BERCHA-BESSEYA-LAURI METHOD (N of V. of Burch, O. of A. Bessey, O. N. Lowry) — a method of quantitative definition of Riboflavinum (vitamin B 2 ) in blood.
The method is based on extraction of various forms of Riboflavinum from erythrocytes and whole blood, the subsequent acid hydrolysis of a flavinadenindinukleotid (FAD) and flyuorimetrichesky determination of the general content of Riboflavinum.
Value of definition of vitamin B 2 and its physiological role in an organism — see. Riboflavinum .
2 — 2,5 ml of oxalic blood centrifuge definition of Riboflavinum in erythrocytes at 1500 — 2000 rpm within 1 hour. The liquid layer together with a small amount of a deposit is sucked away, and the suspension which remained in a test tube is centrifuged within 1 hour at t ° 4 ° again. The liquid layer is merged, 0,5 ml of erythrocytes transfer a pipette to other test tube; the pipette is rinsed by 1% NaCl solution (0,5 ml) which is merged in a centrifugal test tube, add 9 ml of 10% of solution trichloroacetic to - you, mix and leave in the dark for 30 — 60 min. Then contents are mixed again and centrifuged at 3000 rpm within 10 min. Tsentrifugat transfer to a test tube with a ground stopper, placed in the thermostat at t ° 37 ° on 20 hours for hydrolysis of FAD, then neutralized (8 ml of a gidrolivat transfer to the test tube containing 2 ml 4M of solution K 2 HPO 4 , mix and leave on 1 — 2 hour). After neutralization solution is poured out in a ditch of a flyuorimetr and measure intensity of fluorescence with primary light filter having a maximum of absorption apprx. 420 nanometers and secondary — a maximum of absorption apprx. 560 nanometers then solution is merged in a test tube again, add 0,1 ml of standard working solution of Riboflavinum (20 mkg of Riboflavinum to 1 ml) and measure intensity of fluorescence, add 0,5 ml of 10% of solution of hydrosulphite (for clearing of fluorescence) and make the third measurement.
Calculation is made on a formula:
where And — amount of Riboflavinum in 1 ml of standard solution (in mkg), B — the volume of the analyzed test (in ml), R 1 — first measurement of intensity of fluorescence; R 2 — second measurement and R 3 — third measurement.
Normal the content of Riboflavinum in erythrocytes makes apprx. 20 mkg of %.
1 ml of oxalic blood directly after capture bring determination of the general content of Riboflavinum in whole blood in a centrifugal test tube, add 9 ml of 10% of solution trichloroacetic to - you, mix and leave in the dark for 60 min. Then centrifuge at 3000 rpm. Nadosadochny (transparent) liquid is placed in the thermostat at t°37 — 38 ° on 20 hours for hydrolysis then neutralize (add 1 ml to 4 ml of a hydrolyzate 4M of K2HPO4 solution and leave in the dark on 1 — 2 hour). Intensity of fluorescence is measured in the way described above.
Content of Riboflavinum in whole blood normal makes apprx. 12 mkg of %.
Definition of various fractions of Riboflavinum to blood
the First analysis stage — extraction of Riboflavinum of 10% by solution trichloroacetic to - you — is carried out in the cold (t ° 4 °) to avoid hydrolysis of FAD. After centrifuging take 4 ml of cold extract, immediately neutralize 1 ml 4M of solution K 2 HPO 4 also measure intensity of fluorescence (receive value I).
The remained volume of a tsentrifugat is placed in the thermostat on 20 hours at t°37 — 38 ° for hydrolysis of FAD, cooled, neutralized and define intensity of fluorescence (value II). Calculation of maintenance of FAD is made on a formula:
Division on 0,85 is connected with the fact that value I is formed from intensity of fluorescence of a flavinmononukleotid (FMN), free Riboflavinum and 15% of FAD.
The fraction II more labiln also changes depending on degree of security with Riboflavinum more than FAD.
During the studying of security of an organism with vitamin B 2 it is necessary to consider that determination of content it in erythrocytes is more valuable; content of Riboflavinum in leukocytes is much stabler; it long remains on datum level even at insufficient receipt with food. In blood serum the content of vitamin B 2 so it is not enough that on a usual flyuorimetr it is not defined.
Bibliography: Biochemistry and physiology of vitamins, Methods of definition of vitamins, the lane with English, under the editorship of Η. M. Sisakyana, etc., Saturday. 4, page 113, M., 1951, bibliogr.; Biochemical methods of a research in clinic, under the editorship of A. A. Pokrovsky, page 481, M., 1969; In u of with h H. Century of Fluorimetric assay of cocarboxylase and derivatives, in book: Methods in enzimol., ed. by S. P. Colo-wick a. N. O. Kaplan, v. 3, p. 946, N. Y., 1957.
V. Ya. Maximov.