RU1767743C - Method of reagent preparing for determination of activated partial thromboplastin time - Google Patents

Abstract

FIELD: medicinal industry. SUBSTANCE: waste of soybean lecithin production are dissolved in chloroform, and ethanol is added at the ratio biomaterial: chloroform: ethanol = 1:8:(37-40). Supernatant liquid is evaporated, and residue is dissolved in petroleum or diethyl ether, and the product is precipitated with acetone. Formed precipitate is dried and then is dissolved in 5% sorbitol solution and lyophilized. EFFECT: preparing of low-cost reagent, enhanced sensitivity of reagent with respect to 1-st phase of blood coagulation, reagent can be stored at +4 C for one year. 3 tbl

Description

 The invention relates to the medical industry and relates to a method for producing a reagent for determining activated partial thromboplastin time (APTT) from waste products of soya lecithin used as an emulsifier for fat emulsion for intravenous administration.

 A known method of producing a reagent for determining APTT from a reagent for determining the prothrombin index - thromboplastin obtained by aqueous extraction of human brain tissue.

 The disadvantages of this method is its low technology due to the source of raw materials and the preparation of a reagent with insufficiently high activity and short shelf life.

 The purpose of the invention is the expansion of the range of reagents for the determination of APTT with high sensitivity and an increase in its shelf life.

 This goal is achieved by the fact that as a source of phospholipid (PL) use waste products of soya lecithin, which are dissolved in chloroform, add ethanol, and 1 wt.h. phospholipids take 8 vol.h. chloroform and 37-40 vol.h. ethanol, then the precipitate is discarded, the supernatant is evaporated, the dry residue is dissolved in petroleum or diethyl ether and the target product is precipitated with acetone, followed by drying, dissolution in sorbitol solution and lyophilization.

The method is as follows: soybean phospholipids, insoluble in ethanol during the extraction of soya lecithin from defatted phosphatides (Puchkova S.M., Shanskaya A.I., Nedachina N.A. Method for producing soya lecithin. Aut. St. N 773993, C. A 61 K 35/78), dissolved in chloroform at the rate of 8 ml of chloroform per 1 g of phospholipids. Then, with stirring, 96% ethanol (37-40 ml per 1 g of PL) is added. After 1-3 hours, the precipitate was separated by filtration and discarded. The filtrate was evaporated in vacuo at 30-50 ^C. The dry crude product is dissolved in petroleum or diethyl ether (1 ml per 1 g of PL) and the desired product is precipitated with acetone at the rate of 1: 5. The precipitate was separated by centrifugation, dried in vacuo and dissolved in a 5% sorbitol solution (pH 7.4). A solution of phospholipids of the optimal concentration (0.1-0.2%) in a 5% solution of sorbitol is subjected to freeze drying and, after reduction, is used as a reagent for the determination of APTT.

Example 1. 10 g of phospholipids insoluble in ethanol after direct extraction of soya lecithin from fat-free phosphatides are dissolved in 80 ml of chloroform. Inactive with respect to coagulation impurities precipitated by adding a thin stream with stirring 370 ml of ethyl alcohol. After holding for 1 h, the precipitate is filtered off and discarded. The filtrate was evaporated to dryness in vacuo using a rotary evaporator at 30-35 ° ^C. The dry substance is dissolved in 10 ml of petroleum ether, the solution was poured under stirring in 50 ml of acetone and the precipitate formed phospholipids separated by centrifugation at 2500 rev / min 4 ^{° C} and dried vacuum. Get 2.68 g of the final product, which is 26.8% of the original. The resulting phospholipids have the following characteristics: phosphorus 3.4%; acid number 54.0 mg KOH / g; oxidation indicators: the amount of dienes 0.41%, trienes 0.11%, peroxide value 0.02 mg% l ₂ ; fractional composition according to TLC: the content of lysophosphatidylcholine (LYSOFH) 1.6%; phosphatidic acid (FC) 4.0; phosphatidylinositol (PI) 13.8; phosphatidylserine (PS) 3.1% phosphatidylcholine (FC) 21.4%; phosphatidylethanolamine (FEA) 33.8%; neutral lipids 6.5%; unidentified phospholipids 15.8%; Σ _acid / Σ _neutral 0.40; iodine number calculated by the fatty acid composition according to GLC, 141.1.

To obtain the APTT reagent, the PL is dissolved in a 5% sorbitol solution (pH 7.4) to a concentration of 0.15%. The solution is poured into vials 2 ml each, frozen at (-58 ± 2) ^{° C,} quenched for 15 days and freeze-dried. The duration of the sublimation process is 18 hours. The final temperature of the drug during dryness 19 ^C. The drying time of 36 hours.

EXAMPLE EXAMPLE 2 50 g of soy lecithin production waste, air-dried and stored at 4 ^{° C} for 5 months, was dissolved in 400 ml of chloroform to the solution with stirring in a thin stream poured 2 liters of ethyl alcohol. After 2 hours, the precipitate is filtered off and discarded. The filtrate is evaporated using a rotary evaporator at 40-50 ^about C, the dry substance is dissolved in 50 ml of diethyl ether. The ether solution is poured into 250 ml of acetone with stirring. The precipitate was centrifuged at 2500 rev / min, 4 ^{° C} and dried in vacuo. Obtain 11.35 g of the final product, which is 22.7% of the original.

Characteristics of the obtained phospholipid: phosphorus 3.00%; acid number 52.2 mg KOH / g; the amount of dienes 1.03%; trienes 0.27%; peroxide value 0.28 mg% I ₂ ; fractional composition according to TLC: lyso FC 2.9%; FI 18.1%; FS 12.4%; FC 16.1%; PEA 33.6%; unidentified PL 16.7%; Σ _acid / Σ _neutral 0.43%; iodine number calculated by the fatty acid composition according to GLC, 145.3.

Phospholipids are dissolved in a 5% sorbitol solution (pH 7.4) to a concentration of 0.15%. The solution is poured into vials 2 ml each, frozen at (-58 ± 2) ^{° C,} quenched for 15 days and freeze-dried. The duration of the sublimation process is 19 hours. The final temperature of the drug during the final drying ^of 20 ° C. The drying time of 35 hours. According to the proposed method produced a series of reagent 4 and studied their properties. In the process of testing the technology, requirements for raw materials and for individual stages of the process were formulated.

 The ratio of 1 wt.h. phospholipids: 8 vol.h. chloroform: 37-40 vol.h. 96% ethanol is optimal and allows you to get a reagent - a substitute for 3 platelet factors with procoagulant properties and sensitive to coagulation disorders of the studied plasma.

PRI me R 3. 20 g of insoluble in ethanol phospholipids after extraction of soya lecithin from defatted phosphatides, dried in air and stored at 4 ^about C for 2 months. dissolved in 160 ml of chloroform, 760 ml of 96% ethanol are poured into the solution with stirring in a thin stream. After holding for 1 h, the precipitate is filtered off and discarded. The filtrate was evaporated to dryness in vacuo using a rotary evaporator at 30-35 ° ^C. The dry substance is dissolved in 20 ml of petroleum ether, the solution was poured with stirring into 100 ml of acetone and the precipitate formed phospholipids separated by centrifugation at 2500 rev / min 4 ^{° C} and dried vacuum. Obtain 5.14 g of the final product, which is 25.7% of the original. The resulting phospholipids have the following characteristics: phosphorus 3.2%; acid number 53.6 kg KOH / g; oxidation indicators: the number of dienes 0.65%, trienes 0.23%; the peroxide value of 0.08 mg.% I ₂ , the fractional composition according to TLC: the content of lysophosphatidylcholine 1.3%; phosphatidylinositol 14.9%; phosphatidic acids 3.2%; phosphatidylserine 4.5%; phosphatidylcholine 20.9%; phosphatidylethanolamine 33.7%; unidentified phospholipids 14.9%; Σ _acid / Σ _neutral 0.4; iodine number calculated by the fatty acid composition according to GLC, 143.8. Next, proceed as in example 1.

 With a decrease in the amount of ethyl alcohol to 35 vol.h. and lower by 1 wt. including phospholipids, the low-active phospholipids remaining in the solution are fed into the final product and reduce its procoagulant activity. With an increase in the amount of ethanol over 40 vol.h. per 1 part by weight phospholipids decreases the yield of the final product without increasing its specific activity.

 The exact concentration of the solution of the final product, lying within 0.1-0.2%, is selected empirically on the basis of specific activity data. A decrease in the concentration of sorbitol solution below 5% leads to a deterioration in the appearance of the dried reagent, a decrease in storage stability and a decrease in specific activity.

The lyophilized reagent is reconstituted by adding a well-mixed suspension of kaolin (5 mg per 1 ml of distilled water), after which the vial is shaken vigorously for 3 minutes. Recycled thus reagent is stable at ⁴ ° C for a week, and at room temperature for one day.

 Received by the proposed method, the APTT reagent was evaluated based on the determination of the APTT of donors and patients with hemophilia.

 APTT tests were conducted on plasma obtained from donors and from patients with hemophilia. Blood is taken by venipuncture and stabilized with a 3.8% solution of basic sodium citrate (9 parts of blood: 1 part of stabilizer). After mixing and centrifugation for 10 min at 4000 rpm, a plasma is taken with a pipette, which should be used within 2 hours after selection.

The heated at ³⁷ ° C centrifuge tube was placed 0.1 ml of thoroughly mixed APTT reagent were incubated at ³⁷ ° C for 3 min. Then add 0.1 ml of the investigated plasma and incubated for 2 min with periodic shaking. After the incubation period 0.1 ml of pre-warmed to 37 ^{° C} a 0.025 M solution of calcium chloride and simultaneously start the stopwatch, from the measurement of clotting time. The determination is repeated 2 times and the arithmetic mean value is taken.

 The APTT of the same donor plasma samples was determined using APTT reagents obtained by the proposed method (from soybean PL waste), by the prototype (from human brain tissue), and the Reanal reagent (Hungary) widely used in practice ) The results are shown in table 1.

 As can be seen from Table 1, the donor APTT determined by different reagents did not differ significantly, i.e. the procoagulant ability of the proposed reagent from soybean phosphatide waste did not differ from that of reagents from human brain tissue and Reanal reagent.

 When examining hemophilia patients, the APTT index was calculated - the ratio of the patient’s APTT to the APTT of the donor mixture, which showed the depth of the coagulation defect in each case. APTT of the donor mixture, in this case, the APTT of the mixture of at least 5 donors, determined by a reagent from the same vial at the same time as the patient's APTT.

 When conducting this study, the authors did not have a reagent obtained by the prototype, and therefore, the reagent obtained by the proposed method was compared with a reagent company "Reanal". The sensitivity of the two reagents was compared by comparing the APTT indices determined by different reagents for the same patient. Part of the data is presented in Table 2.

 As can be seen from table 2, the degree of violation of APTT depends on the level of coagulation FUSh, with an increase in the activity of the latter, the APTT index decreases. A change in the level of FUS from 2 to 10% corresponds to a change in the APTT index with the Reanal reagent from 2.79 to 1.40, while the proposed reagent gives a change from 5.29 to 1.56, which indicates its greater sensitivity .

± S

), соевый реактив 3,12 ±0,21 (t=3,55, различие статистически достоверно).In the study of a sample of 32 hemophilia patients using the APTT reagent from soybeans and Reanal reagent, data were obtained confirming the greater sensitivity of the proposed reagent. So, on average for the group, the APTT index with Reanal reagent was 2.26 ± 0.12 (

± S

), soy reagent 3.12 ± 0.21 (t = 3.55, the difference is statistically significant).

The use of freeze drying allowed to obtain a reagent with a number of advantages compared to the prototype. Specific reagent properties do not change during storage for one year at ⁴ ° C, while the reagents provided by the prior art, is stable for 3 months when stored at -20 ° ^C.

 Table 3 shows the APTT (in sec) of donors when using a reagent from soybean phosphatide waste during storage.

 Reducing the requirements for temperature storage and dry form allow transporting the reagent over long distances at any time of the year.

 The APTT reagent obtained by the proposed method determines the donor APTT the same as the reagent from human brain tissue and the Reanal reagent, but is more sensitive to disturbance of the first phase of blood coagulation. The reagent is more stable, its shelf life is increased to a year, and is transportable. The proposed method does not use brain tissue or human blood as a raw material, which excludes the possibility of infection of maintenance personnel, does not require expensive and scarce sources of raw materials, and allows you to dispose of waste products of soya lecithin that are not currently used, which allows you to get a much cheaper reagent for definitions of APTT than a reagent from human brain tissue, and a Reanal reagent.

 It should also be noted that there is almost complete absence of a reagent in the medical institutions of the country for the determination of APTT; therefore, such an important test used worldwide to assess the condition of numerous patients with disorders of the first phase of blood coagulation, for preventive studies before all surgical interventions (along with determining the bleeding time and prothrombin time) is not carried out in our country.

Claims (1)

 METHOD FOR PRODUCING A REAGENT FOR DETERMINING AN ACTIVATED PARTIAL THROMBOPLASTIC TIME, including treating the biological material with organic solvents, concentrating the resulting solution and then dissolving the dry residue, characterized in that, in order to increase the sensitivity of the reagent and increase the shelf life of its specific properties, biological materials are used as biological material production of soya lecithin, which are dissolved in chloroform and then treated with ethanol at ootnoshenii biological material, chloroform and ethanol, respectively equal to 1: 8 - 37: 40, then the residue is dissolved in petroleum or diethyl ether, acetone, after drying, the resultant powder was dissolved in 5% sorbitol solution and lyophilized.

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