RU2222811C2 - Method for detecting plasmatic renal clearance - Google Patents

Abstract

FIELD: medicine, nephrology. SUBSTANCE: the present innovation deals with both clinical and experimental trials. One should detect D- xylose concentration due to micromethod 5 min after intravenous injection (P₁), 25 min after injection (P₂), and total plasmatic clearance is detected by the following formula:

, where Y - intravenously injected D-xylose dose; t₁ and t₂ - time intervals to detect D-xylose concentration. The present innovation enables to obtain quantitative characteristics for the state of renal infiltration capacity without measuring urine volume and analysis. EFFECT: higher efficiency of detection. 1 ex

Description

 The invention relates to medicine, namely to nephrology, urology and in clinical and experimental studies, where it is necessary to determine the filtration capacity of the kidneys without measuring and analyzing the composition of urine.

When assessing the "purification" of plasma from any substance (clearance), the decrease in the plasma concentration of the test substance can be of a different nature:
1 - a linear decrease in the concentration of the analyte after a single intravenous administration;
2 - incorrect drop in the concentration level of the analyte with a single intravenous administration
3 - exponential drop. (Shyuk O. Functional studies of the kidneys, 1981; Dederding JP et al. Influence of chronic renal failure on the D-xylose test in man. - Gastroenterol. Clin. Biol., 1988, 12 (6-7), p.542- 547).

 With a linear decrease in the plasma concentration of the test substance per unit time from the corresponding distribution space, the same amount of intravenous substance is lost each time. Plasma clearance is determined by the amount of substance that disappears per unit time from the corresponding distribution space.

This method has the following disadvantages:
- the calculations made in this case are not always accurate;
- the process of determining the distribution space is laborious;
- there is no way to determine the constant of disappearance rate.

 An incorrect drop in the plasma concentration of the test substance is observed, for example, with the introduction of inulin. After intravenous administration, inulin is excreted almost exclusively by the kidneys; therefore, plasma clearance can be calculated for a certain period of time.

This method has the following disadvantages:
- requires maintaining a constant level of inulin in the blood;
- hence, an additional intravenous injection is necessary to ensure the constancy of the level of inulinemia under the mentioned conditions;
- the allocation of inulin is not always adequate to the severity of the pathological process.

 Prototype. The method for determining the plasma clearance after a single intravenous injection of a sterile inulin solution is the closest to our proposed, therefore, it is taken as a prototype (Shyuk O. Functional study of the kidneys, 1981).

 The principle of the prototype method is that after a single intravenous injection of a sterile inulin solution at appropriate intervals, its concentration in the blood is determined. Studies are carried out as follows: first, 0.3 ml of blood is taken (control), then a sterile solution of inulin is administered once intravenously at a rate of 30 mg per kg of weight and blood is taken again at appropriate time intervals after 5, 25, 45, 65 minutes up to the complete disappearance of inulin from the blood.

The calculation is performed according to the formula C _tr = Y / 8, where Y is the intravenously administered dose, S is the space (area) of the distribution of the limited curve of plasma concentrations of inulin in the blood and the X axis. To determine S, the space limited by the plasma concentration curve and the X axis is divided to simple geometric shapes (triangles, trapezoids) that they form. The areas of each figure are calculated and summed.

This method has the following disadvantages:
- one of the conditions is the sampling until the complete disappearance of inulin from the blood, which complicates the study;
- inulin is not exponentially excreted by the kidneys, and the curve for the decrease in plasma concentration is incorrect;
- for some pathological conditions of the kidneys, the results are not always adequate to the severity of the pathological process.

 With an exponential drop, the plasma concentration of the test substance for the same period of time decreases in all cases by the same fraction of the initial concentration.

 The aim of the invention is to obtain a quantitative characteristic of the state of the filtration capacity of the kidneys without measuring the volume and analysis of urine.

SUMMARY OF THE INVENTION
The proposed method is based on the fact that after a single intravenous administration of a solution of D-xylose, the decrease in its plasma concentration is exponential. This makes it possible to determine the rate of disappearance and, accordingly, the plasma clearance. The proposed method is as follows. First, 0.3 ml of blood is taken from a vein for control. Then, a sterile 20% solution of D-xylose is administered intravenously at the rate of 125 mg per kg of body. After 5 minutes and 25 minutes after the administration of D-xylose, 0.3 ml of blood is taken. The concentration of D-xylose in the blood is determined by the micromethod developed by Hasanova P.O. and Shamov I.A., ID 02930 of industry value.

 Micromethod for determining the content of D-xylose in the blood.

 The principle of the method is based on the formation of furfural upon heating of D-xylose in the presence of glacial acetic acid. Furfural with parabromaniline gives a pink color, the intensity of which is determined by FEC or spectrophotometer at a wavelength of 540 nm.

 Reagents: 1) P-bromaniline, 2) glacial acetic acid, x / h, 3) thiourea, 4) trichloroacetic acid (crist.), 5) NaOH (crist.), 6) D-xylose.

Reagent Preparation
1. 2% parabromaniline reagent. Thoroughly dissolve 500 mg of thiourea in 100 ml of glacial acetic acid and add 2 g of p-bromaniline. Store in a dark container, shelf life 1 week.

 2. 16% solution of TCA (trichloroacetic acid). Weigh 8 g of TCA and place in a 50 ml flask and add distilled water to the mark.

 3. 1% NaOH solution. Weigh 2 g of NaOH into a 50 ml volumetric flask and add distilled water to the mark. Store in a cool place, shelf life 1 week.

 4. Standard solution 100 g / l (0.1 mg / ml). Weigh 10 mg of D-xylose, place in a 100 mg flask and pour distilled water to the mark. Store in a cool place, shelf life 1 week.

Measurement Conditions
The room should not have drafts and the temperature should be at least +19 ^o C, because glacial acetic acid crystallizes at +18 ^o C.

 Analysis (see table).

По данному способу общий плазматический клиренс определяют по формуле:

,
где Y - внутривенно введенная доза Д-ксилозы;
t₁ и t₂ - промежутки времени, через которые определяют концентрацию Д-ксилозы;
Р₁ - концентрация Д-ксилозы в крови через 5 минут после внутривенного введения;
P₂ - концентрация Д-ксилозы в крови через 25 минут после внутривенного введения.The contents of the tubes are mixed. The sample and control are centrifuged at a speed of 3000 rpm, carefully take 0.2 ml of a clear supernatant is placed in a clean tube. 0.1 ml NaOH and 0.4 ml distilled water are added to the sample and control, and 1 ml p-bromaniline reagent is added to all tubes. All tubes are placed in a boiling water bath for 20 minutes, then placed in a dark place for 80 minutes. The intensity of the obtained color is measured on a FEK or spectrophotometer: sample against control, standard solution against distilled water. The calculation of the content of D-xylose is carried out according to the formula:

According to this method, the total plasma clearance is determined by the formula:

,
where Y is the intravenously administered dose of D-xylose;
t ₁ and t ₂ - time intervals over which determine the concentration of D-xylose;
P ₁ - the concentration of D-xylose in the blood 5 minutes after intravenous administration;
P ₂ is the concentration of D-xylose in the blood 25 minutes after intravenous administration.

 Normally, the decrease in the plasma concentration is exponential, i.e., over the same period of time, the concentration of the substance decreases by the same amount. This is due to the fact that the rate of excretion is a constant value.

 With pathology, respectively, the severity of the pathological process, the rate of excretion decreases, xylosemia lasts longer, and the plasma clearance decreases accordingly. Since the intravenous administration of D-xylose was not introduced into the pharmacopeia, although oral administration is widely used to study the intestinal absorption capacity of patients, the new method was tested on dogs. In the future, after introducing the method of intravenous administration of D-xylose into the pharmacopeia, the study is planned to be carried out on patients.

Concrete example
As an example of a specific implementation, studies conducted on dogs were taken (laboratory journal, experimental protocols from 09/21/99; 10/10/99; 10/20/99; 10/28/99; 12/12/99). The studies were carried out in three stages: the control period - before giving anesthesia, the period after anesthesia and after a complete transverse transection of the spinal cord. The functional state of the kidneys was judged by a general analysis of urine, creatinine and urea in the blood.

 As an example, we cite a study conducted on a dog weighing 8 kg (5 ml of a 20% solution of D-xylose was administered intravenously).

 Control period; urinalysis: color straw yellow, protein no, specific gravity 1025, white blood cells 0-1, red blood cells no, blood creatinine 86.3 μmol / l, urea 8.2 ml mol / l, inulin clearance 82 ml / min, total plasma clearance of D-xylose 80 ml / min.

 Studies conducted after giving thiopental sodium anesthesia at a rate of 40 mg per kg of body weight. Urinalysis: color straw yellow, protein 0.3 g / l, specific gravity 1020, white blood cells 4-6, red blood cells 8-10, blood creatinine 90.9 mmol / l, urea 9.3 ml mol / l, total plasma inulin clearance 76.2 ml / min; total plasma clearance of D-xylose 72.3 ml / min.

 Studies conducted 10 days after giving anesthesia showed that the clearance of inulin and D-xylose at the control level, as well as other indicators. Subsequently, the same dog underwent a complete transverse transection of the spinal cord at the level of Tp7-Tp8. Urinalysis: color of meat slops, protein 0.9 g / l, specific gravity 1015. white blood cells 8-10, red blood cells - large numbers, blood creatinine 156.0 mmol / l, urea 10.4 ml mol / l, plasma inulin clearance 70.2 ml / min; plasma clearance of D-xylose 65 ml / min.

 A study conducted 10 days after transection showed a further decrease in clearance of D-xylose and inulin.

 From the above examples it is seen that the proposed method for determining the plasma clearance of D-xylose corresponds to the known method for determining the plasma clearance of inulin and adequately reflects the state of the filtration ability of the kidneys in normal and pathological processes leading to a change in this function.

Distinctive features of the proposed method from the prototype:
1) the distribution space of the prototype is determined by the sum of the area of the geometric figures that are formed by the curve of the plasma concentrations falling and the X axis, which is laborious, the distribution space by the proposed method is determined by the mathematical formula: C _tr = K • DU;
2) the decrease in the plasma concentration of D-xylose, unlike inulin, occurs exponentially, which makes it possible to determine the rate constant of disappearance;
3) D-xylose, unlike inulin, after intravenous administration is rapidly distributed in the body, after 3-5 minutes it is maximally concentrated and its concentration in the blood is allocated accordingly.

 Positive effect: the proposed method does not require a long time to conduct research. The micromethod for determining the concentration of D-xylose in the blood is highly sensitive and readily available. The loading dose is well tolerated, without side effects. This method makes it possible to obtain a quantitative characteristic of the state of the initial stage of urination (filtration) without measuring the volume and analyzing the composition of urine. Determination of the total plasma clearance by this method opens up new possibilities in studying the functional state of the kidneys in experiment and in clinical practice, as well as in studies on the kinetics of drugs in the body.

Claims (1)

A method for determining the plasma clearance of kidneys by the clearance of D-xylose, characterized in that the micromethod determines the concentration of D-cilose 5 minutes after intravenous administration (P ₁ ), 25 minutes (P ₂ ), and the total plasma clearance is determined by the formula

where Y is the intravenously administered dose of D-xylose; t ₁ and t ₂ - time intervals over which determine the concentration of D-xylose.

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