The method of evaluation of the antioxidant potential of low-density lipoprotein

 

(57) Abstract:

The invention relates to clinical biochemistry. The method consists in obtaining LDL from the blood serum by precipitation in the presence of heparin (200 U/ml serum) and manganese chloride (50 mm/ml serum) at 0oWith, washing the precipitated LDL 0,9% solution of sodium chloride, dissolve them in 1 ml of 1 M solution of sodium chloride, measuring them in concentration of protein in the conventional method of Lowry, after which 0.25 ml LDL add 0.5 ml of ethanol, shake, add 0.25 ml of ascorbic acid and 0.5 ml of 10 N KOH (for saponification of samples), then the sample is incubated in a water bath at 70oC for 40 min, cooled, add 2 ml of hexane, thoroughly shaken for 3 min, the upper hexane layer is transferred to other tubes and then determine the concentration of vitamins E and a spectrofluorimetric method. The wavelength for retinol is Ex 340, Em 490 nm for a-tocopherol - Ex 290, Em 334 nm. The results are expressed for vitamin E in mg/mg LDL protein for vitamin a in g/mg LDL protein. The invention provides an informative, easy way, takes a short time study and can be used in clinical biochemical laborte vitamins, and aims to evaluate the antioxidant potential of low-density lipoprotein (LDL) and, thus, the degree of protection against oxidative processes.

According to modern concepts in the pathogenesis of atherosclerosis and many other diseases plays a key role violation of the balance of oxidation and antioxidant system of blood, primarily LDL. Oxidized LDL acquire atherogenic properties, they actively captured by macrophages, which are transformed into foam cells morphological marker of the atherosclerotic process. The degree of oxidation of LDL is determined by determination of the concentration of products of lipid peroxidation. In addition, there are evaluation methods "predisposition" LDL to oxidative modification. One of these is a method for estimating the resistance of LDL to oxidation. The index of stability of LDL to oxidation reflects, on the one hand, the number peroxidation in LDL (peroxide lipids, dieny, oxysterol, malonic dialdehyde and others) and, on the other hand, their antioxidant potential (content in LDL endogenous antioxidants). Of the endogenous antioxidants in LDL the most important is vitamin E (-tocopherol). This Antietam. Vitamin E is first consumed in the oxidation of LDL, and plays a key role in protecting LDL from oxidative modification. Vitamin a (retinol) is found in LDL in much smaller quantity, but also prevents them from oxidative modification. Thus, the concentration of fat-soluble antioxidants in LDL (vitamins E, a and others) it is possible to judge the degree of protection of LDL against oxidation.

Today little known easy methods for assessment of concentrations of fat-soluble vitamins in LDL, and existing methods are very time-consuming and, generally, are combined with a modification of the methods of assessing the concentrations of antioxidants in the serum. They therefore regard to the present stage of research in the field of pathogenesis of atherosclerosis and other diseases seem outdated and less informative. Thus, the method is often used in studies of the concentrations of fat-soluble antioxidants in the serum using liquid chromatography high pressure [1], which is a lengthy, time-consuming and requires expensive equipment. If this method is used to LDL, the latter are usually identified by the method of ultracentrifugation in Arkhangelsk institutions [2].

On the other hand, the known rapid method for isolation of LDL from the blood serum by precipitation with heparin [2, 3], in which LDL from the blood serum are precipitated in the presence of heparin (200 U/ml serum) and manganese chloride (50 mm/ml serum), mix, leave for 30 minutes at 0oC, centrifuged for 30 minutes at 1500 rpm at 0oS, after which the precipitate contains precipitiously LDL.

The prototype (the basis of) the claimed invention was a method of study the concentration of vitamins (a-tocopherol) and A (retinol) in the serum, where to 0.25 ml serum add 0.5 ml of ethanol, shake, add 0.25 ml of ascorbic acid and 0.5 ml of 10 N KOH (for saponification of samples), then the sample is incubated in a water bath at 70oC for 40 minutes, cool, add 2 ml of hexane, thoroughly shaken for 3 minutes, the upper hexane layer is transferred to other tubes and then determine the concentration of vitamins E and a spectrofluorimetric method. The wavelength for retinol is Ex 340, Em 490 nm, for tocopherol - Ex 290, Em 334 nm [4].

The disadvantage of this method is the low information content and not enough objective assessment of the status of the antioxidant system SAE indicated above, less modern and informative in comparison with those in LDL. In addition, LDL and transport are a form of fat-soluble vitamins. Together, these arguments suggest the disadvantages of this method as applied to the assessment of oxidative-antioxidative system LDL.

The inventive method differs from the known fact that instead of blood serum concentrations of fat-soluble vitamins E and a are measured directly in the LDL. LDL produce quick heparin-precipitating method by adding 1 ml serum 200 IU of heparin (from the Pharmacopoeia of a solution containing 5000 UNITS/ml), mixing, add 75 l of 1 M solution of chloride of manganese, stirring. The sample was left for 30 minutes at 0oC, centrifuged at 0oWith 30 minutes, the precipitate washed with 1 ml of 0.9% solution of sodium chloride, centrifuged, precipitated LDL dissolved in 1 ml of 1 M solution of sodium chloride. In the resulting measure LDL protein according to the conventional method of Lowry and co-authors (1951) and 0.25 ml LDL determine the concentration of vitamins E and a in the above spectrofluorimetric method [3]. The results are expressed for vitamin E in mg/mg LDL protein for vitamin a in g/mg LDL protein.

The advantage of the oxidant system LDL, as-tocopherol and retinol are the main fat-soluble anioxidants these particles, preventing oxidative changes in these particles.

A comparative study between the known method [4] to determine the concentration of vitamins E and a in the serum and the proposed method for determining the concentrations of vitamins E and a in LDL obtained by precipitation with heparin, from the same sera in 68 men. Thirty-eight of them had disorders of lipid metabolism - of dyslipoproteinemia type II (AGP), and 30 were normolipidemia. In addition, all these men had studied the rate of resistance of LDL to oxidation in vitro. The following results were obtained. In men with AGP as the resistance of LDL to oxidation and the concentration of vitamins E and a in the serum were significantly (p<0,01) reduced ((10,30,7) and (0,550,05) g/ml serum, respectively) compared with men without AGP ((13,010,8) and (0,690,07) g/ml serum, respectively). We investigated the concentrations of vitamins E and directly in LDL (the claimed method) in these same patients. It turned out that men with AGP levels of vitamins E and a in LDL were also lower (p<0.05) and was (1,350,22) mg/mg LDL protein and (56,79,9) g/mg is respectively).

In addition, this study found a strong positive correlation (r= +0,79, p<0,05) between the known and the claimed methods of determination of fat-soluble antioxidants (vitamins E and a). Thus, the application of both methods reveals a significant decrease in the concentration of vitamins E and a in the serum and LDL in patients with AGP compared with men with normolipidemia. Obtained using the proposed method, the data are consistent with the index of stability of LDL to oxidation in men surveyed and do not contradict the data of world and national literature. Indeed, according to the performance of the proposed method, the concentration of-tocopherol in LDL significantly higher (26-28 times) than retinol, which indicates the predominance of vitamin E in LDL as the primary protective factor against their oxidation.

Thus, the inventive method is informative and diagnostically significant, as it allows to determine the concentrations of vitamins E and a themselves directly in LDL, that is, to assess the antioxidant capacity of LDL.

The advantages of this method are the relative ease of technical execution, as the work is carried out by one in the 4 hours.

The method can be used in clinical biochemical laboratories to assess the antioxidant capacity of LDL by determining the concentrations of fat-soluble vitamins E and a in LDL.

Sources taken into account:

1. Milne, D. B., Bottnen J. Retinol, -tocopherol, lycopene and-carotene simultaneously determined in plasma by liquid chromatography. // Clin. Chem. -1986. - V. 32. -P. 874-876.

2. Lindgren F. T., A. Silvers, Jutagir R. et al. A comparison of simplified methods for lipoprotein quantification using the analitic ultracentrifuge as a standart //Lipids - 1977.- V. 12, N 3.- R. 278-282.

3. Burstein M., Scholnik H. R. Lipoprotein-polyanion-metal interactions // Adv. Lipid Res. - 1973. - V. 11.- P. 63-108.

4. Taylor S. L., Lamden, M. P., Tappel A. L. Sensitive fluorometric method for tissue tocopherol analysis. //Lipids - 1976. - V. 11. - No. 7. - R. 530-538.

The method of evaluation of the antioxidant potential of low-density lipoprotein (LDL) by determining the concentration of vitamins E and directly in LDL, which includes adding to 0.25 ml of the analyzed biological sample 0.5 ml of ethanol, shake, then add 0.25 ml of ascorbic acid and 0.5 ml of 10 N KOH, incubation of the sample in a water bath at 70oC for 40 min, cool down, add 2 ml of hexane, thorough shaking for 3 min, fence upper hexane layer, transferring it to other tubes and then determine whether content a biological sample using LDL, selected heparinisation method in the presence of manganese chloride, centrifuged, washed with 0.9% solution of sodium chloride, dissolved in 0.5 ml of 1 M solution of sodium chloride and measured them with protein concentration.

 

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