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Determination of plasma heparin by polybrene neutralization
ANALYTICAL
BIOCHEMISTRY
138, 319-323 (1984)
Determination of Plasma Heparin by Polybrene Neutralization PATRICIA
V. NASH
AND THORIR
D. BJORNSSON
Division of Clinical Pharmacology, Departments of Pharmacology and Medicine, Duke University Medical Center, Durham, North Carolina 27710 Received August 22, 1983 A method for determining heparin activity (in unit/ml) in human plasma is described. The method is based on neutralization of heparin by Polybrene, a polymerized quaternary ammonium salt. It uses serial incubations of plasma with increasing amounts of Polybmne in conjunction with thrombin-induced coagulation times to &fine the amount of Polybrene required to neutral& heparin in the sample. Reference curves involve linear relationships between amounts of Polybrene required to neutralize known amounts of heparin. Coefficients of variation for the assay vary from 4 to 10% over the range of 0.05 to 1.0 unit/ml. KEY WORDS: heparin; heparin assays; anticoagulants; Polybrene; coagulation tests; pharmacokinetics.
Because of the heterogeneity of heparin, a (4,12,16-18), they have not been fully optinatural glycosaminoglycan, its plasma levels mized and their stability and accuracy not and pharmacokinetics have been determined established. The purpose of the present work by measuring the activity of the drug in plasma is to optimize the Polybrene neutralization by various bioassays and chemical neutralassay of heparin (PN-heparin assay)’ and to ization assays (l-3). Most heparin assays are determine the stability of the assay in stored based on coagulation tests; these include ac- plasma samples. tivated partial thromboplastin time (4-6), thrombin time (4,7,8), and activated Factor MATERIALS AND METHODS X assay (9,lO). Other more chemically oriMaterials. Heparin sodium injection (USP, ented heparin assays include metachromatic 1000 units/ml from beef lung) was obtained methods using toluidine blue or azure A (3,11), from the Upjohn Company (Kalamazoo, and neutralization methods using protamine Mich.). Polybrene (P-45 15) was purchased ( 12- 15) or Polybrene (4,12,16- 18). Polybrene from Sigma Chemical Company (St. Louis, has been found to be superior to protamine MO.), and lyophilized, bovine thrombin [USP, as a neutralizing agent ( 12). More recent hep 1000 US (NIH) units per vial (Thrombostat)] arin assay methods use chromogenic or fluofrom Parke-Davis (Morris Plains, N. J.). A rogenic substrates (19-2 1). fibrometer coagulation timer (Baltimore BioRecently, it has become apparent that there logical Laboratories, Cockeysville, Md.) was are significant differences between the various used to determine the coagulation times. heparin assays in vivo (4,22-25) and that the Sample collection. Blood samples used for various bioassays exhibit pronounced interthe standardization of the PN-heparin assay subject variation in heparin sensitivity (26,27). for determining plasma heparin activity were It is therefore desirable to have a chemically collected by venipuncture from healthy volbased assay of heparin that has been standardized and characterized for pharmacoki’ Abbreviations used: PN-heparin assay, Polybrene netic studies. Although neutralization assays neutralization assayof heparin; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid. using Polybrene have been described before 319
0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form nsawd
320
NASH AND BJORNSSON
unteers. The blood was immediately mixed with 3.2% (w/v) sodium citrate solution to yield 10% (v/v) &rated blood, which was centrifuged at 1OOOgfor 15 min. The plasma was transferred to silanized glass containers and kept on ice until used for preparing the samples for the standard curves. Samples containing unknown amounts of heparin are treated in the same way. Preparation of samples. Standard dilutions of the commercial heparin preparation were made with 0.9% (w/v) sodium chloride to yield heparin standards ranging in concentration from 0.5 to 10 units/ml. Fresh heparin standards are made every 1 to 2 months; during this period of time no differences are observed in heparin activity. Series of plasma samples were prepared containing 10% (v/v) of the different heparin standards, resulting in plasma heparin activity between 0.05 and 1.0 unit/ ml of sample. Polybrene standards were prepared in 0.0325 M Hepes buffer, pH 7.4, from a stock solution of 0.5 mg/ml, and concentrations ranged from 1 to 25 &ml in l-fig/ ml increments. Fresh Polybrene standards are made weekly, since Polybrene was found to be unstable in solution over longer periods of time. Both heparin and Polybrene standards are kept at 4°C between use. The thrombin powder was first reconstituted with 0.0325 M Hepes buffer, pH 7.4, to give a concentration of 50 NIH units/ml, and then stored in l-ml aliquots at -80°C until use. Immediately prior to use, the frozen thrombin solution was thawed rapidly and diluted with 0.0325 M Hepes buffer, pH 7.4, and 0,025 M calcium chloride in the same Hepes buffer, to give a final concentration of 3 NIH units/ml thrombin and 0.0 125 M calcium chloride. The final thrombin concentration was adjusted so as to produce a control thrombin clotting time in the absence of added heparin of between 11 and 13 s. Usually this requires thrombin concentrations of about 2 units/ml, ranging from 1.5 to 3 units/ml. The thrombin solution was kept on ice throughout the assay. Polybrene titration of heparin. The determination of heparin activity in plasma by the
PN-heparin assay is based on a standard curve describing the linear relationship between known amounts of heparin and the amounts of Polybrene required to neutralize that hep arin. The standard curve is constructed on the basis of results from five separate heparin series, using plasma from five normal subjects, where each series contains five different heparin concentrations. The reaction mixture consists of 150 ~1 of control plasma for the standard curves or unknown plasma samples, 15 ~1 of a heparin standard for the standard curves, and 50 ~1 of a Polybrene standard or Hepes buffer. This solution is mixed gently and incubated at 37°C for 5 min. The thrombin solution (100 ~1) is then added to initiate the fibrin gel formation. For each heparinized plasma sample to be assayed, a series of mixtures is prepared containing increasing amounts of Polybrene. Since the volume of each Polybrene standard used is only 50 ~1, the actual amount of Polybrene added is l/20 of the amount/l ml of that standard. The coagulation times decrease exponentially with increasing amounts of Polybrene present until all heparin has been neutralized, alter which there is no further reduction in the coagulation time. For each heparinized plasma sample, four to five mixtures are measured with coagulation times above the titration point, and at least two samples are measured after neutralization has been achieved. The choice of initial Polybrene standard and increments in Polybrene standards to use is based on expected plasma heparin activity, or coagulation time in a sample without Polybrene. Following the determination of the coagulation times in the necessary number of samples, the slope (p) of the relationship between the natural logarithms of the coagulation times and the amounts of Polybrene used is determined using linear least-squares regression analysis. For each plasma sample assayed for heparin, the neutralization point (P,) is then calculated where the best-fitted line intersects with the coagulation time observed after neutralization has been reached (CT,,), using the expression
POLYBRENE
NEUTRALIZATION
RESULTS 0 0 0 a
so - “, 60 ‘t 50 - ‘\\
40
0 2 unrflmi HEPARIN 04 ““,,/d”EPAR,N 06vn,t/mlHEPARIN 08unrt/mlWPARIN
0 ‘\\\ *
30 20’ 0
a
0.4
0.6
a
bh, IO
0.2
AMOUNT
0.8
I.0
OF POLYBRENE ADDED (pg)
FIG. 1. Polybrene neutral&&ion of plasma heparin activity in samples containing four different concentrations of heparin, 0.2,0.4, 0.6, and 0.8 unit/ml. For any given sample the addition of increasing amounts of Polybrene results in an exponential decrease in thrombin clotting times until neutralization is achieved. The slopes represent the beat-fitted lines of the relationships between the clotting times and amounts of Polybrene added; the horizontal lines represent the clotting times after neutralization. The neutralization point is indicated by the point where these two lines intersect.
p = InCT,, - InCT, n P
’
where CT0 is the back-extrapolated coagulation time with no Polybrene present, and other symbols are as defined above.
321
ASSAY OF HEPARIN
AND DISCUSSION
Figure 1 shows typical semilogarithmic plots of thrombin coagulation times in plasma samples containing different amounts of heparin vs amounts of Polybrene present. The coagulation times decrease exponentially with increasing amounts of Polybrene present until neutralization is achieved. Table 1 shows data for a reference curve constructed using plasma from five normal subjects and five heparin concentrations in each subject. The relationship between ‘neutralizing amounts of Polybrene and plasma heparin activity is linear. The average intercept and slope values were -0.008 + 0.019 pg and 0.98 1 _+ 0.032 pg/ unit/ml, respectively, with an average coefficient of determination for the standard curve of 0.995 + 0.006; regression analysis of the mean data results in essentially the same values (Fig. 2). Since the volume of plasma used in the assay is only 150 ~1, the slope value of the calibration curve multiplied by 6.667 gives the actual amount of Polybrene required to neutralize 1 unit of heparin. The lower level of sensitivity of the PN-heparin assay is about 0.05 unit/ml, where samples are determined with a coefficient of variation of about 10%.
TABLE 1 REPRESENTATIVE
PARAMETERS OF A CALIBRATION USING SAMPLEIS OBTAINED
CURVE FOR THE PN-HEPARIN FROM Rn NORMAL SUBJECTS
ASSAY CONSTRUCTED
Plasma heparin activity (unit/ml)
(JTn (9
0.2
0.4
0.6
0.8
1.0
12.3 0.3
12.6 0.3
12.6 0.5
12.7 0.6
12.7 0.7
P (rcg/unit/ml)
0.161 0.034
0.196 0.028
0.190 0.023
0.162 0.014
0.132 0.013
r2
0.972 0.024
0.955 0.036
0.932 0.025
0.894 0.042
0.942 0.034
prl (rs)
0.206 0.016
0.370 0.027
0.569 0.022
0.769 0.039
0.988 0.042
cv (%)
7.8
7.3
3.9
5.1
4.2
Note.
Values presented are means 2 SD.
322
NASH AND BJORNSSON
2 z
0.2 0.4 0.6 08 1.0 PLASMA HEPARIN ACTIVITY (unit/ml)
FIG. 2. Typical calibration curve illustrating the linear relationship between the neutralizing amounts of Polybrene and heparin activity added to plasma. Data are from Table 1. Each data point represents a mean of five neutralization curves obtained from as many normal subjects; the vertical lines represent one standard deviation. The slope represents the best-fitted slope of the calibration curve. Note that the neutralizing amount of Polybrene is determined by the amount of heparin present in a 150-~1 sample representing a heparin concentration in unit/ml.
The effects of two related assay variables, i.e., the thrombin concentration used and observed coagulation times after neutralization, were evaluated. It had been observed that using the same thrombin concentration for reference curves from different subjects resulted in different reference curves, This inter-subject difference disappeared when thrombin concentrations were adjusted to yield control coagulation times in the range 11 to 13 s. Within each set of plasma samples, increasing thrombin concentrations will result in shorter coagulation times, decreased slope of the reference curve, and a lower intercept. This is illustrated in Fig. 3A. Overall, inter-subject differences were less important than thrombin interbatch differences for adjusting thrombin concentrations. The effects on the assay of different methods of storage of plasma samples containing heparin were evaluated. It was found that freezing (quick freezing in dry ice/acetone and storage at -80°C) resulted in significant changes in the PN-heparin assay; the coagulation times, neutralizing amounts of Polybrene, and slopes
of the standard curves decreased continually with storage time. A representative set of data is shown in Fig. 3B. Anticoagulation with acid citrate (2 parts 0.1 M citric acid plus 3 parts 0.1 M sodium citrate) or sodium oxalate (0.1 M) did not affect these changes. However, refrigeration (4°C) of heparinized plasma samples over 24 h resulted in an average of -0.09 + 7.77% change in the slopes of five reference curves obtained from normal subject (range -12.54 to 6.57%), indicating that plasma samples can be stored at 4’C for 24 h prior to analysis. Refrigeration over 48 h, however, resulted in an average of 7.3 1 + 7.54% change (range-2.29 to 13.85%) in the slopes of these reference curves. In summary, a reproducible and sensitive assay of heparin in plasma has been optimized. The assay is based on neutralization by Polybrene of negative charges on heparin molecules. Thus, the PN-heparin assay is a chemically based assay, although it utilizes coagulation times as endpoints. Citrated plasma samples for this assay can be stored overnight in a refrigerator. Plasma heparin activity ranging in concentration from 0.05 to 1.0 unit/ ml is determined with a coefficient of variation varying from 4 to 10%. Plasma heparin activity most commonly encountered during continuous iv infusion of heparin in patients with venous thromboembolism is between 0.2 and 0.4 unit/ml (8,28). This assay has been used
RASMA HEPARINACTlVlTY(wtlml)
FIG. 3. Effects of thrombin concentration (A) and storage at -80°C (B) on relationships between neutralizing amounts of Polybrene and plasma heparin activity. The lines represent the best-fitted lines for each set of data.
POLYBRENE
NEUTRALIZATION
in pharmacokinetic studies in normal subjects (4,29) and patients (28). These studies have revealed a marked difference between the in vivo levels of plasma heparin determined by this assay and a bioassay based on activated partial thromboplastin time and suggested that the pharmacokinetics of heparin should be defined on the basis of a chemically based assay of heparin.
ASSAY OF HEPARIN
10. Simon, T. L., Hyers, T. M., Gaston, J. P., and Harker, L. A. (1978) &it. J. Haematof. 39, 111-120. 1 I. Klein, M. D., Drongowski, R. A., Linhardt, R. J., and Langer, R. S. (1982) Anal. B&hem. 124,5964.
12. Godal, H. C. (1960) Stand. J. Clin. Lab. Invest. 12, 446-451.
Refn, I., and Vestergaard, L. (1954) Stand. J. C/in. Lab. Invest. 6, 284-287. 14. Hjort, P., and Stormorken, H. (1957) Stand. J. Clin. Lab. Invest.. Suppl. 29, l-86. 15. Hirsh, J., Van Aken, W. G., GaBus, A. S., Dollery, C. T., Cade, J. F., Yung, W. L. (1976) Circulation 13.
ACKNOWLEDGMENTS This work was supported by NIH Grant HL24343. T.D.B. is a Nanaline H. Duke Scholar.
323
53,691-695.
16. Godal, H. C. (1961) Stand. J. Clin. Lab. Invest. 13, 153-166. 17. Teien, N., and Bjomson, J. (1976) Scand. J. Haemalol. 17,29-35.
REFERENCES 1. Bjommon, T. D. (1983) in Recent Advances in Chnical Pharmacollogy (Turner, P., and Sband, D. G., eds.), Vol. 3, pp. 129-I 55, ChurchilI Livingstone, Edinburgh. 2. E&s, J. W. (1980) Clin. Pharmacokin. 5, 204-220. 3. Jaques, L. B. (1977) in Methods OfBiochemical Analysis (Glick, D., ed.), Vol. 24, pp. 203-312, WileyInterscience, New York. 4. Bjomsson, T. D., Wolfram, K. M., and Kitchell, B. B. (1982) Clin. Pharmacol. Ther. 31, 104-l 13. 5. Marder, V. J. (1970) Thromb. Diath. Haemorrh. 24, 230-239. 6.
Cipolle, R. F., Seifert, R. D., Neilan, B. A., Zaske, D. E., and Haus, E. (1981) Clin. Pharmacol. Ther. 29, 387-393.
Blombiick, Olsson, Stand., 8. Penner, J. 7.
B., Blombaick, M., Olsson, P., WilliamG., and Senning, A. (1959) Acta Chir. Suppl. 245,259-264. A. (1974) Amer. J. Clin. Pathol. 61, 645-
653.
9. Yin, E. T., Wessler, S., and Butler, J. V. (1974) J. Lab. Clin. Med. 81, 298-310.
18. Grann, V. R., Homewood, K., andGolden, W. (1972) Amer. J. Clin. Pathol. 58, 26-32. 19. Teien, A. N., and Lie, M. (1977) Thromb. Res. 10, 399-410. 20. Lie Larsen, M., Abildgaard, U., Teien, A. N., and Gjesdal, K. (1978) Thromb. Res. 13,285-288. 21. Mitchell, G. A., Gargiulo, R. J., Huseby, R. M. (1978) Thromb. Rex 13,47-52. 22. Levy, S. W., and Jaques, L. B. (1978) Thromb. Res. 13,429-44
1.
Bounameaux, H., Marbet, G. A., L&mle, B., Eichlisberger, R., and Duckert, F. ( 1980) Amer. J. Clin. Pathol. 74, 68-73. 24. Talstad, I. (1980) Thromb. Rex l&485-491. 25. Barrowcliffe, T. W., Johnson, E. A., EggIeton, C. A., and Thomas, D. P. (1977) Thromb. Res. 12,2723.
36.
Bjomsson, T. D., and Wolfram, K. M. (1982) Eur. J. Clin. Pharmacol. 21, 491-497. 27. Whitfield L. R., and Levy, G. (1980) Clin. Pharmacoi. Ther. 28, 509-516. 28. Bjomsson, T. D., Nash, P. V., and Andersen, J. (1982) Clin. Pharmacol. Ther. 31, 204. 29. McGowan, F. X., Nash, P. V., and Bjomsson, T. D. (1983) &it. J. Clin. Pharmacol. 16, 677-682. 26.
BIOCHEMISTRY
138, 319-323 (1984)
Determination of Plasma Heparin by Polybrene Neutralization PATRICIA
V. NASH
AND THORIR
D. BJORNSSON
Division of Clinical Pharmacology, Departments of Pharmacology and Medicine, Duke University Medical Center, Durham, North Carolina 27710 Received August 22, 1983 A method for determining heparin activity (in unit/ml) in human plasma is described. The method is based on neutralization of heparin by Polybrene, a polymerized quaternary ammonium salt. It uses serial incubations of plasma with increasing amounts of Polybmne in conjunction with thrombin-induced coagulation times to &fine the amount of Polybrene required to neutral& heparin in the sample. Reference curves involve linear relationships between amounts of Polybrene required to neutralize known amounts of heparin. Coefficients of variation for the assay vary from 4 to 10% over the range of 0.05 to 1.0 unit/ml. KEY WORDS: heparin; heparin assays; anticoagulants; Polybrene; coagulation tests; pharmacokinetics.
Because of the heterogeneity of heparin, a (4,12,16-18), they have not been fully optinatural glycosaminoglycan, its plasma levels mized and their stability and accuracy not and pharmacokinetics have been determined established. The purpose of the present work by measuring the activity of the drug in plasma is to optimize the Polybrene neutralization by various bioassays and chemical neutralassay of heparin (PN-heparin assay)’ and to ization assays (l-3). Most heparin assays are determine the stability of the assay in stored based on coagulation tests; these include ac- plasma samples. tivated partial thromboplastin time (4-6), thrombin time (4,7,8), and activated Factor MATERIALS AND METHODS X assay (9,lO). Other more chemically oriMaterials. Heparin sodium injection (USP, ented heparin assays include metachromatic 1000 units/ml from beef lung) was obtained methods using toluidine blue or azure A (3,11), from the Upjohn Company (Kalamazoo, and neutralization methods using protamine Mich.). Polybrene (P-45 15) was purchased ( 12- 15) or Polybrene (4,12,16- 18). Polybrene from Sigma Chemical Company (St. Louis, has been found to be superior to protamine MO.), and lyophilized, bovine thrombin [USP, as a neutralizing agent ( 12). More recent hep 1000 US (NIH) units per vial (Thrombostat)] arin assay methods use chromogenic or fluofrom Parke-Davis (Morris Plains, N. J.). A rogenic substrates (19-2 1). fibrometer coagulation timer (Baltimore BioRecently, it has become apparent that there logical Laboratories, Cockeysville, Md.) was are significant differences between the various used to determine the coagulation times. heparin assays in vivo (4,22-25) and that the Sample collection. Blood samples used for various bioassays exhibit pronounced interthe standardization of the PN-heparin assay subject variation in heparin sensitivity (26,27). for determining plasma heparin activity were It is therefore desirable to have a chemically collected by venipuncture from healthy volbased assay of heparin that has been standardized and characterized for pharmacoki’ Abbreviations used: PN-heparin assay, Polybrene netic studies. Although neutralization assays neutralization assayof heparin; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid. using Polybrene have been described before 319
0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form nsawd
320
NASH AND BJORNSSON
unteers. The blood was immediately mixed with 3.2% (w/v) sodium citrate solution to yield 10% (v/v) &rated blood, which was centrifuged at 1OOOgfor 15 min. The plasma was transferred to silanized glass containers and kept on ice until used for preparing the samples for the standard curves. Samples containing unknown amounts of heparin are treated in the same way. Preparation of samples. Standard dilutions of the commercial heparin preparation were made with 0.9% (w/v) sodium chloride to yield heparin standards ranging in concentration from 0.5 to 10 units/ml. Fresh heparin standards are made every 1 to 2 months; during this period of time no differences are observed in heparin activity. Series of plasma samples were prepared containing 10% (v/v) of the different heparin standards, resulting in plasma heparin activity between 0.05 and 1.0 unit/ ml of sample. Polybrene standards were prepared in 0.0325 M Hepes buffer, pH 7.4, from a stock solution of 0.5 mg/ml, and concentrations ranged from 1 to 25 &ml in l-fig/ ml increments. Fresh Polybrene standards are made weekly, since Polybrene was found to be unstable in solution over longer periods of time. Both heparin and Polybrene standards are kept at 4°C between use. The thrombin powder was first reconstituted with 0.0325 M Hepes buffer, pH 7.4, to give a concentration of 50 NIH units/ml, and then stored in l-ml aliquots at -80°C until use. Immediately prior to use, the frozen thrombin solution was thawed rapidly and diluted with 0.0325 M Hepes buffer, pH 7.4, and 0,025 M calcium chloride in the same Hepes buffer, to give a final concentration of 3 NIH units/ml thrombin and 0.0 125 M calcium chloride. The final thrombin concentration was adjusted so as to produce a control thrombin clotting time in the absence of added heparin of between 11 and 13 s. Usually this requires thrombin concentrations of about 2 units/ml, ranging from 1.5 to 3 units/ml. The thrombin solution was kept on ice throughout the assay. Polybrene titration of heparin. The determination of heparin activity in plasma by the
PN-heparin assay is based on a standard curve describing the linear relationship between known amounts of heparin and the amounts of Polybrene required to neutralize that hep arin. The standard curve is constructed on the basis of results from five separate heparin series, using plasma from five normal subjects, where each series contains five different heparin concentrations. The reaction mixture consists of 150 ~1 of control plasma for the standard curves or unknown plasma samples, 15 ~1 of a heparin standard for the standard curves, and 50 ~1 of a Polybrene standard or Hepes buffer. This solution is mixed gently and incubated at 37°C for 5 min. The thrombin solution (100 ~1) is then added to initiate the fibrin gel formation. For each heparinized plasma sample to be assayed, a series of mixtures is prepared containing increasing amounts of Polybrene. Since the volume of each Polybrene standard used is only 50 ~1, the actual amount of Polybrene added is l/20 of the amount/l ml of that standard. The coagulation times decrease exponentially with increasing amounts of Polybrene present until all heparin has been neutralized, alter which there is no further reduction in the coagulation time. For each heparinized plasma sample, four to five mixtures are measured with coagulation times above the titration point, and at least two samples are measured after neutralization has been achieved. The choice of initial Polybrene standard and increments in Polybrene standards to use is based on expected plasma heparin activity, or coagulation time in a sample without Polybrene. Following the determination of the coagulation times in the necessary number of samples, the slope (p) of the relationship between the natural logarithms of the coagulation times and the amounts of Polybrene used is determined using linear least-squares regression analysis. For each plasma sample assayed for heparin, the neutralization point (P,) is then calculated where the best-fitted line intersects with the coagulation time observed after neutralization has been reached (CT,,), using the expression
POLYBRENE
NEUTRALIZATION
RESULTS 0 0 0 a
so - “, 60 ‘t 50 - ‘\\
40
0 2 unrflmi HEPARIN 04 ““,,/d”EPAR,N 06vn,t/mlHEPARIN 08unrt/mlWPARIN
0 ‘\\\ *
30 20’ 0
a
0.4
0.6
a
bh, IO
0.2
AMOUNT
0.8
I.0
OF POLYBRENE ADDED (pg)
FIG. 1. Polybrene neutral&&ion of plasma heparin activity in samples containing four different concentrations of heparin, 0.2,0.4, 0.6, and 0.8 unit/ml. For any given sample the addition of increasing amounts of Polybrene results in an exponential decrease in thrombin clotting times until neutralization is achieved. The slopes represent the beat-fitted lines of the relationships between the clotting times and amounts of Polybrene added; the horizontal lines represent the clotting times after neutralization. The neutralization point is indicated by the point where these two lines intersect.
p = InCT,, - InCT, n P
’
where CT0 is the back-extrapolated coagulation time with no Polybrene present, and other symbols are as defined above.
321
ASSAY OF HEPARIN
AND DISCUSSION
Figure 1 shows typical semilogarithmic plots of thrombin coagulation times in plasma samples containing different amounts of heparin vs amounts of Polybrene present. The coagulation times decrease exponentially with increasing amounts of Polybrene present until neutralization is achieved. Table 1 shows data for a reference curve constructed using plasma from five normal subjects and five heparin concentrations in each subject. The relationship between ‘neutralizing amounts of Polybrene and plasma heparin activity is linear. The average intercept and slope values were -0.008 + 0.019 pg and 0.98 1 _+ 0.032 pg/ unit/ml, respectively, with an average coefficient of determination for the standard curve of 0.995 + 0.006; regression analysis of the mean data results in essentially the same values (Fig. 2). Since the volume of plasma used in the assay is only 150 ~1, the slope value of the calibration curve multiplied by 6.667 gives the actual amount of Polybrene required to neutralize 1 unit of heparin. The lower level of sensitivity of the PN-heparin assay is about 0.05 unit/ml, where samples are determined with a coefficient of variation of about 10%.
TABLE 1 REPRESENTATIVE
PARAMETERS OF A CALIBRATION USING SAMPLEIS OBTAINED
CURVE FOR THE PN-HEPARIN FROM Rn NORMAL SUBJECTS
ASSAY CONSTRUCTED
Plasma heparin activity (unit/ml)
(JTn (9
0.2
0.4
0.6
0.8
1.0
12.3 0.3
12.6 0.3
12.6 0.5
12.7 0.6
12.7 0.7
P (rcg/unit/ml)
0.161 0.034
0.196 0.028
0.190 0.023
0.162 0.014
0.132 0.013
r2
0.972 0.024
0.955 0.036
0.932 0.025
0.894 0.042
0.942 0.034
prl (rs)
0.206 0.016
0.370 0.027
0.569 0.022
0.769 0.039
0.988 0.042
cv (%)
7.8
7.3
3.9
5.1
4.2
Note.
Values presented are means 2 SD.
322
NASH AND BJORNSSON
2 z
0.2 0.4 0.6 08 1.0 PLASMA HEPARIN ACTIVITY (unit/ml)
FIG. 2. Typical calibration curve illustrating the linear relationship between the neutralizing amounts of Polybrene and heparin activity added to plasma. Data are from Table 1. Each data point represents a mean of five neutralization curves obtained from as many normal subjects; the vertical lines represent one standard deviation. The slope represents the best-fitted slope of the calibration curve. Note that the neutralizing amount of Polybrene is determined by the amount of heparin present in a 150-~1 sample representing a heparin concentration in unit/ml.
The effects of two related assay variables, i.e., the thrombin concentration used and observed coagulation times after neutralization, were evaluated. It had been observed that using the same thrombin concentration for reference curves from different subjects resulted in different reference curves, This inter-subject difference disappeared when thrombin concentrations were adjusted to yield control coagulation times in the range 11 to 13 s. Within each set of plasma samples, increasing thrombin concentrations will result in shorter coagulation times, decreased slope of the reference curve, and a lower intercept. This is illustrated in Fig. 3A. Overall, inter-subject differences were less important than thrombin interbatch differences for adjusting thrombin concentrations. The effects on the assay of different methods of storage of plasma samples containing heparin were evaluated. It was found that freezing (quick freezing in dry ice/acetone and storage at -80°C) resulted in significant changes in the PN-heparin assay; the coagulation times, neutralizing amounts of Polybrene, and slopes
of the standard curves decreased continually with storage time. A representative set of data is shown in Fig. 3B. Anticoagulation with acid citrate (2 parts 0.1 M citric acid plus 3 parts 0.1 M sodium citrate) or sodium oxalate (0.1 M) did not affect these changes. However, refrigeration (4°C) of heparinized plasma samples over 24 h resulted in an average of -0.09 + 7.77% change in the slopes of five reference curves obtained from normal subject (range -12.54 to 6.57%), indicating that plasma samples can be stored at 4’C for 24 h prior to analysis. Refrigeration over 48 h, however, resulted in an average of 7.3 1 + 7.54% change (range-2.29 to 13.85%) in the slopes of these reference curves. In summary, a reproducible and sensitive assay of heparin in plasma has been optimized. The assay is based on neutralization by Polybrene of negative charges on heparin molecules. Thus, the PN-heparin assay is a chemically based assay, although it utilizes coagulation times as endpoints. Citrated plasma samples for this assay can be stored overnight in a refrigerator. Plasma heparin activity ranging in concentration from 0.05 to 1.0 unit/ ml is determined with a coefficient of variation varying from 4 to 10%. Plasma heparin activity most commonly encountered during continuous iv infusion of heparin in patients with venous thromboembolism is between 0.2 and 0.4 unit/ml (8,28). This assay has been used
RASMA HEPARINACTlVlTY(wtlml)
FIG. 3. Effects of thrombin concentration (A) and storage at -80°C (B) on relationships between neutralizing amounts of Polybrene and plasma heparin activity. The lines represent the best-fitted lines for each set of data.
POLYBRENE
NEUTRALIZATION
in pharmacokinetic studies in normal subjects (4,29) and patients (28). These studies have revealed a marked difference between the in vivo levels of plasma heparin determined by this assay and a bioassay based on activated partial thromboplastin time and suggested that the pharmacokinetics of heparin should be defined on the basis of a chemically based assay of heparin.
ASSAY OF HEPARIN
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ACKNOWLEDGMENTS This work was supported by NIH Grant HL24343. T.D.B. is a Nanaline H. Duke Scholar.
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