RU2039983C1 - Method for determining leukocytic elastase activity in plasma - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves diluting plasma separated from cellular sediment to 15-20 times lower concentration, introducing into a spectrophotometer cuvette, adding tris-HCl-buffer heated up to 28-30 C and then Boc-Ala-O- Np substrate solved in acetonitrile. Increased optical density of the sample is recorded after incubation at wave length being equal to 347.5-410.0 nm during 8-10 min. Enzyme activity is calculated from a formula. EFFECT: enhanced accuracy in determining elastase activity.

Description

 The invention relates to medicine, namely to methods for determining the activity of leukocyte elastase in blood plasma.

 It is known that many diseases or pathological conditions accompanied by an inflammatory reaction of the body (peritonitis, septicemia, respiratory distress syndrome, multiple organ failure, etc.) proceed against the background of activation of proteolytic enzymes of both plasma and cellular origin. The most powerful destructive factor in inflammation is leukocyte elastase, a serine trypsin-like proteinase with a neutral pH optimum, which can break down many proteins, glyco- and lipoproteins: elastin and collagen fibers, fibronectin, coagulation factors, fibrinolysis, kallikrein-komplein system and. A direct consequence of the degranulation of leukocytes and the release of elastase into the extracellular space are the most dangerous complications: impaired respiratory function of the lungs and thrombohemorrhagic syndrome, caused by direct damage to the endothelium of the pulmonary capillaries and destruction of plasma factors.

 Known immunological methods for determining the amount of leukocyte-released elastase based on enzyme-linked immunosorbent assay (ELISA) (Neuman SG Gunzer, N. Hennrich, H. Lang PMN-elastase "assay": enzyme Immunoassay for Human Polymorphonuclear Elastase Complexed with α-1-Proteinase Inhibitor J Clin Chem Clin Biochem, 1984, 22 (10): 693-697; Kramer DDM Maeller. Bardor AMMSimon, W. Tilgen, E. Sehichel, D. Petzoldt, Measurement of free human leukocyte elastase dnd human leukocyte elastase / α , proteinase inhibitor complexes by an ELISA. J. Imand. Method. 1990.131 (1). 41-48.

 These methods are based on the quantitative identification of either an elastase complex with its specific plasma inhibitor (α-1-proteinase inhibitor, α-1PI), or an elastase antigen in both a free and an inhibitor-bound state.

 Enzyme-linked immunosorbent assays for determining elastase, currently used, are very long-term and cannot be used in express diagnostics. The duration of the determination is associated with multi-stage processing of the tablet, its long incubation and washing at each stage of the analysis. The duration of this method of analysis from 5 to 8 hours

 Enzyme-linked immunosorbent assays require expensive biological products: a leukocyte elastase preparation, its mono- and polyvalent antibodies, α-1PI and mono- or multivalent antibodies to this inhibitor, conjugate of antibodies with a reporter enzyme. In addition, ELISA plates and a substrate are required for reaction with a reporter enzyme. As a prototype, a method has been adopted for spectrophotometric determination of the activity of free leukocyte elastase in blood plasma, including the introduction of a substrate into the blood plasma, which is used as Ala Tur-Leu-Val-pNA bis, and spectrophotometric determination of its cleavage rate in the presence of a buffer solution (Nagamatsy YI Yamamoto, A. Fukuda, M. Ohta, Y. Tsuda, Y. Okada. Determinatiou of leukocyte elastase comcentration in plasma and serum. By a simple method using a specific cynthetic Substrate. Haemostasis 21 (6); 338-345).

 However, the known method has disadvantages, as it does not allow to evaluate the activity of elastase present in the plasma, free and bound, but only some part of the leukocyte elastase that is not associated with the inhibitor.

 The objective of the invention is to provide a method for more rapid and complete determination of the activity of both free and associated leukocyte elastase.

 The essence of the invention lies in the fact that in the method for determining the activity of leukocyte elastase in blood plasma, comprising adding a buffer solution and substrate to a previously diluted physiological blood plasma solution, followed by spectrophotometric determination of the rate of substrate cleavage, the blood plasma is diluted 15-30 times, while Tris-HCl is used as a buffer solution, and Boc as a substrate. Ala ONp diluted in acetonitrile.

 The method allows to determine the activity of both free and associated with elastase inhibitors, i.e. up to 80-100% of all elastase in biological fluid, for example, in blood plasma. This makes it possible to clarify the diagnosis, give a forecast of the development of complications and take timely measures aimed at suppressing the activity of this enzyme with the help of medications.

 The time for determining elastase is reduced in comparison with known methods by almost 100 times and is about 6 minutes In this case, the minimum amount of the required blood plasma volume is 10 μl.

 Simplicity and speed make it possible to use the method in express diagnostics.

 The cost of determination is reduced by several thousand times, which makes the method cost-effective.

 The method can be used to determine the activity of elastase in any biological fluid, including blood plasma, urine, etc.

 The method is as follows.

 For work, a two-beam spectrophotometer with a thermostatically controlled cell holder of any type is used.

Blood plasma separated from the cell pellet by centrifugation in an amount of 0.1-0.3 ml is diluted 15-30 times with physiological saline. The spectrophotometric cuvette 3 ml contribute 30-70 .mu.l of diluted plasma was added warmed to 28-30 ^{° C} Tris-HCl buffer, substrate dissolved in acetonitrile Boc. Ala ONp. The sample is thoroughly mixed directly in the cuvette and registration of the increase in its density on a spectrophotometer at a wavelength of 347.5-410.0 nm for 8-10 minutes is started. An optical control is a sample containing 30-70 μl of physiological saline and the volume of Tris-HCl buffer and substrate is the same as in the experimental sample.

, где Δ прирост оптической плотности за минут;
К пересчетный коэффициент;
n разведение плазмы;
t время измерения активности;
V объем разведенной плазмы крови.The calculation of the activity of the enzyme is carried out according to the formula:

where Δ increase in optical density in minutes;
K conversion factor;
n plasma dilution;
t time for measuring activity;
V volume of diluted blood plasma.

 PRI me R 1. Patient N. diagnosis: chronic renal failure on chronic hemodialysis. Blood was taken for analysis 2 hours after the start of dialysis.

To 100 μl of blood plasma was added 1.4 ml of physiological saline (dilution 15 times) and thoroughly mixed. Then, in one trial made cuvette 50 ul of diluted plasma and heated to 30 ^C. Tris-HCl buffer. In another control cuvette, 50 μl of physiological saline and Tris-HCl buffer were added in a volume equal to the volume in the experimental cuvette. The contents of both cuvettes were thoroughly mixed. After some time of holding the cuvettes in a thermostated cuvette holder of the spectrophotometer, a Boc.Ala ONp substrate solution was added simultaneously to both cuvettes. The contents of the cuvette were again thoroughly mixed and the density was recorded in the experimental cuvette against the control at a wavelength of 347.5 nm for 8-10 minutes.

где Δ прирост плотности за 1 мин, равный 0,020;
t время измерения, равное 1 мин;
15 разведение плазмы;
0,050 объем плазмы в опытной пробе.The calculation of activity was performed according to the maximum increase in density according to the formula:

where Δ increase in density in 1 min, equal to 0.020;
t measurement time equal to 1 min;
15 dilution of plasma;
0.050 plasma volume in the experimental sample.

0,9 Е/мл
Если плазма крови больного была разведена в 30 раз, то максимальный прирост плотности опытной пробы составил 0,011.So,

0.9 U / ml
If the patient's blood plasma was diluted 30 times, then the maximum increase in the density of the experimental sample was 0.011.

4,3 Е/мл
П р и м е р 2. Предлагаемым способом была измерена активность лейкоцитарной эластазы, предварительно связанной с α-1-протеиназным ингибитором.Recalculation according to the formula:

4.3 U / ml
PRI me R 2. The proposed method was measured the activity of leukocyte elastase, previously associated with α-1-proteinase inhibitor.

A leukocyte elastase preparation with an activity of 0.7 U / ml (according to MeOSuc Ala Pio ValpNA) and 2.9 U / ml (according to Boc Ala ONp),
5 μl of leukocyte elastase gave the following density increments: for MeOSuc Ala Ala PiO ValpNA for 1 min 0.032 for Boc Ala ONp for 1 min 0.022
To suppress the activity of leukocyte elastase, the following incubation mixture was prepared:
25 μl of elastase + 5 μl of Tris-HCl buffer + 15 μl of α-1-PI. The mixture was incubated for 15 minutes at 30 ^C. Residual activity was assessed by elastase MeO Suc Ala Ala Pio ValpNA.

9 μl of the incubation mixture containing 5 μl of elastase gave a density increase of 0.011, which corresponded to 33% of the initial activity. Thus, 3 μl of α-1 PI inhibited the activity of 5 μl of elastase by 67%
To identify full activity associated with the α-1 PI elastase into test cuvette was added 9 l of an incubation mixture containing 5 l of elastase, and heated to 30 ^C. Tris-HCl buffer. 9 μl of physiological saline and the same volume of Tris-HCl buffer as in the experimental sample were added to the control cuvette. The control and experimental cuvettes were incubated in a thermostated cell holder at ³⁰ ° C, then simultaneously both cuvettes were added substrate Boc Ala ONp. After thorough mixing, a density increase over 8 minutes was recorded.

3/1 Е/мл
Таким образом, способ позволил выявить всю активность лейкоцитарной эластазы.The maximum increase in optical density in 1 min was 0.024, which corresponded to

3/1 U / ml
Thus, the method allowed to identify all the activity of leukocyte elastase.

Claims (1)

 METHOD FOR DETERMINING THE ACTIVITY OF LEUCYTIC ELASTASIS IN BLOOD PLASMA, including dilution of blood plasma with physiological solution, addition of a buffer solution and substrate thereto, followed by spectrophotometric determination of the rate of substrate cleavage, characterized in that the blood plasma is diluted 15 to 20 times, three are used as a buffer solution HCI, and as a substrate, Boc Ala-O-Np, diluted in acetonitrile.