Method for biochemical detecting the degree of chronic hepatitis activity

FIELD: medicine, biochemistry.

SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.

EFFECT: higher accuracy of detection.

3 ex

 

The invention relates to medicine, namely biochemistry, and can be used, in particular, to determine the degree of activity of chronic hepatitis.

In practical medicine known methods for determining the activity of chronic hepatitis based on clinical data (Grigoriev PA, Yakovenko EP Diagnosis and treatment of diseases of the digestive system. - M.: Medicine, 1996. - 515 S.; W. Sherlock, J. Dooley. Diseases of the liver and biliary tract. - M.: GEOTAR, 1999. - 859 S.).

The disadvantages of these methods are:

the high degree of subjectivity;

not enough good reproducibility.

The closest method to the present invention is a method for determining the activity of chronic hepatitis, based on the assessment of the concentration of bile acids serum (Axelson M, Mork Century, Aly A., Waldivs G. Concentration of bile acids in plasma from patients with liver disease // J. Lipid. Res. - 1999. - Vol.40, No. 6. - P.1788-1792).

This method has the following disadvantages: the method is not sufficiently accurate, as it has a fairly wide “grey zone”, within which the accuracy of the interpretation of serum bile acids to determine the degree of activity of chronic hepatitis low.

The aim of the presented invention is to improve the accuracy of determining the degree of activity of chronic hepatitis.

The goal is to image the shadow is achieved by what in the serum determine the level of lactoferrin and bile acids and their ratio equal 5-17 define chronic hepatitis high activity.

Isolated determination of bile acids in serum in some cases does not allow us to diagnose chronic hepatitis high degree of activity, as high levels of bile acids is characteristic of cirrhosis of the liver (Grigoriev PA, Yakovenko AP, 1996). The definition only lactoferrin in this case also is not informative, because the lower level of this protein is observed in a number of pathological conditions (Nikolaev A.A., 1994). Simultaneous determination of serum levels of lactoferrin and level of bile acids with subsequent determination of the ratio between them can significantly increase the efficiency of the method for determining chronic hepatitis high degree of activity.

Thus, the advantage of the proposed method in higher diagnostic accuracy in the determination of chronic hepatitis high degree of activity.

The positive effect of the proposed method achieved an accuracy assessment of serum levels of lactoferrin and bile acids.

The proposed method was successfully tested in the first Alexander Mariinsky Regional clinical hospital from January 2001 to January 200, 128 patients with chronic diffuse liver diseases.

Below are the results of testing.

Example 1.

Sick And in So M, 39 years old (case history No. 15947), was in the clinic with 3.10.2002 on 29.10.2002, he started with the following complaints: dull pain in the right hypochondrium, poor appetite, weight loss, weakness, itchy skin.

Objectively: the patient is in satisfactory condition, the skin with a yellowish shade, sclera subcarina, telangiectasias on the face, neck, chest, Palmar erythema. The lungs and heart without features. The abdomen is soft, slightly painful in the right hypochondrium. The size of the liver by karlovu: 13-11-8 cm, palpation edge of the soft, rounded. Spleen palpation and percutere not increased.

To determine the level of bile acids serum was used enzymatic method.

The method is based on the enzymatic oxidation reaction of 3-alpha-hydroxy group of bile acids in the oxo group under the action of the enzyme 3-alpha hydroxysteroiddehydrogenase and further transfer of the hydrogen atom using the enzyme diaphorase and OVER on nicrosini-tetrazole with the formation of colored products. The colour intensity is directly proportional to the concentration of bile acids in serum. The scheme of the reaction is as follows:

1) Sample 20 ál of serum or standard solution of bile acids are placed in a test tube.

2) Add Reagent a from a set of 0.5 m is.

3) Exposure at 37°C for 5 minutes.

4) Add Reagent B from a set of 0.5 ml.

5) Exposure at 37°C for 15 minutes.

6) the Addition of the stop reagent and 0.2 ml.

7) Colorimetric detection at 530 nm.

In parallel with the analysis of samples conducted a study of standard dilutions of bile acids to construct the calibration curves.

The level of bile acids in the serum was of 113.2 mmol/L.

The level of lactoferrin serum was determined by enzyme-linked immunosorbent assay.

In the present work, we used enzyme-linked immunosorbent assay, when the insoluble carrier of adsorbed protein (antigen or antibody) and then all of immunochemical interactions occur on the media.

As a solid phase, we used 96-well plates firms “Linbro”. The wells were made with 200 µl of specific antibodies (2-5 μg), were incubated for 16-20 hours and washed using buffered physiological solution (pH of 7.2)containing 0.05% tween-20. After washing, the wells were made in 200 μl of serial dilutions of the standard antigen, usually containing from 0.1 to 200 ng/ml protein, analyzed samples and detergent solution (control).

After incubation and washing was brought to 200 μl of the antibody to the investigated antigen covalently linked to the enzyme (conjugate) in dilution, which was set empirically. After the incubatee and washing was performed enzymatic reaction and spectrophotometry. The calibration graph was built in each experience. The sensitivity of the method in our work was 6 ng/ml.

Conjugation of antibodies with the enzyme spent periodate oxidation. Dissolve 5 mg of peroxidase-6 (Sigma) in 1 ml of distilled water, was added 2 ml of freshly prepared 0.1 M solution of periodate sodium and was stirred for 30-40 minutes at room temperature. Aldehyde peroxidase solution were dialyzed against 10 ml of carbonate buffer (pH 9,5) was added 10 mg of specific antibody, dissolved in the same buffer, and then stirred at room temperature for 2-3 hours. To the mixture was added 0.1 ml of 0.4% solution of sodium borohydride and left for 2 hours at room temperature. For stabilization of the conjugate was added to bovine serum albumin to 1%glycerol to 40% and stored at -20°C until use.

Serum levels of lactoferrin was 917,2 ng/ml.

The ratio was calculated by the formula:

the level of lactoferrin (ng/ml)/level of bile acids (µmol/ml).

The ratio was 917,2/of 113.2=8,1. As of 8.1 is included in the range 5-17, a patient diagnosed with chronic hepatitis high degree of activity.

Held further clinical and instrumental examination, including x-ray, ultrasound and other methods of research has completely verific is encoded diagnosed.

Example 2.

Patient M in centuries, 22 years old (case history No. 7539), was in the clinic with 8.02.2002 on 27.02.2002, was Admitted with complaints of periodic aching pain in the right hypochondrium. Patients consider themselves to 1999, when he suffered acute viral hepatitis “B”.

Objectively: the patient is in satisfactory condition, the skin is a normal color. The lungs and heart without features. The abdomen is soft, with moderately painful palpation in the right upper quadrant and epigastrium. The size of the liver by karlovu: 9-8-7 cm, palpation edge of the soft, rounded. Spleen palpation and percutere not increased.

In viral marker range: HbsAg (+), HbeAg (-), aHCV (+), aHDV (-).

The level of bile acids in serum, determined by the method described above (see example 1), 37.7 μmol/ml, and the level of lactoferrin, determined by ELISA (see example 1), was 1690,0 ng/ml.

The ratio was 1690,0/37,7=44,8. As 44,8 not included in the range 5-17, then the patient at diagnosis with a high degree of activity was excluded. Diagnosis: chronic hepatitis coinfection with viral etiology (b+C), the minimum degree of activity.

Held further clinical and instrumental examination, including x-ray, ultrasound and other methods of research, allowed us to verify the diagnosis.

Example .

Patient W and O. D., 52 years old (case history No. 13471), was in the clinic with 21.08.2001 on 22.09.2001, Complaints: dull pain in the right hypochondrium, skin itching, weakness, fatigue, drowsiness in the daytime, the increase in abdominal volume, nausea, dry mouth, gingival, nasal bleeding, hemorragie on the skin, cramps in the extremities, arthralgia, tremor of the hands, head, swollen feet, a small amount of urine.

Objectively: the state of heavy, lethargic, skin yellowish color, with traces of scratches, telangiectasias on the body, hemorrhages on the limbs, body, sclera subcarina, “liver” language, Palmar erythema. Edema of the lower extremities. The lungs and heart without features. Belly increased in volume at the expense of free fluid in the abdomen, painful to palpation in the right upper quadrant. The size of the liver by karlovu: 10-7-7 cm, palpation region of dense, uneven. Palpated the lower pole of the spleen on the inhale.

The level of bile acids in serum, determined by the method described above (see example 1), was 98,3 µmol/L. the Level of lactoferrin, determined by ELISA (see example 1) was 302,1 ng/ml.

The ratio was 302,1/98,3=3,07. Because of 3.07 is not in the range 5-17, then the patient at diagnosis hepatitis high degree of activity was excluded. Diagnosis: cirrhosis of the liver.

The course is built in the further clinical and instrumental study including x-ray, ultrasound and other methods of research, allowed us to verify the diagnosis.

The proposed method achieved a positive effect: increases the accuracy of determining the degree of activity of chronic hepatitis. This allows to prescribe an adequate treatment, which increases its efficiency, and therefore, improves the prognosis for the patient.

The proposed method can be used in clinical practice to determine the degree of activity of chronic hepatitis.

The biochemical method of determining the degree of activity of chronic hepatitis by assessing the level of bile acids in the serum, characterized in that the serum determine the level of lactoferrin and bile acids and their ratio equal 5-17 define chronic hepatitis high activity.



 

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