gastro-intestinal tract is not revealed. Conducted scratch tests with food allergens, reactions assessed as negative. Blood examination by way of setting up a load test with a set of food allergens (52 food item is oduct, charts and tables 2.1, 2.2, 2.3, 2.4 - attached) revealed a decrease (compared with a negative control) area under the curve TDS - incubation time when a load test with antigen pork (decrease of 56%, which corresponded to Δ AUC=44%) and antigen pears (decrease by 44%, Δ AUC=56%), without reaching, however, the values of the positive control (Δ AUC in response to histamine = -3%), which regarded as a manifestation not of Allergy and food intolerance. Observed a less pronounced reduction Δ AUC (within 99-60%) when testing 11 food (fish, carp, walleye, buckwheat and rice grits, corn flour, potatoes, cabbage, walnuts, melon, black currant, cherry), regarded as indifferent action. There was also increased (within ΔAUC≥100%) rate of TDS-test 39 food.

Based on the results, the patient was recommended eating only those foods that contributed in the test in vitro to increase or at least not contributed to the reduction of TDS in the blood, reflecting, apparently, their positive influence on the redox balance and the level of nonspecific resistance of the organism. The use of such individual diet for 4 weeks led to a complete restoration of normalnogo being patient, to increase efficiency, good endurance food.

Due to the implementation of the proposed differences that inherently are new criteria for evaluating the effect on a living organism test substance, the diagnosis of food Allergy or food intolerance, provides the following advantages of the proposed method: 1) the method is simple and affordable method that may be feasible not only in immunological or allergic, but in any clinical laboratory;

2) there is no danger of provoking the patient's side or allergic reactions; 3) the method allows for the simultaneous testing of a practically unlimited number of allergens;

4) methods of blood testing using TDS-reactions to allergens can be identified and differentiated not only an allergic reaction, but also non-immunological allergies or food intolerances;

5) determination of the quantitative characteristics of changes of indicators curve TDS - incubation time as a criterion for the evaluation of food intolerance can improve the diagnostic accuracy in comparison with the prototype, where such criteria are not available.

1. The method of determining the effects on the body of the food product, which consists in the fact that the blood sample is determined thiol-disulfide ratio (TDS), incubated it with a single dose of antigen specific food product, while after 30 min, 1 h and 3 h after the start of incubation, re-add successively increased in 2, 4 and 8 times, respectively, the dose of antigen and determine TDS at intervals of 30 min, 1 h, 3 h and 24 h of incubation, construct a plot of the values of TDS from the incubation time and calculate the area under the curve values from TDS the incubation time.

2. The method according to claim 1, characterized in that when the reduction of the area under the curve TDS on the time of incubation to the values obtained by incubation with histamine as antigen (positive control), determine the allergic effect of the tested food product.

3. The method according to claim 1, characterized in that when the reduction of the area under the curve TDS on the time of incubation in comparison with the negative control, define intolerance test food product.

4. The method according to claim 1, characterized in, is that with the increase in the area under the curve TDS on the time of incubation in comparison with the negative control, determine the beneficial effect of the tested food product.



 

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