SU1208511A1 - Method of determining hydrophobic compounds in biological fluids - Google Patents

Description

The invention relates to biochemistry and medicine and can be used to determine the hydrophobic compounds in biological fluids, for example, when making control of the transformation and accumulation in the human body of drugs for their accumulation in biological fluids.

The purpose of the invention is to increase the detection sensitivity.

Example K First, bacterial luciferase is obtained as follows.

The culture of Photobacterium fischeri No. 6 tiry, growing on solid medium of the next comp; ava5 wt.%: sea water 38, peptone; m with peptone broth 8; agar-agar 2; distilled water the rest, sown in a liquid medium composition, wt.%: 0,75 O, 1; (NH) jHP04 0.05; 0, G1 | peptone 1; NaC1 28; distilled water else, pH 7.4; after 11 hours, 200 ml of the culture was sterile transferred to a 10 liter fermenter with the same medium and fermented for 9 hours while controlling and maintaining the pH of the medium (7.0) and the level of dissolved oxygen (10% of saturation) constant 22 C. At the end of the fermentation, the cells are separated from the culture fluid.

by centrifugation at 25,000 x

j

X 30 min and destroy the lysis in potassium-sodium phosphate buffer 0.02 M, pH 7.0 with dithiothreitol using Triton X-100 as a detergent in the amount of 0.5% by weight of the cells for 40 min at 17 ° C followed by sonication 22 kHz x 2 min. (3 0.6 A, device UDZ-l). The buffer is added in a ratio of 4: 1 (volume / weight of the cells). Large particles are removed by centrifugation at 105,000 x 1 h. Fractional precipitation of proteins with ammonium sulfate in the range of 40–75% of saturation is carried out. The precipitate is dissolved in 50 MP ./Na - phosphate buffer (composition is the same), applied to a Sephadex X 10 column for rapid dialysis, then the active fraction is applied to a Sephadex o -100 column to remove low-grade ballast and - high molecular weight compounds. The resulting drug lucife

five

The times are converted to volatile buffer triethylamine-HC 1 0.01 M, pH 8.8 and then lyophilized. The final product luciferase has a purification degree 120 times its activity with nicotinamide adenine dinucleotide and a specific activity of 2.010 quantum / s mg protein. It is stable when stored for two years.

In a patient, mixed saliva is sampled and the effect of saliva on the enzymatic activity is determined. The luminescence intensity of a 1 ml sample was measured on a photometer (

but"

control / which you received0

five

0

five

0

five

they are dbbavleniem 0.3 mp water x 0.7 mp 0.02 M potassium sodium phosphate buO

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Phera, pH 7.0, containing 5 10 M octanal, 2 IO-M reduced nicotinamide adenine dinulkeotide (NADH), 5 -10 M flavin mononucleotide (FMN) and 0.1 mg / ml luciferase, B to the experimental sample instead of water, add 0.3 ml of mixed saliva, the value of P, experience. Aminopirine is administered to the patient at a dose of 0.15 mg / kg of body weight and after 2.4, 20 and 24 hours the luminous intensity is measured. The magnitude of the inhibitory i effect is estimated by

 Formula X is 100%.

. The data are given in table.1.

As can be seen from Table 1, after taking aminopyrin, accumulation of the drug is observed in the saliva and the inhibitory effect increases. The degree of utilization of the drug can be monitored by reducing the amount of inhibition of luminescence.

Example2. In a patient, mixed saliva is sampled before and after the administration of 0.5 g / kg of ethazol weight, and the effect of 0.03 ml of mixed saliva on the luminescence of the enzyme system containing 0.02 M potassium sodium phosphate buffer is measured. Octanal, 10 MNADH, 0.02 mg / ml luciferase and 10 M FMN (final sample volume 1 ml.

The evaluation of the inhibitory effect is carried out as in Example 1.

The results of the analysis of the effect of the drug are given in Table 2.

As can be seen from Table 2, the introduction of etazol leads to an increase in the inhibition of the enzymatic luminescent system by samples of mixed

saliva. From these data, effective transformation of the drug compound in the human body is visible.

An example of an RHL. A patient takes a sample of mixed saliva before and after the administration of 0.5 g of etazole, and the effect of 0.2 ml of mixed saliva is measured.

l mV

% inhibition

420

I, mV% inhibition

932 177 251 326 484 783 81 73 65 48 16

VNIIPI Order 284/56

Lishal PSh Patent, Uzhgorod, Proektna St., 4.

114

on the luminescence of the enzyme system (final volume 1 mp) containing 5 octanal, 5 NDCN, 0.4 mg / ml luciferase and 5 10 M FMN. The evaluation of the inhibitory effect is carried out as in Example 1.

The results of the analysis of the action of ethanol are given in Table 3.

Table 1

222,285,386,411

47

32

Circulation 778 Subscription

Claims (1)

METHOD FOR DETERMINING hydrophobic compounds in biological fluids by addition of sample to the medium containing the bacterial luciferase, flavin mononucleotide, aldehyde, and phosphate buffer 'subsequent registration decrease luminescence, characterized in that, in order to improve sensitivity, as the aldehyde using ^octanal? at a concentration of 10 ^S -5 · 10 M, the incubation medium is further added in a concentration of reduced nicotinamide adenine dinucleotide -5'10 Yu M, and luciferase and flavin mononucleotide used in concentrations 0,020,4 mg / ml and 1O '-5 ^s - 10 ~ ⁵ M respectively.