THROX3USIS QPergamon
RESEARCH 13; 733-733 Printed Press Ltd.1978.
in Great
Britain
0049-384S/7P/llOl-0733
502.oo~c
A FLUORESCENT SUBSTRATE ASSAY FOR PLASMINOGEN Sharon P. Pochron, Gary A. Mitchell, Ileana Albareda Rolf M. Huseby and Robert J. Gargiulo DADE Division American Hospital Supply Corporation, Miami, Florida 33152 U.S.A. in revised form 3.8.1978. (Received 27.7.1978; Accepted by Editor W.H. Seegers) ABSTRACT A rapid and sensitive method for the detection of plasminogen levels in plasma is presented using the synthetic substrate D-valineleucine-lysine-5-amidoisophthalic acid, dimethyl ester. Plasma plasminogen is assayed fluorometrically following conversion of plasminogen to plasmin using streptokinase for enzyme activation. The plasmin activity measured by this procedure is comparable to results obtained with standard plasmin activity assay procedures (u-casein) and to immunologic determinations of plasminogen concentration. The substrate used is extremely sensitive to the detection of plasmin and makes possible the use of a small sample volume. This eliminates interference from inhibitors without additional sample preparation, which in turn increases the ease and rapidity of the assay. The accuracy and precision observed demonstrate its suitability for routine clinical use.
INTRODUCTION Abnormal levels of plasma plasminogen have been reported in a variety of diseases and in certain physiological states. Increased levels have been documented in acute bacterial infections, inflammatory conditions, thrombophlebitis, surgery, myocardial infarction, pregnancy and after long-term use of oral contraceptives, while plasminogen levels are reduced in diffuse intravascular coagulation and cirrhosis (1). Currently, plasma plasminogen is measured by a variety of procedures which include caseinolytic (2,3), fibrin plate (4,5) and chromogenic substrate (6,7,8) assays, and immunologic methods based on the techniques of Mancini et al. (9) and Becker (10). A fluorescent substrate assay for plasminogen is described in this paper. The method, which may be used for routine measurement of olasma plasminogen is an extension of previous work reported by our laboratory (11,12). MATERIALS AND METHODS Fluorometric Assay Buffer:
0.05 M tris(hydroxymethyl)aminomethane, 0.05 M
734
SUBSTRATE
FOR PLASMIKOGEN
ASSAY
Vol.l3,No.5
glycine, 0.01% Brij-35, pH adjusted to 8.0 with 1 N HCl. Activation Buffer: 0.05 M tris(hydroxymethyl)aminomethane, 0.1 M glycine, pH adjusted to 7.5 with 1 N HCl. Caseinolytic Assay Buffer: 0.06 M tris(hydroxymethyl)aminomethane, 0.09 M NaCl, PH adjusted to 7.5 with 1 N HCl. Perchloric Acid, 0.5 M: deionized water.
42.8 ml of 70% perchloric acid diluted to 1.0 1 with
Hydrochloric Acid, N/6:
1 N HCl diluted 1:6 in deionized water.
Sodium Hydroxide, N/6: Sodium Chloride, 0.15 M: deionized water.
1 N NaOH diluted 1:6 in deionized water. 8.768 g NaCl dissolved and diluted to 1.0 1 in
Streptokinase: Varidase*, lyophilized, 100,000 units/vial, from Lederle Laboratories, Pearl River, New York 10965. Diluted to 10,000 units/ml with activation buffer and stored in aliquots at -20C. The solution was thawed and used without further dilution in the caseinolytic assay; for the fluorometric assay, the solution was diluted in activation buffer to 2000 units/ml. Bovine Albumin, 30% solution: Reheis Chemical Co., Chicago, Illinois 60690. Diluted 1:2.5 (12%) in caseinolytic assay buffer and used to prepare a 1:2 dilution of plasminogen reference material. Plasminogen Reference Material: 9.7 CTA units/ml in 50% glycerol, obtained from Dr. David Aronson, National Institutes of Health, Bethesda, Maryland 20014. Diluted 1:2 in 12% bovine albumin (4.85 CTA units/ml, 6% albumin). H-D-valine-leucine-lysine-5-amidoisophthalic acid, dimethyl ester, di-trifluoroacetate: mol. wt. 777.8, from Enzyme Systems Products, Inc., P.O. Box 1191, Indianapolis, Indiana 46206. The substrate was dissolved in fluorometric assay buffer to a concentration of 0.8 mM/l and warmed to 37C before use. A Km value of 3.1 x 10-4 M (37C) was determined within the assay system using the Lineweaver-Burk equation. a-Casein: From Worthington Biochemical Corp., Freehold, New Jersey 07728. 1.4 g a-casein was dissolved in 100 ml caseinolytic assay buffer. M-PartigenTM Plasminogen Kit: radial immunodiffusion test from Behring Diagnostics, American Hoechst Corp., Somerville, New Jersey 08876. Turner Model 430 Spectrofluorometer: G.K. Turner Associates, Palo Alto, California 97303. Added EauiDment: temoerature control for samDle cell holder; model EV-200 recorder from Heath Schlumberger, Benton~Harborj Michigan 49022. The excitation and emission wavelength and slit width settings were 335 x 430 nm and 60 x 15 nm respectively. The recorder chart speed was 1 inch/min. Disposable TPX plastic cuvets, 10 x 8 x 50 mm were used.
*Reg. TM of American Cyanamid Company, Wayne, New Jersey.
Yo1.13,Xo.j
ST,-BSTFUTE
FOR
PLASMIBOGES
ASSAY
735
Gilford Instrument Laboratories, Inc., Gilford Model 2403Spectrophotometer: Oberlin, Ohio 44074. Wavelength setting was 275 nm. Rectangular 10 x 10 x 45 mn quartz cuvets with 4 ml capacity were used. Blood Specimens were collected in plastic tubes containing one volume of 3.8% trisodium citrate for each nine volumes of blood. The specimens were centrifuged at 1,500 to 2,000 9 for 10 to 15 minutes to obtain the plasmas which were stored at -20C and thawed prior to assay. Samples used for precision studies were stored in aliquots at -20C to avoid repeated freezing and thawing. Eleven of the 15 samples used in this study were obtained from laboratory workers with normal prothrombin and partial thromboplastin times. Three of the samples, numbers 3, 9 and 14, were lyophilized plasmas used as controls throughout our studies. Sample number 15 was a post-mortem plasma supplied by Dr. John A. Penner, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan. The fluorescent substrate assays were performed with 10 ~1 of plasma sample or diluted reference plasminogen added to 0.5 ml streptokinase solution (2000 units/ml). The reagents were vortex-mixed, incubated for 15 minutes at 37C to convert the plasminogen to its active form, plasmin, and stored at 4C until assayed. Two hundred microliters of each of these activation mixtures was transferred to a cuvet containing 2.0 ml of substrate solution pre-warmed to 37C. The plasmin activity was determined by kinetic measurement of the released 5-aminoisophthalic acid,dimethyl ester, over a 2-4 minute interval. The plasmin activity of each sample expressed in CTA units/ml was calculated by comparison of the relative fluorescent rate of the sample with that of the reference plasminogen (4.85 CTA units/ml) as follows: Plasmin, CTA units/ml =
X 4.85 Relative Rate of Sample Relative Rate of Reference Plasminogen
The radial imunodiffusion tests were performed according to the manufacturer's instructions using a minimum diffusion time of 48 hours. The caseinolytic assays were performed according to the method described by Johnson et al. (3) with some modification to permit the assay of plasma samples. Prior to activation with streptokinase, plasma inhibitors were destroyed by incubation of 0.1 ml of plasma sample or diluted reference plasminogen with 0.1 ml N/6 HCl for 15 minutes at room temperature (13). The acid was neutralized with 0.1 ml N/6 NaOH and 0.1 ml streptokinase (10,000 U/ml) was added. The mixtures were incubated for 15 minutes at 37C followed by the addition of a solution containing 2.1 ml caseinolytic assay buffer and 2.5 ml 1.4% a-casein prewarmed to 37C. The change in absorbance at 275 nm over a 30 minute period was determined by removal of 2 ml aliquots at 2 and 32 minutes. Reactions were stopped with 3.0 ml 0.5 M perchloric acid. After standing a minimum of 30 minutes at room temperature, the tubes were centrifuged for 5 minutes at approximately 2000 9, and the supernates filtered through Whatman No. 42 filter paper. The absorbance of reagent blanks using either assay buffer or 6% albumin in place of the sample was 3.01 which was subtracted from the absorbance values of all samples. The plasmin activity in CTA units/ml was obtained, as in the fluorescent substrate assay, by comparison of the sample absorbance with that of the reference plasminogen.
736
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FOR PLASMlNOGEN
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Vol.l3,No.5
RESULTS A detailed sumnary of the plasma plasminogen values obtained using the three assay techniques employed in this study are presented in Table 1. Plasminogen levels are expressed in CIA units/ml for the fluorescent substrate (FS) and caseinolytic (CAS) assays, which measure activity, and in mg/dl for the radial irtnnunodiffusion(RID) assay, which measures concentration. A normal population mean and standard deviation were calculated for each method using the values obtained for the 11 normal plasma samples. The measured mean values for the fluorescent substrate and caseinolytic assays showed remarkably good agreement. The mean plasminogen activity for the fluorometric assay was 3.70 f 1.13 compared to 3.77 + 1.01 CTA units/ml for the caseinolytic assay. The mean value obtained for the radial immunodiffusion assay, 13.2 t"3.6 mg/dl, compares well with the normal mean reported by the manufacturers of the test, 12 mg/dl. TABLE I Sununary of Results Obtained in Fluorescent Substrate Assay for Plasminogen Compared with Two Standard Methods Sample No.
FS Assay CTA Units/ml
CAS Assay CTA Units/ml
RID Assay mg/dl
z 7
5.8 5.5 5.3 4.5 3.8 3.7 3.6
9*
:::
:: 12 13 14* 15"
;:;
:.; 218
;*: 2:o 0.1
z 2:6 0.3
20.4 19.2 19.2 14.6 12.4 13.2 12.8 11.7 12.1 11.7 11.1 9.8 a.5 7.2 1.6
3.70 1.13
3.77 1.01
13.2 3.6
: 3" 4
a
Mean S.D.
5.3 5.5 6.0 4.5 3.9 3.8 3.9 3.5
*Samples not used in calculation of the normal population mean. (See Blood Specimens, MATERIALS AND METHODS section).
The correlation between the three methods is demonstrated when the data in Table I is compared by least squares analysis (Fig. 1). The correlation coefficients are 0.98 for the fluorescent versus caseinolytic assay and 0.99 for the fluorescent versus radial immunodiffusior assay.
a.0,
??
J
??
5.0. ?? ? ?
m=
0.33
b i-0.2-V I
1.0
2.0 3.0 PlasminoCn. CTA Units/ml C*SElNOlYTlC
=
10 tlasminogw!.
ASSAY
0.w
20 mwdl
RADIAL IMMUNOOlFFUSfON ASSAY
FIG.
1
Correlation between the fluorescent substrate assay and two reference methods for determining plasminogen.
The precision of the fluorescent substrate method for the determination of plasminogen is shown in Table II. Three plasma samples, each containing a different level of plasminogen activity, were used in the study. The mean relative fluorescence rates (RFR) are shown along with the standard deviations (SD) and coefficients of variation (CV%). Both the within-day and between-daycoefficients of variation were under 5% at all plasminogen levels tested, well within acceptable limits for clinical laboratory determinations.
TABLE II Precision of Relative Fluorescent Rates (RFR) Obtained in Fluorescent Substrate Assay of Plasminogen in Plasma Within-day, n = 5 Sample A B C Between-day, n = 5 A B C
Mean RFR
SD
cvz
0.44 0.73 1.23
0.015 0.011 0.019
3.4 1.5 1.6
0.46 0.76 1.30
0.021 0.020 0.040
4.5 2.6 3.1
739
SC'BSTRATE FOR PLASMINOGEN
ASSAY
Vol.l3,No.5
To establish further the validity of the fluorescent substrate assay, analytical recovery of purified plasminogen added to plasma was also determined and the results are presented in Table III. The plasminogen activities, expressed in CTA units/ml, of a plasma sample and a solution of purified plasminogen were determined separately. The plasma sample with increasing amounts of the purified plasminogen solution was then re-assayed. The studies included enzyme levels over the entire range of the assay, from very low to very high plasminogen activities. Recovery values exceeded 95% at all test levels. More importantly, the recovery of plasminogen at the lower end of the assay range where analytical accuracy might be expected to suffer compared well with values for samples containing higher plasminogen levels. TABLE III Analytical Recovery of Purified Plasminogen Added to Plasma Samples Tested
Plasma ~1 1: 10 10 10
CTA Units/ml
Purified Plasminogen Pl : 5 ::
Recovery %
Determined
Calculated
0.9 2.6 3.4 4.3 6.1
_____ 3.5 4.4 6.2
____ -__97.1 97.1 98.4
DISCUSSION Satisfactory procedures for determination of plasma plasminogen have not been available for the clinical laboratory. The long incubation time required for the radial immunodiffusion test does not lend itself to rapid determinations of plasminogen levels, particularly in those disease states where an expedient diagnosis is critical. Alternatively, the caseinolytic method is both laborious and time consuming and as a result is even less suited to normal clinical laboratory operations than the radial immunodiffusion procedure. Mattler and Bann (E) who used a chromogenic substrate assay for determining plasma plasminogen reported that the substrate assay values which they obtained were low when compared to caseinolytic results, but no explanation was presented for their findings. This discrepancy was not observed in the results of our fluorescent and caseinolytic assays. The fluorescent substrate assay reported offers distinct advantages over current methodologies. The substrate used is extremely sensitive to the detection of plasmin activity allowing the use of less than 4 ~1 of patient plasma. Pretreatment of plasma samples to destroy the known plasmin inhibitors is not required because the dilution of the plasma sample sufficiently eliminates or minimizes inhibitor interference. As little as 0.001 CTA units plasmin can be detected by this assay. Few manipulations are required, data reduction is minimal, and total assay time is considerably less than when other assays are used. Assay precision for plasma samples was better than ~5% C'j while the accuracy was established by comparison with two reference methods. It should be noted that the excellent correlation observed between the fluorescent substrate and radial immunodiffusion assays may be influenced by the fact that nearly all of the samples tested were obtained from appar-
ently normal donors, where differences in activity vers;s concentration would be expected to be minimal. While abnormal plasminogen levels have been reported, existing procedures are neither rapid nor easily performed and there are few reports of 2xtensive clinical studies. Thus, the diagnostic significance of the altered levels is not well documented in all cases. The fluorescent substrate assay described in this paper is readily adaptable to routine clinical us2 and should facilitate clarification of the role of plasninogen in health and disease. REFERENCES 1.
Lackner, H. and Javid, J.P. The clinical sign1'ficance of the plasminogen level. Am.J.Clin.Path. 60. 175, 1973.
2.
Remrlert, L.F. and Cohen, P.P. Partial purification and properties of a proteolytic enzyme of human serum. J.Biol.Chem. 131, 431, 1949.
3.
Johnson, A.J., Kline, D.L. and Alkjaersig, Pi. Assay methods and standard preparations for plasmin, plasminogen and urokinase in purified systems, 1967-1968. Thromb.Diath.haemorrh. (Stuttg.) 2l_, 259, 1969.
4.
The fibrin plate method for estimating Astrup, T. and Mullertz, S fibrinolytic activity. Arch.Biochem. 9, 346, 1952.
5.
Lassen, M. Heat denaturation of plasminogen in the fibrin plate method. Acta.Physiol.Scand. c. 371, 1952.
6.
Clavin, S.A., Bobbitt, J.L., Shuman, R.T. and Smithwick, E.L. Jr. Us2 of peptidyl-4-methoxy-2-naphthylamides to assay plasmin. Anal.Biochem. g, 355, 1977.
7.
Teger-Nilsson, A.C., Friberger, P. and Gyzander, E. Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate. Scan.J.Clin.Lab.Invest. 2, 403, 1977.
8.
Mattler, L.E. and Bang, N.U. Serine orotease specificity for peptide chromogenic substrates. Thrombos.Haemostas. (Stuttg.) 38_, 776, 1977.
9.
Mancini, G., Carbonera, A.O. and Heremans, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem. Z_, 235, 1965.
10.
Becker, W. Determination of antisera titres using the single radial immunodiffusion method. Immunochem. 6, 539, 1969.
11.
Mitchell, G.A., Hudson, P.M., Huseby, R.M., Pochron, S.P. and Gargiulo, R.J. Fluorescent substrate assay for antithrombin III. Thromb.Res. 12, 219, 1978.
12.
Mitchell, G.A., Gargiulo, R.J., Huseby, R.M., Lawson, D.E., Pochron, S. P. and Sehuanes, J.A. Assay for plasma heparin using a synthetic peptide substrate for thrombin: Introduction of the fluorophore aminoisophthalic acid, dimethyl ester. Thromb.Res. (In press).
13.
Alkjaersig, N., Fletcher, A.P. and Sherry, S. The mechanism of clot dissolution by plasmin. J.Clin.Invest. 3&, 1086, 1959.
RESEARCH 13; 733-733 Printed Press Ltd.1978.
in Great
Britain
0049-384S/7P/llOl-0733
502.oo~c
A FLUORESCENT SUBSTRATE ASSAY FOR PLASMINOGEN Sharon P. Pochron, Gary A. Mitchell, Ileana Albareda Rolf M. Huseby and Robert J. Gargiulo DADE Division American Hospital Supply Corporation, Miami, Florida 33152 U.S.A. in revised form 3.8.1978. (Received 27.7.1978; Accepted by Editor W.H. Seegers) ABSTRACT A rapid and sensitive method for the detection of plasminogen levels in plasma is presented using the synthetic substrate D-valineleucine-lysine-5-amidoisophthalic acid, dimethyl ester. Plasma plasminogen is assayed fluorometrically following conversion of plasminogen to plasmin using streptokinase for enzyme activation. The plasmin activity measured by this procedure is comparable to results obtained with standard plasmin activity assay procedures (u-casein) and to immunologic determinations of plasminogen concentration. The substrate used is extremely sensitive to the detection of plasmin and makes possible the use of a small sample volume. This eliminates interference from inhibitors without additional sample preparation, which in turn increases the ease and rapidity of the assay. The accuracy and precision observed demonstrate its suitability for routine clinical use.
INTRODUCTION Abnormal levels of plasma plasminogen have been reported in a variety of diseases and in certain physiological states. Increased levels have been documented in acute bacterial infections, inflammatory conditions, thrombophlebitis, surgery, myocardial infarction, pregnancy and after long-term use of oral contraceptives, while plasminogen levels are reduced in diffuse intravascular coagulation and cirrhosis (1). Currently, plasma plasminogen is measured by a variety of procedures which include caseinolytic (2,3), fibrin plate (4,5) and chromogenic substrate (6,7,8) assays, and immunologic methods based on the techniques of Mancini et al. (9) and Becker (10). A fluorescent substrate assay for plasminogen is described in this paper. The method, which may be used for routine measurement of olasma plasminogen is an extension of previous work reported by our laboratory (11,12). MATERIALS AND METHODS Fluorometric Assay Buffer:
0.05 M tris(hydroxymethyl)aminomethane, 0.05 M
734
SUBSTRATE
FOR PLASMIKOGEN
ASSAY
Vol.l3,No.5
glycine, 0.01% Brij-35, pH adjusted to 8.0 with 1 N HCl. Activation Buffer: 0.05 M tris(hydroxymethyl)aminomethane, 0.1 M glycine, pH adjusted to 7.5 with 1 N HCl. Caseinolytic Assay Buffer: 0.06 M tris(hydroxymethyl)aminomethane, 0.09 M NaCl, PH adjusted to 7.5 with 1 N HCl. Perchloric Acid, 0.5 M: deionized water.
42.8 ml of 70% perchloric acid diluted to 1.0 1 with
Hydrochloric Acid, N/6:
1 N HCl diluted 1:6 in deionized water.
Sodium Hydroxide, N/6: Sodium Chloride, 0.15 M: deionized water.
1 N NaOH diluted 1:6 in deionized water. 8.768 g NaCl dissolved and diluted to 1.0 1 in
Streptokinase: Varidase*, lyophilized, 100,000 units/vial, from Lederle Laboratories, Pearl River, New York 10965. Diluted to 10,000 units/ml with activation buffer and stored in aliquots at -20C. The solution was thawed and used without further dilution in the caseinolytic assay; for the fluorometric assay, the solution was diluted in activation buffer to 2000 units/ml. Bovine Albumin, 30% solution: Reheis Chemical Co., Chicago, Illinois 60690. Diluted 1:2.5 (12%) in caseinolytic assay buffer and used to prepare a 1:2 dilution of plasminogen reference material. Plasminogen Reference Material: 9.7 CTA units/ml in 50% glycerol, obtained from Dr. David Aronson, National Institutes of Health, Bethesda, Maryland 20014. Diluted 1:2 in 12% bovine albumin (4.85 CTA units/ml, 6% albumin). H-D-valine-leucine-lysine-5-amidoisophthalic acid, dimethyl ester, di-trifluoroacetate: mol. wt. 777.8, from Enzyme Systems Products, Inc., P.O. Box 1191, Indianapolis, Indiana 46206. The substrate was dissolved in fluorometric assay buffer to a concentration of 0.8 mM/l and warmed to 37C before use. A Km value of 3.1 x 10-4 M (37C) was determined within the assay system using the Lineweaver-Burk equation. a-Casein: From Worthington Biochemical Corp., Freehold, New Jersey 07728. 1.4 g a-casein was dissolved in 100 ml caseinolytic assay buffer. M-PartigenTM Plasminogen Kit: radial immunodiffusion test from Behring Diagnostics, American Hoechst Corp., Somerville, New Jersey 08876. Turner Model 430 Spectrofluorometer: G.K. Turner Associates, Palo Alto, California 97303. Added EauiDment: temoerature control for samDle cell holder; model EV-200 recorder from Heath Schlumberger, Benton~Harborj Michigan 49022. The excitation and emission wavelength and slit width settings were 335 x 430 nm and 60 x 15 nm respectively. The recorder chart speed was 1 inch/min. Disposable TPX plastic cuvets, 10 x 8 x 50 mm were used.
*Reg. TM of American Cyanamid Company, Wayne, New Jersey.
Yo1.13,Xo.j
ST,-BSTFUTE
FOR
PLASMIBOGES
ASSAY
735
Gilford Instrument Laboratories, Inc., Gilford Model 2403Spectrophotometer: Oberlin, Ohio 44074. Wavelength setting was 275 nm. Rectangular 10 x 10 x 45 mn quartz cuvets with 4 ml capacity were used. Blood Specimens were collected in plastic tubes containing one volume of 3.8% trisodium citrate for each nine volumes of blood. The specimens were centrifuged at 1,500 to 2,000 9 for 10 to 15 minutes to obtain the plasmas which were stored at -20C and thawed prior to assay. Samples used for precision studies were stored in aliquots at -20C to avoid repeated freezing and thawing. Eleven of the 15 samples used in this study were obtained from laboratory workers with normal prothrombin and partial thromboplastin times. Three of the samples, numbers 3, 9 and 14, were lyophilized plasmas used as controls throughout our studies. Sample number 15 was a post-mortem plasma supplied by Dr. John A. Penner, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan. The fluorescent substrate assays were performed with 10 ~1 of plasma sample or diluted reference plasminogen added to 0.5 ml streptokinase solution (2000 units/ml). The reagents were vortex-mixed, incubated for 15 minutes at 37C to convert the plasminogen to its active form, plasmin, and stored at 4C until assayed. Two hundred microliters of each of these activation mixtures was transferred to a cuvet containing 2.0 ml of substrate solution pre-warmed to 37C. The plasmin activity was determined by kinetic measurement of the released 5-aminoisophthalic acid,dimethyl ester, over a 2-4 minute interval. The plasmin activity of each sample expressed in CTA units/ml was calculated by comparison of the relative fluorescent rate of the sample with that of the reference plasminogen (4.85 CTA units/ml) as follows: Plasmin, CTA units/ml =
X 4.85 Relative Rate of Sample Relative Rate of Reference Plasminogen
The radial imunodiffusion tests were performed according to the manufacturer's instructions using a minimum diffusion time of 48 hours. The caseinolytic assays were performed according to the method described by Johnson et al. (3) with some modification to permit the assay of plasma samples. Prior to activation with streptokinase, plasma inhibitors were destroyed by incubation of 0.1 ml of plasma sample or diluted reference plasminogen with 0.1 ml N/6 HCl for 15 minutes at room temperature (13). The acid was neutralized with 0.1 ml N/6 NaOH and 0.1 ml streptokinase (10,000 U/ml) was added. The mixtures were incubated for 15 minutes at 37C followed by the addition of a solution containing 2.1 ml caseinolytic assay buffer and 2.5 ml 1.4% a-casein prewarmed to 37C. The change in absorbance at 275 nm over a 30 minute period was determined by removal of 2 ml aliquots at 2 and 32 minutes. Reactions were stopped with 3.0 ml 0.5 M perchloric acid. After standing a minimum of 30 minutes at room temperature, the tubes were centrifuged for 5 minutes at approximately 2000 9, and the supernates filtered through Whatman No. 42 filter paper. The absorbance of reagent blanks using either assay buffer or 6% albumin in place of the sample was 3.01 which was subtracted from the absorbance values of all samples. The plasmin activity in CTA units/ml was obtained, as in the fluorescent substrate assay, by comparison of the sample absorbance with that of the reference plasminogen.
736
SZiBSmTE
FOR PLASMlNOGEN
ASSAY
Vol.l3,No.5
RESULTS A detailed sumnary of the plasma plasminogen values obtained using the three assay techniques employed in this study are presented in Table 1. Plasminogen levels are expressed in CIA units/ml for the fluorescent substrate (FS) and caseinolytic (CAS) assays, which measure activity, and in mg/dl for the radial irtnnunodiffusion(RID) assay, which measures concentration. A normal population mean and standard deviation were calculated for each method using the values obtained for the 11 normal plasma samples. The measured mean values for the fluorescent substrate and caseinolytic assays showed remarkably good agreement. The mean plasminogen activity for the fluorometric assay was 3.70 f 1.13 compared to 3.77 + 1.01 CTA units/ml for the caseinolytic assay. The mean value obtained for the radial immunodiffusion assay, 13.2 t"3.6 mg/dl, compares well with the normal mean reported by the manufacturers of the test, 12 mg/dl. TABLE I Sununary of Results Obtained in Fluorescent Substrate Assay for Plasminogen Compared with Two Standard Methods Sample No.
FS Assay CTA Units/ml
CAS Assay CTA Units/ml
RID Assay mg/dl
z 7
5.8 5.5 5.3 4.5 3.8 3.7 3.6
9*
:::
:: 12 13 14* 15"
;:;
:.; 218
;*: 2:o 0.1
z 2:6 0.3
20.4 19.2 19.2 14.6 12.4 13.2 12.8 11.7 12.1 11.7 11.1 9.8 a.5 7.2 1.6
3.70 1.13
3.77 1.01
13.2 3.6
: 3" 4
a
Mean S.D.
5.3 5.5 6.0 4.5 3.9 3.8 3.9 3.5
*Samples not used in calculation of the normal population mean. (See Blood Specimens, MATERIALS AND METHODS section).
The correlation between the three methods is demonstrated when the data in Table I is compared by least squares analysis (Fig. 1). The correlation coefficients are 0.98 for the fluorescent versus caseinolytic assay and 0.99 for the fluorescent versus radial immunodiffusior assay.
a.0,
??
J
??
5.0. ?? ? ?
m=
0.33
b i-0.2-V I
1.0
2.0 3.0 PlasminoCn. CTA Units/ml C*SElNOlYTlC
=
10 tlasminogw!.
ASSAY
0.w
20 mwdl
RADIAL IMMUNOOlFFUSfON ASSAY
FIG.
1
Correlation between the fluorescent substrate assay and two reference methods for determining plasminogen.
The precision of the fluorescent substrate method for the determination of plasminogen is shown in Table II. Three plasma samples, each containing a different level of plasminogen activity, were used in the study. The mean relative fluorescence rates (RFR) are shown along with the standard deviations (SD) and coefficients of variation (CV%). Both the within-day and between-daycoefficients of variation were under 5% at all plasminogen levels tested, well within acceptable limits for clinical laboratory determinations.
TABLE II Precision of Relative Fluorescent Rates (RFR) Obtained in Fluorescent Substrate Assay of Plasminogen in Plasma Within-day, n = 5 Sample A B C Between-day, n = 5 A B C
Mean RFR
SD
cvz
0.44 0.73 1.23
0.015 0.011 0.019
3.4 1.5 1.6
0.46 0.76 1.30
0.021 0.020 0.040
4.5 2.6 3.1
739
SC'BSTRATE FOR PLASMINOGEN
ASSAY
Vol.l3,No.5
To establish further the validity of the fluorescent substrate assay, analytical recovery of purified plasminogen added to plasma was also determined and the results are presented in Table III. The plasminogen activities, expressed in CTA units/ml, of a plasma sample and a solution of purified plasminogen were determined separately. The plasma sample with increasing amounts of the purified plasminogen solution was then re-assayed. The studies included enzyme levels over the entire range of the assay, from very low to very high plasminogen activities. Recovery values exceeded 95% at all test levels. More importantly, the recovery of plasminogen at the lower end of the assay range where analytical accuracy might be expected to suffer compared well with values for samples containing higher plasminogen levels. TABLE III Analytical Recovery of Purified Plasminogen Added to Plasma Samples Tested
Plasma ~1 1: 10 10 10
CTA Units/ml
Purified Plasminogen Pl : 5 ::
Recovery %
Determined
Calculated
0.9 2.6 3.4 4.3 6.1
_____ 3.5 4.4 6.2
____ -__97.1 97.1 98.4
DISCUSSION Satisfactory procedures for determination of plasma plasminogen have not been available for the clinical laboratory. The long incubation time required for the radial immunodiffusion test does not lend itself to rapid determinations of plasminogen levels, particularly in those disease states where an expedient diagnosis is critical. Alternatively, the caseinolytic method is both laborious and time consuming and as a result is even less suited to normal clinical laboratory operations than the radial immunodiffusion procedure. Mattler and Bann (E) who used a chromogenic substrate assay for determining plasma plasminogen reported that the substrate assay values which they obtained were low when compared to caseinolytic results, but no explanation was presented for their findings. This discrepancy was not observed in the results of our fluorescent and caseinolytic assays. The fluorescent substrate assay reported offers distinct advantages over current methodologies. The substrate used is extremely sensitive to the detection of plasmin activity allowing the use of less than 4 ~1 of patient plasma. Pretreatment of plasma samples to destroy the known plasmin inhibitors is not required because the dilution of the plasma sample sufficiently eliminates or minimizes inhibitor interference. As little as 0.001 CTA units plasmin can be detected by this assay. Few manipulations are required, data reduction is minimal, and total assay time is considerably less than when other assays are used. Assay precision for plasma samples was better than ~5% C'j while the accuracy was established by comparison with two reference methods. It should be noted that the excellent correlation observed between the fluorescent substrate and radial immunodiffusion assays may be influenced by the fact that nearly all of the samples tested were obtained from appar-
ently normal donors, where differences in activity vers;s concentration would be expected to be minimal. While abnormal plasminogen levels have been reported, existing procedures are neither rapid nor easily performed and there are few reports of 2xtensive clinical studies. Thus, the diagnostic significance of the altered levels is not well documented in all cases. The fluorescent substrate assay described in this paper is readily adaptable to routine clinical us2 and should facilitate clarification of the role of plasninogen in health and disease. REFERENCES 1.
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