A Diffusion-Precipitation Assay for Measuring ~eparinconcentrations S t e ~ k H. n wentlandl, Chrlstlna A. Morgan, Nersa M. Cepero, and Isabel Lorenro Houston Baptist University. Houston. TX 77074
In this article we report a simple, reliable dye-binding method of measuring heparin concentrations. Heparin is one member of a family of glycosaminoglycans (GAG'S),and is typically isolated from pig intestinal mucosa. I t is used extensively in the clinical control of blood coagulation. A key step in the coagulation cascade is the conversion of the protein fibrinogen to fibrin, which then reacts further to form a clot. This conversion is brought about by the proteolvtic enzvme thrombi, which itself is formed through a s h e s of ;nzymatic reactions. To modulate coagulatio< the circulatine blood rotei in antithrombin 3 (AT3) binds to thrombinand slowly deactivates it. But upon heparin administration a heparin-AT3 complex forms, which deactivates thrombin rapidly and thuseffectively inhibits coagulation. Heparin may be viewed as a biochemical polymer with the repeating unit of 1.-iduronic acid bonded to 2-amino-2deoxv-D-elucose.both of which are sulfated (Fie. 1). Modifialong the cation of both moieties occurs a t various chain. The negatively charged sulfate groups, appropriately positioned, are necessary for the binding to (and hence activation of) AT3. The chain length of heparin is variable. Its binding affinity for AT3 increases with increasing length, since AT3 is more likely to bind to the optimal arrangement of sulfate groups. But this correlation is by no means exact. Reference 1 is a comprehensive review of heparin structure, biosynthesis, and action. and Heparin has been analyzed by hoth coawlation (2,3) 14-13) techniaues: reference 14 is a aood renoneozvmatic --.-~ - ~ view ofboth types of methods. ~ h activated e partid thromboplastin time (APW) test is a commonly used clinical coagulation test. Of the nonenzymatic techniques, dye-bindine methods are used for simple, rapid estimations. In these methods, heparin is added to a sdution of a tbiazine dye such as toluidine blue (Fig. 2), azure A, or methylene blue. The highly negative heparin binds to these extensively conjugated dyes and shifts their absorption maximum (metachromasia (15)). The intensity of this new absorption is then correlated with heparin concentration. But this correlation is not always cpanlitative, for the heparin-dye complex can have a variahle absorption maximum. Furthermore, this metachromatic shift is not soecific for heparin; other GAG'S f o m similar complexes (5j. ~lternatel; the heparin-dye comnlex is Dreci~itatedand the decrease in unreacted dye in the supernatani quantitated spectrophotometrically. But can flocculate (6); thus special means must the precipitate - . be employed to prevent interference by tbe precipitate (16,
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In. The method we now report is based on the formation of a
heparin-dye precipitate. In our method, heparin solutions are diffused radially into an agarose gel slab impregnated with toluidine blue. The heparin binds to the toluidine blue to form a circular precipitate, the area of which is proportional to the concentration of heparin. Since no specific wavelength is measured, the variability in metachromatic 1
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446
Journal of Chemical Education
oso; Figure 1. Major repeating unit of heparin, consisting of L-Muronlcacid (left (rlgM molety. N- and C E molety. C2 sulfated)and 2-aminc-2deo~~iucose sulfated).me two moteties are joined by a 1.4-glycosldic bond (0.
Figure 2. ToluMlne blue (14).
shifts is not a oroblem. Since no clear b o u n d m between supernatant and precipitate is needed, flocculation is of no concern. This diffusion techniaue has ~reviouslvbeen aoplied to measuring fibrinogen cincentrakon (18);and correlation between spot area and concentration in electrophoretic systems has previously been demonstrated (11). Materials
Bovine serum albumin (Fraction V), toluidine blue 0, heparin (Grade 11, porcine, sodium salt, 165 USP K-1 unitslmg), agarhse (Type I, low EEO), and tris (buffer grade) were obtained from the Sigma Chemical Company. Human serum and plasma (citrated) were obtained from the authors. Procedure Tris buffer used throughout this experiment was 0.05 M, pH 8.6. A mold for the agarose gel was made by damping a Plexiglas spacer (1116 in. thick) between two clean 3- X 3-in. glass plates. The spacer was a 114-in.-widestrip that extended along the sides and bottom of the plates. To 10 mL of tris buffer waa added 1W mg agarme, and the mixture heated at the boiling point until the agarase dissolved. Three milliliters toluidine blue solution (15 mg11W mL tris huffer) was added while the agarme was cooling, and the resulting solution was quickly stirred and poured into the mold before gelation took place. The mold was set aside for at least 10 min; then the top plate and spacer were removed. Using a plastic syringe fitted with a cut-off 15-gauge syringe needle, six holes were placed symmetrically in the gel slab. Four
Figure 3. Heparin-tobidin%blue precipitation spots on humidified (wet)and (dry) aagsmse gel dabs using standard heparin solutions of 1.5, 3.0. 4.5, 6.0, 7.5. and 9.0 mg/mL his buffer.
hi&ut
heoarin . . . ~and .9.0 mdmL .. tris huffed . standard solutions (1.5.3.0.6.0. and two unknown aolutions were applied using a Drummond rnrcn,dispenser delivering 3.33 rL. and the plate was placed in a humidified chamber overnight (to keep the %arose from drying out). After this time six circular spots were formed by the precipitation of heparin with toluidine hlue. The gel may he used at this point (wet) or after it has dried to a thin film (dry). Three diameters (120' apart) from each spot were measured to the nearest 0.1 mm with a vernier caliper. The average was calculated and used to compute the area of the soot. Soot area was dotted aeainst heoarin concentration, and the heparin concentrations in the unknowns were determined from the resulting calibration eurvc. ~
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Figure 4. Calibration curves and correlation coefficients of spot area vs. agarose gel slabs. heparin concentraion hom humidified (X) and dried out (0) Data was obtained fromU?eget slabs in Figure 3.
Results and Dlscusrlon
Correlation between spot area and heparin concentration was determined by regression analysis using both wet and dry gels. Standard heparin solutions of 1.5, 3.0.4.5, 6.0, 7.5, and 9.0 mg/mL tris buffer were used in these runs. Although there was conaiderahle gel-to-gel variation in spot area for each concentration, the correlation coeff'icienm obtained from individual gels were usually greater than 0.99. Figure 3 shows the results obtained for the best drv ael and best wet gel. The measurements from these two gels were used to construct the calibration curve of spot area vs. concentration shown in Figure 4. Similar correiations (typically greater than 0.99) were obtained from standard h e ~ a r i nsolutions of these same concentrations prepared in albumin solution (4.3 mg/100 mL water), human serum, and citrated human plasma. Thus this method is applicable for measuring heparin concentration in tris buffer, albumin solution, serum, and plasma, on either wet or dry gels. However this method requires that standards and unknowns be run on the same gel, and for optimum results, that the heparin solutions be fresh. In single-blind experiments, the accuracy of this method was assessed by measuring the concentrations of known heparin solutions and comparing them to their true values. These solutions were prepared in tris buffer and in citrated human plasma as described above. Accuracy of 6% is obtainedin both media (see table). The lower limit of detection is slightly less than 1.0 mg/mL, and the upper limit of linearity is slightly greater than 9.0 mg/mL. This figure was calculated by us using thedata reported just as ws calculated the accuracy of our method.
Accuracy of Heparin Assay
Trw Val-
hbprin Concentralton (mg/mL) in Tris Buffer Experimemal % Errw
2.50 4.00 5.00 6.00 6.67 7.50
Value 2.34 3.69 4.76 5.48 6.28 7.35
Avg. % Error (f SD) = 6.0
6.4 8.0 4.8 8.7
5.6 2.0
* 2.4
Heparin CMceMralion
(mg/mL) in Huma? Plasma
True Value
Experimental Value
1.00 1.75 2.25 3.00 3.25 3.75
1.06 1.87 2.08 3.15 3.13 3.98
%
Error 6.0 6.8 7.6 5.0 3.7 6.1
Avg. % Ermr (+So)= 5.9 f 1.4
Summary
The method reported above is simple, reliable, and avoids some troublesome problems associated with dye-binding methods denendina on soectroohotomet~.I t reauires no instrumentition axh is easily adapted to iarge numbers of students. The accuraw of this method (6%) is cornoarable to that of a ~~ectrophotbmetric tohidine-blue method ( 5 7 0 ) ~ reported in the literature (19). While experiments based on our method are appropriate for all undergraduate levels, it is particularly suited for upper level biochemistry and allied health students, who have been exposed to the theory involved and are professionally interested in the clinical imdications. Even thoueh this method is applicable to measiring heparin in buffeFand in human serum or nlasma. the threat of infectious diseases prompts us to strbngly rkcommend avoiding human blood components altogether.
Volume 67
Number 5
May 1990
447
