Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES UNIVERSITY | METHODS | PLASMA PREKALLIKREIN


Determination of prekallikrein in plasma with Chromogenic Substrate S-2302

Measurement Principle
The activation of plasma prekallikrein is mediated by the Hageman factor on negatively charged surfaces and in presence of HMW kininogen. A number of methods have been described for the activation of prekallikrein. This method is related to the use of a prekallikrein activator composed by ellagic acid, phospholipid, Hageman factor and HMW kininogen, which was commercially available from Chromogenix. Different batches of this prekallikrein activator, not available any more, were assayed against the first PKA international standard and resulted in an activity of about 100-120 IU per vial. In the Reagents section the main characteristics of the prekallikrein activators have been reported. However, the assay should be validated with respect to the particular activator used. Following the activation, the plasma kallikrein formed catalyses the hydrolysis of p-nitroaniline (pNA) from the chromogenic substrate H-D-Pro- Phe-Arg-pNA (chromogenic substrate S-2302). The rate at which pNA is released is measured photometrically at 405 nm. This can be followed on a recorder (initial rate method) or read after stopping the reaction with acetic acid (acid stopped method). The concentration of prekallikrein is calculated by using standards prepared from normal plasma.

F XII Contact activation
FXIIa
Prekallikrein FXIIa + HMW kininogen
Kallikrein
H-D-Pro-Phe-Arg-pNA + H2O Kallikrein
H-D-Pro-Phe-Arg-OH + pNA

Reagents

  1. Chromogenic Substrate S-2302, 25 mg    Art. No. 82 03 40
    Reconstitute the substrate S-2302 (MW: 611.6) with 20 ml of distilled water.
  2. Plasma Prekallikrein Activator
    Prepare a solution of plasma prekallikrein activator (PKA) with an activity of 20-24 IU/ml. This component should contain ellagic acid, phospholipid and a plasma fraction composed by Hageman factor and high molecular weight kininogen. (Meier H et al. 1977; Wiggins RC et al. 1977; Friberger P et al. 1979; Kerry PJ et al. 1985). In alternative refer to Claeson G et al. 1978; Friberger P et al. 1979; Shibuya Y et al. 1991, to use other prekallikrein activators.
  3. Tris Buffer, pH 7.8 (25C)
    Tris 6.1 g (50 mmol/l) Distilled water 800 ml Adjust the pH to 7.8 at 25C by adding an appropriate amount (approximately 38 ml) of 1 mol/l HCl. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for six months at 2-8C.
  4. Normal plasma
    Blood should be taken from at least 20 healthy donors. 10-30 ml of citrated blood (9 vol blood and 1 vol 0.1 mol/l sodium citrate) are taken from each donor (the first ml of blood is discarded). Plasma is prepared by centrifugation at 2000 x g for 20 minutes at 15-25C. Equal amounts of plasma from the donors are mixed and dispensed in small volumes. In order to avoid low-temperature activation of prekallikrein the plasma should be kept at 15-25C and used as soon as possible or quickly frozen at -20C or below. After thawing at +37C the plasma should be kept at 15-25C and used as soon as possible. Frozen plasma is stable for three months at -20C or below. Avoid refreezing.
  5. Acetic acid, 20%
    Acetic acid is used in the acid-stopped method.

Specimen collection
Blood (9 vol) is mixed with 0.1 mol/l sodium citrate (1 vol) and centrifuged at 2000 x g for 20 minutes at 15-25C. In order to avoid low-temperature activation of prekallikrein plasma should be kept at 15-25C for not more than 24 hours or immediately frozen at -20C or below. After thawing at 37C the plasma should be kept at 15-25C and used as soon as possible. Frozen plasma may loose some prekallikrein on freezing or thawing, but will remain stable for three months at -20C or below. Avoid refreezing.

Standard curve
The normal plasma has a prekallikrein concentration of 100% and is diluted according to the table below.

Prekallikrein
%

Normal plasma
ml

Buffer
ml

25

100

300

50

200

200

75

300

100

100

-

-

125

see below

-

Method

Sample dilution

Buffer

3000 l

Test plasma or standard

50 l

To obtain the 125% standard, mix 125 ml normal plasma with 6 ml buffer.
the test tube method or the Microplate method can be performed by the acid-stopped or the initial rate method.

Acid stopped method

 

A

B

Prekallikrein activator

200 l 50 l

Incubate at 37C

3-4 min 3-4 min

Diluted sample

200 l 50 l

Incubate at 37C

2 min* 2 min*

Substrate (37C)

200 l 50 l

Incubate at 37C or read the initial rate

2 min 2 min

Acetic acid 20%

200 l 50 l

A: test tube method
B: microplate method

*The incubation time depends from the prekallikrein activator used. 2 min is the incubation time with a PKA with the characteristics described in the reagent section.

For the acid-stopped method: read the absorbance at 405 nm within 4 hours. If the plasma is icteric, hemolytic or lipemic, plasma blanks should be determined. Plasma blank is prepared by adding the reagents in reverse order starting with the acetic acid, without incubation. Subtract the absorbance of the blank from the absorbance of the corresponding sample.

For the initial rate method in test tubes: transfer sample immediately after addition of the chromogenic substrate to a 1 cm semi-microcuvette (preheated at 37C) for measurement of the absorbance change at 405 nm.

Calculation
Plot A or DA/min for the standards against their concentration of prekallikrein on linear graph paper. Read the prekallikrein value for the corresponding A or DA/min for the unknown test sample from the standard curve.

Bibliography

  1. Claeson G et al. Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. Haemostasis 7, 76-78 (1978).
  2. Friberger P et al. Determination of prekallikrein in plasma by means of a chromogenic tripeptide substrate for plasma kallikrein. In: KININS II. Biochemistry, Pathophysiology and Clinical Aspects. Eds. Fujii S, Moriya H & Suzuki T. Plenum Publishing Corp, 67-82 (1979).
  3. Sakuragawa N et al. Changes of prekallikrein in the cases with disseminated intravascular coagulation syndrome. In KININS II. Systemic Proteases and Cellular Function. Eds Fujii S, Moriya H & Suzuki T. Plenum Publishing Corp, 185-193, (1979).
  4. Aasen AO et al. Studies on components of the plasma kallikrein-kinin system in plasma samples from normal individuals and patients with septic shock. Advances in Shock Research 4. Ed A. Lener. Alan R. Liss Inc. 1-10 (1980).
  5. Gallimore MJ et al. Further studies on components of the plasma kallikrein system in plasma samples from cancer patients and normal individuals. In Progress in Chemical Fibrinolysis and Thrombolysis. Vol. V. Ed Davidson J F, 256-258 (1980).
  6. Gallimore MJ et al: Simple chromogenic peptide substrate assays for determining prekallikrein, kallikrein inhibition and kallikrein like activity in human plasma. Thromb Res 25, 293-298 (1982).
  7. Friberger P et al. Chromogenic substrates for kallikreins and related enzymes. Agents and Actions. Suppl 9, 83-90 (1982).
  8. Kerry PJ et al. Standardization of prekallikrein activator (PKA): the 1st International Standard for PKA. Br J Haematol 60, 345-352 (1985).
  9. Friberger P and Gallimore MJ. Description and evaluation of a new chromogenic substrate assay kit for the determination of prekallikrein in human plasma. In KININS IV, 543-547 (1986).
  10. De La Cadena R et al. Evaluation of a microassay for human plasma prekallikrein. J Lab Clin Med 109, 601-607 (1987).
  11. Gallimore MJ and Friberger P. Prekallikrein activator, Letter to the Editor. Thromb Haemost 52, 366 (1984).
  12. Shibuya Y et al. Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate. Clin Chim Acta 200, 119-128 (1991).
 

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