BESSEY METHODS (The lake of A. Bessey) — quantitative methods of determination of content of vitamin A and carotinoids in blood serum, vitamin C — in leukocytes, and also activity of alkaline and acid phosphatases in blood serum.
Definition of vitamin A and carotinoids in blood serum
Definition of vitamin A and carotinoids in blood serum (in A. A. Anisova's modification). The method is based on alkaline hydrolysis of ethers of vitamin, extraction of vitamin A and carotene with the subsequent spektrofotometrichesky measurement of light absorption by solution before destruction of vitamin A ultraviolet rays.
Normal the content of vitamin A in blood serum fluctuates from 30 to 70 mkg of %, and carotene — from 80 to 230 mkg of %. Decrease in content of vitamin A in blood is described at a hyperthyroidism, a peptic ulcer of a stomach and a duodenum. It is combined with the normal or a little increased level of carotene (usually the lower the level of vitamin A, the is higher the level of carotene in blood). This carotenemia, on a nek-eye to the data, is connected with involvement in pathological process of a liver and is the sign indicating disease severity. At a serious general illness (cancer, tuberculosis, infections) ability of a liver to deposit vitamin A is broken, permeability of kidneys increases and already available hypovitaminosis amplifies.
Content of polyneuramin of blood goes down also at parenchymatous damages of a liver and does not change at obturatsionny jaundice. Increase in level of vitamin A in blood usually takes place at a hypothyroidism, Itsenko's diseases — Cushing and some other states.
Technology of definition. Take 1 ml of blood in a centrifugal test tube (from a finger) and centrifuge 10 min. at 3000 rpm; 0,12 ml of blood serum transfer to a test tube, add the same number of 1 N of spirit caustic heat, mix and put for hydrolysis in the water bath for 20 min. at t ° 60 °. Test tubes cool within 5 — 10 min. (on ice), add to everyone 0,12 ml of xy-lol-ligroin or xylol-octa-new mix (1: 1) for extraction of vitamin. Then test tubes cool again and centrifuge 8 — 10 min. The top coat (a xylol-ligroino-vy or a xylol - the octane extract containing vitamin A and carotinoids) is transferred to a quartz mikrokyu-veta; in control to a microditch pour pure a xylol - ligroin or a xylol - octane mix. Definition of carotinoids is carried out at the wavelength of 460 nanometers, definition of vitamin A — by double measurement of optical density of solution at the wavelength of 328 nanometers (before and after radiation of tests by ultraviolet rays). The studied tests irradiate with an ultraviolet light with wavelength 310 — 410 nanometers within 45 — 60 min.
Calculation. The maintenance of carotinoids is calculated on a formula:
X = 480·E460,
where X — the required maintenance of carotinoids in microgram-percent; 480 — Bessey's coefficient for carotene; E460 — the optical density of solution of carotene.
Content of vitamin A is calculated on a formula:
X = 637 · (E1 — E2),
where X — required size (in mik-rogramm-percent); 637 — Bessey's coefficient for vitamin A; E1 — the optical density of solution of vitamin A before radiation, and E2 — after radiation.
See also Retinolum .
Definition of redoxon in leukocytes
Definition of redoxon in leukocytes (in A. A. Anisova's modification). The method is based on allocation of leukocytes and extraction of redoxon from them with the subsequent spektrofotometrichesky determination of content of the last.
Normal leukocytes contain from 20 to 30 mg of % ascorbic to - you.
At assessment of the received values ascorbic to - you it is necessary to consider that decrease in its contents in leukocytes at insufficient receipt with food occurs later, than in urine and a blood plasma. The high level of redoxon in leukocytes remains at contents it in blood not lower than 0,2 mg of %. Clinical value of redoxon — see. Ascorbic acid .
Technology of definition. From 0,5 ml of 3,8% of solution of sodium citrate bring 0,2 ml of blood in a conic test tube (from a finger), mix and put for 5 — 7 min. on cold. Then centrifuge during 1,5 — 2 hours at 150 — 200 rpm. The Pasterovsky pipette carefully suck away the top liquid (muddy) coat containing a suspension of elements of blood, transfer to other test tube and centrifuge 10 min. at 2500 — 3000 rpm. Nadosadochny liquid is deleted, and to the deposit consisting of leukocytes add 0,04 ml of 5% of solution trichloroacetic to - you and frozen. In 5 — 7 min. the test tube is taken out, defrozen in a stream of a cold water (for destruction of a stroma of leukocytes and fuller extraction of redoxon) and centrifuged 10 min. at 2500 — 3000 rpm. Select a micropipet 0,03 ml of the solution containing redoxon add 0,01 ml of the combined reactant (100 volume parts of 2,2% of solution of dinitrophenylhydrazine in 10 N solution a chamois to - you, 5 parts of 5% of aqueous solution of thiourea and 5 parts of 0,6% of CuSo solution 4 · 5H 2 O in 0,1 N solution a chamois to - you). The test tube (closed by a ground stopper) is placed for 4 hours in the thermostat at t ° 38 °, then bring 0,05 ml of the solution cooled 65% in a test tube a chamois to - you and in 20 min. fotometrirut on the spectrophotometer at the wavelength of 520 nanometers.
Calculation of contents ascorbic to - you make according to the standard curves made for pure ascorbic to - you.
Definition of activity of phosphatases in blood serum
Definition of activity of alkaline (K.F.188.8.131.52) and acid (K. F. 184.108.40.206) phosphatases in blood serum [across Bessey — Lauri — to Brock (O. of A. Bessey, O. N. of Lowry, M. of J. Brock, 1946)]. Ability of phosphatases to hydrolyze radio communication in p-nitrophenyl phosphate, with release of the p-nitrophenol giving yellow coloring in alkaline condition is the basis for a method. Intensity of the last characterizes a degree of activity of enzymes.
Definition of activity of phosphatases is successfully applied to differential diagnosis of diseases of a liver, bone system and a prostate. Substantial increase of activity of an alkaline phosphatase in cases of a liver is noted at mechanical jaundices, at acute hepatitises, cholangites, cirrhoses and especially at a yellow hepatatrophia. The moderate giperfosfatazemiya is one of early satellites of metastasises of malignant new growths. Increase in fosfatazny activity is characteristic of rickets, and the degree of activity usually corresponds to disease severity. Substantial increase of activity of an alkaline phosphatase is found at osteomalacy, at Pedzhet's disease, at a hyper parathyroidism and other diseases of bone system.
Definition of activity of acid phosphatase in blood serum is used for diagnosis of diseases of a prostate, and in particular cancer when there is a sharp increase in its activity. Besides, on change of activity of acid phosphatase it is possible to judge efficiency of therapeutic use of estrogen.
Technology of definition. Bring 1 ml of the corresponding substrate buffered solutions in test tubes (0,05 M the glycine buffer and 5,5·10 - 3 M solution of p-nitrophenyl phosphate, pH 10,5 — for an alkaline phosphatase and 0,05 M the citrate buffer, 5,5·10 - 3 M solution p-nitrofe-nilfosfata, pH 4,8 — for acid phosphatase). Then contents of test tubes are warmed up in the thermostat of 5 — 10 min. at t ° 37 °; in pilot test tubes add serum (to test on an alkaline phosphatase of 0,1 ml, on acid 0,2 ml), 30 min. at t 37 mix and incubate °. After an incubation in test on an alkaline phosphatase add 10 ml 0,02 N of caustic liquor, and to test on acid phosphatase — 4 ml 0,1 N of NaOH solution; mix and fotokolorimetrirut at the wavelength of 405 nanometers (390 — 420 nanometers) in a ditch with thickness of a layer of 1 cm against the corresponding control. Control is put for each serum. Calculation. Activity of enzyme is expressed or quantity of the micromoths of p-nitrophenol released under the influence of 1 ml of blood serum in 1 min. at t ° 37 ° or in conventional units (the amount of enzyme, a cut releases 0,05 micromoths of p-nitrophenol in experimental conditions).
See also Phosphatases .
Bibliography: Biochemistry and physiology of vitamins, Methods of definition of vitamins, the lane with English, under the editorship of H. M. Sisakyana, etc., Saturday. 4, M., 1951; Biochemical methods of a research in clinic, under the editorship of A. A. Pokrovsky, page 162, etc., M., 1969.
V. Ya. Maximov.