RAOULIA-CRIEPI METHOD (Y. Raoul, O. Crepy) — a quantitative method of definition of niacin and its amide (Niacinum, PP vitamin) in blood. River — To. the m is used for control of concentration niacin (see) and its amide in blood of the patients accepting nicotinic to - that and niacinamide as medicine. The method is offered in 1937
to Priptsip R. — To. the m is that Niacinum forms at interaction with cyanic bromine and n-aminofenilsulfonamidom (reaction of Keniga) the connection painted in yellowish-orange color; intensity of coloring of solution is proportional to concentration of Niacinum in the analyzed sample and can be measured photometric.
Course of definition
1. Hemolysis and sedimentation: add 35 ml of a distilled water to 5 ml of blood, stir up, place for 1 hour in ice or the refrigerator at — 10 °, then carefully heat to receiving homogeneous liquid and leave for 1 hour at the room temperature then add 10 ml of 5% of solution of aluminum sulfate, stir up, leave to stand 30 min., add 1 g of calcium carbonate in powder, stir up, lumps make small a glass rod, then boil until foam, caused by release of carbon dioxide gas, does not reach a neck of a flask; filter, precisely measure the volume of a filtrate.
2. Hydrolysis and neutralization: the filtrate is placed in a porcelain casserole and evaporated on the boiling water bath up to the volume of 10 ml, filtered, add 10 ml to a filtrate concentrated salt to - you and boiled within two hours; after cooling liquid is transferred to a volumetric flask on 25 ml, add 1 — 2 drop bromtimolovy blue (0,04% spirit solution) and neutralize 20% solution of caustic soda till greenish color that corresponds to a pH value 6,8, then add 1,5 ml of phosphatic buffer pH 6,8 solution and added the bidistilled water to a tag then solution is filtered.
3. Reaction of Keniga: place 5 ml of the neutralized solution in three test tubes (pilot, single and standard tests). Add 0,2 ml (10 mkg) of the diluted standard solution to the third test tube nicotinic to - you (for its preparation dissolve 100 mg nicotinic to - you in 100 ml of a distilled water, before the use part 50 mkg/ml to concentration), add 0,2 ml of a distilled water to two other test tubes; all three test tubes immerse in the water bath at 80 ° for several minutes, then in the first and third test tubes add 1 ml of the cyanic bromine prepared just before the use by addition to bromic water (saturated solution) of 10% of solution of potassium cyanide on drops before decolouration to the 2nd test tube add 1 ml of a distilled water; all three test tubes immerse for 2 min. in the water bath at 80 °, cool under a water jet and leave for 15 min. in the dark; add 0,5 ml of the mix consisting of 3 ml of 10% of n-aminofenilsulfonamida solution in 6% salt to each test tube - those and 2 ml of 50% salt to - you. Kolo-rimetrirut on the photoelectrocolorimeter at 470 nanometers.
Calculation of maintenance of Niacinum in the studied sample is carried out on a formula:
[(E op — E x ) / (E St - E op )] *10 (mkg)
where E op — extinction of pilot test; E x — extinction of single test, E St — extinction of standard test; 10 — contents nicotinic to - you in standard test.
The maintenance of Niacinum in whole blood defined by R. — To. the m, makes normal 0,6 — 0,8 mg / 100 ml.
See also Biochemical methods of a research .
Bibliography: Dzhordzhesku P. and Peunesku E. Biochemical methods of the diagnosis and a research, the lane from Romanians., page 356, Bucharest, 1963; Raoul Y. Dosage de l’azote s^rique n’appartenant ni aux proteides complexes ni aux composes azotes de bas poids moleculaire, Bull. Soc. Chim. biol. (Paris), t. 19, p. 1361, 1937.
L. M. Pimenova.