RU2070323C1 - Method for determination of efficiency of therapy at patients having chronic renal insufficiency - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves precipitation of globulins of serum blood, the process is carried out with the help of ammonium sulfate. Sufficient amount of probe solution is added into supernatant liquid. Intensity of luminescence is registered at wavelength being equal to 540 nm. If said intensity of luminescence decreased then therapy is considered to be effective. EFFECT: enables estimation of efficiency of therapy at its early stages, improves efficiency of the method.

Description

 The invention relates to medicine and can be used to determine the effectiveness of therapy for patients with chronic renal failure (CRF).

 A known method for determining the effectiveness of treatment of patients with chronic renal failure, which includes recording the concentration of creatinine in the blood and the level of glomerular filtration (Ryabov S.I. Natochin B.V. Bondarenko B. Diagnosis of kidney disease. Leningrad: Medicine, 1979. 256 pp.). With a decrease in the concentration of creatinine (Cr) and an increase in the level of glomerular filtration (F), effective therapy is indicated.

 However, at least 2 ml of blood serum is required to determine the concentration of Cr; in addition, the dynamics of these indicators is too slow, which does not allow us to judge the effectiveness of therapy in the early stages of its implementation.

 The objective of the invention is to provide a method for determining the effectiveness of therapy in the early stages of its implementation.

 The problem is solved in that in the method for determining the effectiveness of therapy for patients with chronic renal failure by examining the blood serum according to the invention, globulins are precipitated from the blood serum with ammonium sulfate, a sufficient amount of the DSP-6 probe solution (n-toluenesulfonate 4- / 4 '> is introduced into the supernatant dimethylaminophenyl / ethenyl-1-hexyl pyridinium), the luminescence intensity is recorded at a wavelength of 540 nm and the therapy is considered effective when the luminescence intensity decreases.

 Serum albumin, the main protein component of blood plasma, has a unique ability to modify and bind various ligands. In the literature there is evidence of significant changes in the tertiary and secondary structure of serum albumin (HSA) in various pathological conditions. We found that the modification of albumin, that is, conformational changes in its molecule, occurs when exposed to endotoxins formed in the blood of patients with chronic renal failure. These changes precede an increase in the concentration of free toxins in the blood, which makes it possible to evaluate the effectiveness of therapy in the early stages of its implementation. Assessment of conformational changes in HSA was carried out by the spectrofluorimetric method using a DSP-6 fluorescent probe, which was previously used when working with erythrocyte membranes (Kalnina I.E. Shibaeva T.N. Bluma R.K. Sominsky V.N. Determination of the conditions for the use of DSM probes and DSP-6 for studying erythrocyte membranes // Proceedings of the Academy of Sciences of Latvia, 1989. Issue 7. P. 107 111. We have discovered the property of the DSP-6 probe to bind to HSA and increase its luminosity when interacting with albumin in patients with chronic renal failure. Attempts to measure change in a binding manner CSA abilities using another probe 8-anilino-1-naphthalene sulfonate (ANS) yielded results in liver pathology and failed to apply to renal patients (uremia of various origins) / Miller Yu.I. Use of a fluorescent probe in assessing the binding ability of serum albumin human liver failure //. Laboratory. 1989. Issue 7. S. 20 23 /. Used in this method probe DSP-6 was effective in the study of HSA in renal patients.

The method can be carried out as follows. Globulins are precipitated from the patient’s blood serum by salting out in the presence of 33% ammonium sulfate with constant stirring for 2 hours at room temperature / Vaptsarov I. Iomtov M. Savov S. Dyukmejiev I. Eshkenazi M. Dysproteinemia. Sofia: Medicine and physical education. 1978. 336 p. /. The suspension was centrifuged at 700 rpm for 10 min at room temperature. After appropriate dilution to a final protein concentration of 3 mg / ml, add a DSP-6 fluorescence probe (final probe concentration in the sample 1.0 2.5 10 ^-5 M) to the supernatant, and after exposure for 20 30 minutes, the exposure intensity is recorded fluorescence (I _fl ) at a wavelength of 540 nm, (excitation wavelength of 470 nm) on a Hitachi spectrofluorimeter 650-40 (Japan). When the probe concentration is below 1.0 10 ^-5 M, the intensity of the luminescence of DSP-6 bound to the protein decreases sharply; when the probe concentration is above 2.5 10 ^-5 M it is impossible to work, since at an optical density greater than 0.42 the intensity decreases. The working concentration of the probe was chosen from the dependence of: I _fl protein – probe system on the concentration of the probe at a protein concentration of 3 mg / ml. For measurements, we used a solution with an optical density of 0.42. The working concentration of the protein is selected from the dependence of I _fl probe-protein system on the concentration of albumin. Registration of I _fl was carried out at λ = 540 nm, and not at λ _max. , since on the slope of the spectrum, at a wavelength of 540 nm, differences in I _fl between different patients were detected more clearly. An excitation wavelength of 470 nm is selected as λ _max. in the excitation spectrum of DSP-6 at λ _max. 610 nm emission. The intensity values that we operated on when developing this method are values representing the ratio of the luminous intensity of the probe bound to the patient's protein to the luminous intensity of the probe bound to Reanal albumin (I _rel. ). In each study, the numbers obtained on a spectrofluorimeter were normalized according to the standard, which was Reanal albumin. Fluorimetric analysis of partially purified albumin was carried out for each patient repeatedly, both before therapy and against its background.

 Example 1. Patient A., 27 years old, diagnosis: chronic glomerulonephritis (CGN), active course, CRF-2b, azotemia, metabolic acidosis, secondary hypertension. Therapy: from 11.29.90: trental, furosemide, anaprilin, adelfan (until 14.12.90); from 11.12.90 festival; from 12/18/90 enterosorbent SKNP-1. Table 1 presents the survey data of patient A. for 2 months.

It can be seen from the table that between December 3, 90, December 12, 90 and December 12, 90 there is practically no significant dynamics in the Cr level, the filtration drops by 1.3 times. I _rel. by 12.12.90 already reduced by 1.3 times compared with 03.12.90 and further, against the background of enterosorbent, 4 days after its appointment, I _rel. drops 1.55 times (12/21/90), which indicates effective therapy and that is recorded only 12/26/90: creatininemia decreases, the glomerular filtration rate increases. Further decrease in I _rel. 1.8 times preceding the change in Cr and F in 14 days. The albumin fractions of the blood serum of patient A. were studied by polyacrylamide gel electrophoresis (EP in PAAG). Clear differences in electrophoregrams were obtained between 12/03/90 and 10/01/91: in the serum from 10/01/91 there are no additional bands that are detected on 12/3/90 and which probably correspond to fractions of modified albumin.

 Example 2. Patient B., 45 years old, diagnosis: CGN, exacerbation, secondary hypertension, chronic renal failure-2a, azotemia. Therapy: from 10.17.90: chimes, furosemide, clonidine; from 10.26.90 aminophylline; from 05.11.90 apressin; from 20.11.90 - enterosorbent SKNP-1. Table 2 presents the survey data of patient B. for 1.5 months.

The data in table 2 demonstrate the positive effect of treating patient B. from 10/18/90 to 11/26/90. Relative to 10/31/90, already towards 11/13/90 and especially to 11/26/90, creatininemia decreased, F increased: 1.28 times (11/13/90) and then 2.25 times (11/26/90). The luminescence intensity of the probe with respect to the initial one (1.20) decreases 1.22 times already on 10/31/90, and then 1.58 times on 11/13/90. A sharp increase in I _Rel. registered on 11/19/90 and further on 11/26/90 (1.72 times) precedes a decrease in glomerular filtration and an increase in creatininemia, which is detected only after 15 days, 12/03/90. When EF in PAAG between 10/18/90 and 11/26/90 found the same differences as in the previous example, namely: in the serum from 11/26/90 there are no additional bands detected on 10/18/90.

 Example 3. Patient C., 45 years old, diagnosis: mesangial-proliferative GN, type 2 course, without exacerbation, chronic renal failure-2a, azotemia, secondary hypertension. Therapy: from 02/14/91: kordafen, adelfan, calcium carbonate; from 02.28.91 - biseptol, bromhexine; from 06.03.91 enterosorbent SKNP-1. Table 3 presents the data of the examination of patient B. for 2 months.

The data in the table demonstrate the ineffectiveness of treatment of patient B. from 02.15.91 to 04.15.91: there is no significant dynamics in Cr, F drops 1.2 times. I _rel. in comparison with the initial one, 02.22.91 already increases 1.5 times, preceding a decrease in glomerular filtration, which is recorded only on 03.03.91. Further I _rel. remains almost at the same level, which corresponds to the absence of positive changes in the patient's condition. The electrophoretic pictures of the albumin fractions of this patient on 02.15.91 and 04.04.91 turned out to be almost the same.

From the above examples you can see:
the change in the relative spectral parameter occurs on average 10 15 days earlier than the change in the concentration of Cr and F;
decrease in I _Rel. correlates with effective therapy;
increase in I _rel. indicates the ineffectiveness of therapy.

Using the proposed method allows you to:
evaluate the effectiveness of treatment of patients with chronic renal failure in the early stages of its implementation;
conduct research in serum microdoses (0.03 0.04 ml);
significantly increase sensitivity.

Claims (1)

 A method for determining the effectiveness of the treatment of chronic renal failure, including a study of blood serum during therapy, characterized in that globulins are precipitated from the patient's blood serum with ammonium sulfate, a 4- / 4-dimethylaminophenyl / -ethenyl-1- fluorescent probe is introduced into the supernatant hexyl pyridinium (DSP-6), the luminescence intensity is recorded at a wavelength of 540 nm, and when the intensity is reduced relative to the initial therapy, it is considered effective.

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