m a
Anal. Chem. 1993, 65, 2078-2084
Electrochemical Sensor for Heparin: Further Characterization and Bioanalytical Applications Shu-Ching Mat and Victor C.Yang’ College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065
Bin Fu and Mark E. Meyerhoff Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109- 1055
Further studies regarding the potentiometric response to heparin of polymeric membranes doped with lipophilic quaternary ammonium salts are reported. Among a wide range of membrane formulations examined, optimum response toward macromolecular heparin is achieved using tridodecylmethylammonium chloride as the active membrane component within poly(viny1 chloride) or poly(viny1 chloride)/(vinyl acetate) films plasticized with dioctyl sebacate. Although such membranes are shown to exhibit a typical Hofmeister potentiometric selectivity pattern toward small inorganic and organic anions, a very large and reproducible response to heparin at submicromolar levels is observed in the presence of physiological saline (0.12-0.15 M NaCl), as well as in citrated whole blood. Equal response on a mass basis occurs with fragments of heparin as small as 2500 Da. Complexation of heparin’s anionic sites by macromolecules that bind heparin with high affinity (e.g., protamine and poly@-lysine)) reduces the membrane electrode’s heparin response, indicating that the electrode detects biologically available (unbound) heparin levels in solution. Quantitative potentiometric titrations of protamine with porcine mucosa heparin followed by the sensor yield stoichiometric values in good agreement with the literature. The biomedical utility of the sensor is demonstrated by measuring its response in whole blood from patients undergoing open heart surgery before and after heparin therapy and correlating such response to conventional blood clotting time measurements. Heparin is a complex, highly negatively charged polysaccharide with an average molecular weight of 15 OOO. It was the second major biopolymericdrug introduced (after insulin) more than a half century ago.13 To date, heparin has become the anticoagulant reagent of choice to prevent blood clotting in many clinical procedures. Approximately 33 metric tons of heparin, representing 500 million doses, are used worldwide each year. Currently available heparin assays are based on the measurement of plasma or whole blood clotting time4 or
* To whom correspondence should be addressed.
t Current address: NovaBiomedicalCorp.,200PrmpectSt., Waltham, MA 02254. (1)Danishefsky, I.; Rosenfeld, L.; Kuhn, L.; Lahiri, B.; Whyzmuzis, C. In Heparin andRelatedPolyaaccharidea,Structure anddctiuities; Annals of the New York Academy of Science 556; Ofosu, F. A., et al., Eds.; The New York Academy of Sciences: New York, 1989;p 29. (2)Linhardt, R.J. Chem. Ind. 1991,45. (3)Roden, L.;Feingold, D. S. Trends Biol. Sci. 1986,10, 407.
the detection of the proteolytic activity of certain clotting factors using synthetic substrates6 (e.g., anti-Xa chromogenic assay, etc.). Although these methods are well accepted by clinicians, from an analytical perspective, they are indirect, lack a defined biochemistry (e.g., clotting time assays), and yield results that are not necessarily relevant to the total therapeutic quantity of the heparin drug.4.6 A more specific analytical method for direct monitoring of the heparin molecule, used in conjunction with these existing indirect biological activity measurements, could provide a more complete measure with respect the true status of heparin therapy. Recently? we reported preliminary results demonstrating that specially formulated poly(viny1 chloride) (PVC) membranes doped with heparin ion pairing reagent, tridodecylmethylammonium chloride, exhibit a rather large and reproducible potentiometric response to heparin, with surprising selectivity over other sulfated polymers, and small inorganic anions (e.g., chloride). Herein, we provide more details on this sensing system, particularly with respect to the effect of membrane composition (quaternary ammonium salt, plasticizer, polymer matrix, etc.) and the size/source of the detected heparin species on the magnitude of the potentiometric responses observed. We further explore the potential bioanalytical utility of this sensor, both for studying the interaction of heparin and heparin fragments with other macromolecules and as a potentially valuable new biomedical device for monitoring the levels of heparin in whole blood during open heart surgery and other clinical procedures where heparin is used for systemic anticoagulation.
EXPERIMENTAL SECTION Reagents. Tetramethylammonium bromide, tetramethylammonium chloride, tetramethylammonium perchlorate, and dioctyl sebacate were purchased from Fluka Chemika-Biochemika (Ronkonkoma,NY). Tetrapentylammoniumbromide, tetraoctylammonium bromide, tributyl phosphate, dibutyl phosphate, and low molecular weight poly(viny1chloride)(PVC;MW 90 OOO) were products of Aldrich Chemical Co. (Milwaukee, WI). Triethylphenylammonium iodide and trimethylphenylamonium iodide were obtained from Eastman Chemical Co. (Kingsport, TN), and dimethyldioctadecylamonium bromide and dioctyl phthalate were products of Kodak (Rochester, NY). Hexaoctyltrimethylammonium bromide was obtainedfrom Sigma Chemical Co. (St. Louis, MO). Dicapryl adipate, di-n-hexyl azelate, dipropyleneglycol dibenzoate, tri-n-butyl citrate, 2-ethylhexyl epoxytallate, glycerol triacetate, dioctyl maleate, tri-(noctyl, n-decyl) trimallitate, methyl oleate, isopropyl palmitate, tert-butyl phenyl diphenyl phosphate, butyl octyl phthalate, diisooctyl phthalate, glyceryl triacetyl ricinoleate, dibutyl se(4)Walenga, J. M.; Fareed, J.; Hoppensteadt, D.; Emanuele, R. M. CRC Crit. Rev. Clin. Lab. Sci. 1986,22,361. (5)Yin, E. T.; Weesler, S.; Butler, J. J. Lab. Clin. Med. 1973,81,298. (6) Ma, S.;Meyerhoff, M. E.; Yang, V. C. Anal. Chem. 1992,64,694.
0003-2700/93/0365-2078$04.00/0 @ 1993 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
bacate, isopropylisostearate,and diethyl succinate were products of Polyscience,Inc. (Warrington, PA). Glycerol triacetate, poly(vinyl chloride (VC)/(vinyl acetate) (VAc)/(vinyl alcohol)(VA) terpolymer, poly(viny1 butyrate) (PVB) of different molecular weights,polystyrene (PS),and poly(viny1chloride)/(vinylacetate) (VC/VAc)copolymers were purchased from Scientific Polymer Products, Inc. (Ontario, NY). Tecoflex polyurethane (SG-80A) was from Thermedics, Inc. (Wobum, MA). Siliconerubber (SR) 3140 was a product of Dow Corning (Midland, MI). Solid heparin (sodium salt from porcine intestine mucosa, 169 USP units/mg) was from Hepar Industries, Inc. (Franklin, OH), and injectable heparin (from beef lung, lo00 units/mL) was a product of Upjohn (Kalamazoo, MI). Heparin fragments of different molecular weights, obtained by nitrous acid hydrolysis of heparin, were graciously provided by the Institute of Choay (Paris, France) and Pharmuka Laboratories (Gennevilliers, France). Smaller heparin fragments (tetra- and hexasaccharides F3, F4, and F5), prepared by enzymatic degradation of heparin with flavobacterial heparinase, were kindly provided by Dr. R. Linhardt's laboratory at the University of Iowa.7.8 The disaccharide a-uronic acid[ 144]glucosamine sulfonate, protamine sulfate, and poly(L-lysine)were products of Sigma Chemical Co. All other reagents were of analytical grade. Solutions were prepared with deionized water. Preparation of Polymer Membranes and Evaluation of Membrane Electrode Responses. Polymer membranes were cast using the conventional method for ion-selective electrode (ISE) membrane preparation." Unless otherwise stated, tetrahydrofuran (TI-IF)was used to dissolve membrane components to prepare homogeneouspolymer solutions. Typically,a solution of ca. 1.5 mL (containing 200 mg of ingredients) was added to aglass ring (24-mmi.d.) placed on aglass slide. After the solvent was evaporated at room temperature for 8 h, smaller disks (5mm i.d.) were cut from the larger membrane and were incorporated intoPhillips electrodebodies (IS-561,Glasblaserei Moller, Ziirich, Switzerland). A 15 mM NaCl solution was used as the internal filling solution. The potentiometric response of all electrodes was measured relative to an external Ag/AgCl doublejunction reference electrodeat room temperature (22"C). Sample solutions (saline solution or whole blood samples) were stirred slowly with a magnetic stirrer during the emf measurements. To generate solutions with different heparin levels, small aliquots of a standard, concentrated heparin solution were added to a fixed volume of saline or blood. Titrations of protamine were performed in a similar manner with microvolumes of a stock heparin solution(lo00 units/mL) added to a known concentration of protamine sulfate prepared in 0.12 M NaC1. Potentiometric selectivity coefficientsof the optimizedheparin membrane toward small inorganic anions were determined using the separate solution method.1° Whole Blood Measurements. Whole blood samples were drawn from patients undergoing open heart surgery. Samples for electrode measurements were drawn into citrated Vacutainer tubes (Becton Dickinson & Co., Rutherford, NJ) and stored at 4 "C until the emf measurements were made. At the same time, separate samples of whole blood were drawn into CA 510 blood collectiontubes for immediate measurement of blood activated clotting time (ACT) using a Hemochron system (International Technidyne Corp., Edison, NJ). For correlation studies, three samples of blood were obtained for each measurement during the course of the open heart procedure. Initial blood samples, taken prior to administering heparin to the patient, were used to obtain baseline ACTS and initial potentiometric response values. The second samples were taken after the patient had been systemically heparinized to the extent desired by the clinician in charge of the procedure. The third samples were obtained after administering the heparin antagonist,protamine, (7) Merchant,Z. M.; Kim, Y. S.; Riu, K. G.; Linhardt, R. J. Biochem. J. 1986,229,369. (8)Rice, K. G.; Linhardt, R. J. Carbohydr. Res. 1989,190,219. (9)Cragps, A.;Moody, G. J.; Thomas, J. D. R. J. Chem. Educ. 1974, ~~
61,641. (10)Guilbault, G. G.;Durst, R. A.; Frant, M. S.; Freiser, H.; Hansen, E. H.; Light, T. S.; Pungor, E.; Rechnitz, G. A.; Rice, N. M.; Rohm, T. J.; Simon, W.; Thomas, J. D. R. Pure Appl. Chem. 1976,48,127.
2079
Table I. Effect of Quaternary Ammonium Anion Exchanger Structure on Magnitude of Potentiometric Responses toward Porcine Heparind membrane AE (mV) cationic species
61 Q2 63
Q4 65 Q6
Q7 Q8
69 QlO Qll Ql2
Q13 Q14
Q15 Q16 Q17 a
0.4 -3.6 -4.4
-10.6 -15.4 1.9 0.5
-6.6 2.1
0.2 -17.0 -3.0
-43.4 -40.4
tetradodecylammonium triethylphenylammonium tetrapentylammonium trimethylphenylammonium dimethyldioctadecylammonium
tetraoctylammonium tetramethylammonium (chloride) hexadecyltrimethylammonium
tetraethylammonium trimethylphenylammonium tetramethylammonium (bromide) tetrabutylammonium tridodecylmethylammonium
Aliquat 336 (trioctylmethylammonium) Azure A dye protamine sulfate polybrene
All are PVC membranes preparedwith the followingcomposition:
32 wt % PVC, 65 wt % DOS,and 3 wt % ' quaternary ammonium salt. The AE is the electrode's millivolt change upon the addition of 6.9 units/mL (rial concentration of 40.8 fig/mL) to a 0.15 M NaCl
*
solution.
~~~
to the patient. In all cases, the samples tested by the electrode method were unidentifiablewith respect to the individual patient.
RESULTS AND DISCUSSION Effect of Membrane Formulations on Potentiometric Heparin Response. In our preliminary communication6 we summarized, without providing detailed quantitative data, our general observations regarding the effect of membrane composition on the magnitude potentiometric response to heparin observed when PVC films are doped with various quaternary ammonium species, at different ratios of polymer/ plasticizer/quaternary salt. The preferred membrane composition for heparin sensing (for maximum sensitivity) was reported to be 66 wt 7% PVC, 32.5 w t 7% dioctyl sebacate, and 1.5 wt 7% TDMAC. The choice of this specific formulation evolved from a very large number of careful experiments in which the potentiometric response to heparin was assessed by varying each component in the membrane. In these initial screening studies, electrodes possessing membranes with more typical ratios of plasticizer/PVC were examined (i.e., 2 1ratios used in conventional polymer membrane ion-selective electrodes") in a background electrolyte of 0.12 or 0.15 M NaC1. Beyond testing various quaternary ammonium species, other cationic molecules and polymers with known heparin-binding interactions were also investigated (e.g., Azure A dye, polybrene, protamine). Table I summarizes the results of these exploratory studies. Membranes doped with Azure A, protamine, and polybrene (membranes Q15, -16, and -17, respectively), did not yield stable potentials in the background NaCl electrolyte, apparently because these species are rather hydrophilic and leach rapidly from the polymer membrane matrix into the aqueous test solution. On the other hand, membranes formulated with various quaternary ammonium compounds (membranes Ql-Ql4) did exhibit stable response in saline upon subsequent additions of porcine heparin to the test solution (at a concentration of 6.9 units/mL or 40.8 pg/ mL). For those quaternary ammonium species with short alkyl chains (e.g., membranes Q2-4,Q7, and Q9-121, potentiometric response to heparin was relatively small and in some cases even positive (cationic) in direction. This result correlates well with the observations of Grant et al.12 who (11)Morf, W.E. The Principles of Zon-Selective Electrodes and of Membrane Transport;Elsevier Science Publishing Co.: New York, 1981.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
reported that the affinity of heparin with small aliphatic quaternary ammonium species is weak. For quaternary ammonium species with longer alkyl groups (e.g., membranes Q1, -5, -6, -8,-13, and -14) it was found that the presence of at least one methyl group is critical to achieve significant heparin response. For example, the membrane potential of polymer membrane Q14 (doped with Aliquat 336, Le., trioctylmethylammonium sites) changes more than -40 mV when heparin is added (at a final concentration of 6.9 units/mL (40.8 pg/mL or ca. 2.7 X 10-6M)) to the background 0.15 M NaCl solution, while the potential change for membrane Q6 containing tetraoctylammonium sites is actually slightly positive (+1.9 mV). A similar result is observed when the responses of membranes Q1 and Q13 (tetradodecylammonium vs tridodecylmethylammonium) are compared. Clearly, as stated previously,6 some accessibility to the positively charged nitrogen center is required for the interaction of macromolecular heparin with the quaternary ammonium species a t the surface of the polymer membrane. At the same time, the specific structure of the quaternary ammonium species (i.e., three vs two long-chain alkyl groups, a phenyl vs alkyl group, etc.) doped within the polymeric membrane is also a very critical factor in determining the magnitude of the potentiometric response to heparin (e.g., see relatively small anionic responses to heparin of membranes Q4, Q5, Q8, and QW. The effect of concentration of quaternary ammonium sites on the magnitude of the potentiometric heparin response was also investigated. Unlike conventional ISE polymer membranes," where above a certain critical concentration of membrane ionophore (e.g., valinomycin for K+ sensor, etc.) response to the selected small cations or anions is essentially unchanged (i.e,, Nernstian), varying the concentration of heparin ion pairing agents, Aliquat 336 or TDMAC, in the PVC membranes does significantly affect the resulting membrane electrode's response toward heparin. For a series of PVC membranes prepared with similar levels of PVC (ca. 32 w t 7% ), similar concentration of plasticizer (dioctylsebacate) (ca. 65 wt %), and various concentrations of Aliquat 336 or TDMAC (ranging from 0.05 to 6 w t %), it was found that the electrode's responsetoward heparin is descreased dramatically if the concentration of Aliquat 336 is less than 2 w t % and if the concentration of TDMAC is not within the range of 1.0 and 3.0wt 7% (data not shown). Similar results were obtained in further studies when polymer membranes were prepared with the preferred composition of less plasticizer and more PVC (i.e., ca. 33 wt 5% plasticizer and ca. 65 wt 76 PVCP (see also below). To further optimize the composition of a heparin-sensing membrane, a series of different plasticizers were used to cast PVC films using Aliquat 336 as the heparin-sensing quaternary ammonium exchanger. As summarized in Table 11, Aliquat 336-based membranes plasticized with isopropyl palmitate (IPP;membrane PlO),isopropyl isostearate (IPIS; membrane P16), and dioctyl sebacate (DOS;membrane P20) yield the largest response to heparin in saline solution. Additional studies, however, indicated that IPP and IPIS were not completely compatible with the PVC membranes; Le., a portion of these plasticizers was exuded out of the bulk phase of the membrane and onto the surfaces when the solvent was evaporated. Dioctyl sebacate was therefore chosen as the optimum plasticizer for all subsequent studies. The effect of polymer membrane matrix itself on the potentiometric response to heparin has also been examined. As shown in Table 111, among the various membranes cast with DOS (at low plasticizer levels) and TDMAC (at 1.5 wt (12) Grant, D.; Long, W. F.; Williamson, F. B. Biochem. J. 1992,285,
477.
Table 11. Effect of the Plasticizer on Potentiometric Response of PVC Membrane toward Heparineb plasticizer membrane AE (mV) P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25
-30.0 -28.5 -18.8 -5.7 -29.3 0.0 -29.5 -25.5 -26.0 -42.9 -0.3 -28.1 -32.7 -27.6 -25.2 -49.6
-40.4 -29.4 0.0 -10.4 -12.4 0.0
dicapryl adipate di-n-hexyl azelate dipropylene glycol dibenzoate tri-n-butylcitrate 2-ethyl hexyl epoxytallate glycerol triacetate di(2-ethylhexyl maleate)dioctylmaleate tri(n-octyl,n-decyl) trimallitate methyl oleate isopropyl palmitate tert-butyl phenyl diphenyl phosphate butyl octyl phthalate diisooctyl phthalate glyceryl triacetyl ricinoleate dibutyl sebacate isopropyl isostearate diethyl succinate glycerol triacetate dimethyl phthalate dioctyl sebacate dioctyl phthalate tributyl phosphate dibutyl phthalate o-nitrophenyloctyl ether 5-phenyl phosphate
PVC membraneswere prepared with following composition: -32 plasticizer, and -3 wt % Aliquat 336. The AE is the electrode's potential change toward the addition of 6.9 unita/mL (final concentration of 40.8 pg/mL) of porcine heparin to a 0.15 M NaCl solution. a
*
wt % PVC, -65 wt %
Table 111. Effect of Polymer Membrane Matrix on Potentiometric Response to Heparin*b membrane AE (mV) polymer matrix
c1 c2 c3
-48.0 -48.0 -1.3
c4 c5 C6 c7 C8 c9 c10
-1.2 0.0 0.0 -8.1 -16.5 -19.1 0.0
c11
-48.0
c12 C13
0.0 -60.0
PVC, MW 275 OOO PVC, MW 90 OOO VC (91 % )/VAc (3%)/VA (6%) terpolymer, MW70000 poly(viny1 butyrate), MW 305 OOO poly(viny1butyrate), MW 220 OOO polystyrene, MW 280 OOO PU, Tecoflex SG80A PU, 113090C, 16% HzO uptake VC (81% )/VAc (17 %) copolymer (2% maleic acid),MW 30 OOO VC (90%)/VAc (10%) copolymer, MW 115 OOO PVC/PVOH SRc
0 Polymer membranes were prepared with the following composition: -65 wt % matrix, -32.5 wt % DOS,and 1.5wt % TDMAC. b The AE is the potential change of electrode upon the addition of 1.4 unita/mL (final concentration of 8.29 pg/mL) of heparin to 0.12 M NaC1. This membrane was cast with THF solution of SR and TDMAC only; no plasticizer was added.
76 ), membranes prepared with either PVC (high and low MW; C1, C2) or a block copolymer of PVC and poly(viny1acetate) (VC (90%)/VAc(lO%);Cll)yield thelargestanionicresponse to heparin. Note that the emf responses in these matrix experiments were measured after adding only 1.4 units/mL of porcine heparin (8.3pg/mLor 5.5 X lO-7M) to a background of 0.12 M NaC1, yet the magnitude of the emf changes observed for the PVC- and VC/Vac-based membranes is actually larger than those listed in Tables I and 11, where the response to a much higher concentration of heparin (6.9 units/mL) was used to assess performance. This differencein relative heparin response between these studies emphasizes the critical effect of the plasticizer/polymer ratio on the magnitude of the
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
heparin signal. That is, data for preliminary studies shown in Tables I and I1 were obtained using membranes with the conventional 2:l ratio of plasticizer/polymer whereas membranes used to obtain the data in Table I11 contained a 1:2 ratio of plasticizer to polymer (except for silicone rubber membrane; see below). We have found repeatedly that while PVC membranes formulated with conventional plasticizer/ polymer ratios yield modest heparin response, such response occurs only at concentrations of heparin of >1unit/mL (in saline solution). This may explain why significant positive interference from heparin has not been reported previously for some chloride ion-selective electrodes (formulated with similar quaternary ammonium species in conventional,highly plasticized PVC membranes) used to detect chloride in undiluted blood samples containing low levels of heparin.13 On the other hand, membranes with lower plasticizer/polymer ratios respond to heparin in saline a t much lower levels (4 unit/mL; see Table I and Figure 3a in ref 6). Beyond both high and low molecular weight PVC, and the VC/VAc copolymer,potentially useful response to heparin is also observed with a silicone rubber membrane (C13in Table 111) cast from THF and TDMAC only (no plasticizer). However, unlike the PVC and Vc/VAc matrices, response of the silicone rubber membranes to heparin was reduced dramatically in the presence of endogenous anions and proteins found in plasma and whole blood samples,and further experiments with this membrane were not continued. Interestingly, it has also been found that the optimum membrane formulation for heparin sensitivity is highly dependent on the specific polymer material employedfor membrane casting. For example, as shown in Table 111, membranes prepared with various polyurethanes (C7-C9) exhibit relatively small responses to heparin when formulated with the optimum 1:2 plasticizer/polymer ratio found for PVC-based films. However, if the plasticizer/polymer ratio for these materials is increased to conventional 2:l levels, a much larger heparin response, similar to that observed with PVC films can be achieved (data not shown). All of the above data point to the fact that the potentiometric response to heparin of polymeric films is dependent in a very complex way on the specific structure of the dopant quaternary ammonium species, its concentration in the membrane, and the exact nature and composition of the surroundingmembrane matrix. Indeed, as stated previously,6 preferred selectivity of heparin to its structural analogs and its sulfated glucosamine residues (i.e., sulfated monosaccharides) may be due to the ability of heparin's many sulfate groups to interact simultaneously with a large number of relatively immobile, positively charged trialkylmethylammonium sites at the surface of the hydrophobic membrane, and it is this multidentate charge-charge interaction that inducesthe phase-boundarypotential changes a t this interface (see Figure 1). The equilibrium constant for this type of multisite surface interaction appears to be quite large and influenced greatly by the materials used to prepare the heparin-sensitive membrane. While we previously suggested, on the basis of electrodialysis experiments with fluorescently labeled heparin: that heparin molecules can actually migrate into the bulk of the polymer membrane, efforts to reproduce these earlier observations have yielded contradictory results. After prolonged exposure (24 h) to a strong electric field (120 V) placed across a stack of thin heparin-sensing membranes, a new preparation of fluorescently labeled heparin was observed only to move onto the outer surface of the membrane in contact with the labeled heparin solution (after washing), not into the bulk of any of the interior membranes. This discrepancy in results may be due to the fact that the (13) Byme, T. P. Sel. Electrode Rev. 1988, 10, 107.
CI
-
ct-
+
I
-I
-I CI
2081
Heparin
0
0
-
0
0
-
I 0
0
J.
CI -
Flgure 1. Schematic representation of how low concentrations of macromolecular heparin may interact with quaternary ammonium groups in the surface of a polymeric membrane to yield a large potentiometric anion response in the presence of physiological levels of chloride.
fluorescein-labeled heparin preparation used in the earlier experimentscould have contained small levelsof unconjugated fluorescein, or fluorescein attached to some lower molecular weight fragments of heparin, and it may have been these fluorescent lower molecularweight species that were observed to move into the organic membrane phase. Obviously,there are still many mechanistic questions remaining with respect to how the heparin molecule interacts with the membrane to yield a large change in membrane potential, and the influence of membrane composition on such heparin response. At this point, for further bioanalytical studies, a membrane composition of 66 wt % PVC, 32.5 wt % DOS, and 1.5 wt % TDMAC has proven most useful for all heparin-sensing applications (note: membranes containing Aliquat 336, although providingan equallylarge heparin response in saline, exhibit a significant nonspecific background signal when in contact with plasma or whole blood6). Response t o Small Anions. It is important to note that similar anion-selective membrane electrodes have been reported previouslyin the literature, primarily for the detection of small anions (e.g., SCN-, C104-, NOS-,and even Cl-).l4--17 Indeed, in theory and in practice PVC membranes doped with lipophilic quaternary ammonium sites (dissociated ion exchangers) will always exhibit a typical Hofmeisterselectivity pattern toward small inorganic test anions.llJ8J9 The optimized heparin-responsive membrane described here behaves no differently. The logarithm of the potentiometric selectivity coefficients for citrate, Nos-, B r , HC03-, s 0 k 2 , C032-,OAc, H,PO4-, ClOr, and salicylate are -1.91,0.94,1.6,0.00, -0.98, -0.74, -1.30, -1.00,5.00, and 4.10, respectively. This correlates with the expected Hofmeister series selectivity pattern, with (14) Wegmann, D.; Weiss, H.; Ammann, D.; Morf, W. E.; Pretsch, Sugahara, E. K.; Simon, W. Mikrochim. Acta 1984,3, 1. (15) Choi, K. K.; Fung, K. W. Anal. Chim. Acta 1982,138, 385. (16) Ishibashi, N.; Jyo, A.; Matsumoto, K. Chem. Lett. 1973, 1297. (17) Jyo, A.; Torikai, M.; Ishibashi, N. Bull. Chem. SOC.Jpn. 1974,47, 2862. (18) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888,24, 247. (19) Oesch, U.; Ammann, D.; Pham, H. V.; Wuthier, U.; Zund, R.; Simon, W. J . Chem. SOC.,Faraday Trans. 1 1986,82,1179.
2082
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
Table IV. Comparison of Potentiometric Responses toward Porcine Heparin Fragments of Different Molecular Weights and Intact Beef Lung Heparin. same molar same mass conc AEb concAE heparin (MW) (mV) (mV) porcine mucosa heparin (15 000) heparin (9800) heparin (4500) heparin (2500) beef lung heparin (15 OOO) disaccharide (uronic acid-2-sulfaminoglucose (461)) F3 fragment (1227)d F4 fragment (1333)d F5 fragment (1836)d
-42 -37 -17 0 -53
95 85 75 65
-42‘ -42‘ -42‘ -42‘
oe -17e -27‘ -22e
*
4 The AE was measured in 0.12 M NaCl solution. In solutions of the same molar concentration that were equal to 3.94 X lo-’ M. This concentration of heparin 15 OOO (from porcine intestine mucosa) is equal to a solution with biological activity of 1 unit/mL (6 pg/mL). In solutions of the same weight percent concentration equal to 6 pg/mL. This concentration is equivalent to approximately 1 unit/ mL porcine mucosa heparin (15 OOO) activity. Refer to rep for structures of these tetra and hexasaccharide structures. e Response shown is for 10 pg/mL concentrations of these smaller fragments.
55 45
>
35
E
95 85 75 65 55 45 35
rl 500
the greatest response toward perchlorate. Citrate was tested because it is often used as an anticoagulant reagent in commercial blood collection tubes (e.g., Vacutainer tubes) or bags. Generally, a small amount of concentrated citrated solution is present in blood tubes or bags. The resulting citrate concentration in the whole blood sampled into these containers can approach 0.0125 M. Because of the uncertainties in how to treat a polyanionic molecule such as heparin with respect to classical calculations of potentiometric selectivity, selectivity coefficients relative to the membrane’s heparin response are not determined; however, the fact that a large heparin response is observed a t heparingw > heparinlm > heparinm. On the other hand, almost identical potentiometric response (-42-mV change) was obtained for solutions of these different fragments that were prepared at the same weight concentration (6 pg/mL). The heparin fragments generated by hydrolysis with nitrous acid are simply shortened polysaccharide pieces of the original heparin molecule.27@ Samples of same molar concentration, therefore,result in solutionsof different weight concentration. Samples prepared with the same weight concentration yield solutionsof same totalpolymer sulfate content. As also shown in Table IV, much smaller di-, tetra-, and hexasaccharide fragments of heparin (MW range of 400-1800), formed by the action of heparinase enzyme (see compound fragment identifications in ref 8) have also been tested, and the magnitude of response to these smaller fragments is greatly diminished. Again, these results suggest that the simultaneous ionic interaction of many sulfate groups with cationic tridodecylmethylammonium sites at the surface of the polymer membrane is responsible for the electrode’sresponse to heparin (see Figure 1) and that there is a minimal polysaccharide chain length required to obtain the observed maximum response to the polyanions (somewhere between 2500 and 1800). Furthermore, it appears that most of these ionic interaction is from the sulfate/sulfonate groups on glucosamine residues since the electrode does not yield any potentiometricresponse toward de-N-sulfonated heparin even at relatively high concentrations (e.g., 20 pg/mL; data not shown). Interestingly, as also shown in Table IV, the electrode’s response toward intact beef lung heparin at the same mass concentration is somewhat greater than the sensor’sresponse to porcine mucosa heparin. This is likely due to the difference in sulfate/sulfonatecontent of heparins isolated from different sources. It is known that the beef lung heparin contains 85% of the trisulfated/sulfonated disaccharidesequence illustrated in Figure 3 (a-L-iduronic acid 2-sulfate [1+41 2-deoxy-2sulfamino-a-D-glucose6-sulfate) while porcine intestine mucosa heparin contains only 75 % of this unit on a weight basis.29 This accounts for the fact that the stoichiometry for the interaction of beef lung heparin with protamine is somewhat less than that for porcine heparin (about 20% less of beef heparin is required to neutralize a given amount of prota(27) Dietrich, C. P.; Silva, M. E.; Michelacci, Y. M. J. Biol. Chen. 1973,248,6408. (28) Linker, A.; Hovingh, P. Methods Enzymol. 1972, 28,902. (29) Casu, B. In Heparin and Related Polysaccharides, Structure and Activities; Annals of the New York Academy of Science 656; Ofosu,F. A., et al., Eds.; The New York Academy of Sciences: New York, 1989; P 2.
0~03~Ns03F W e 3. Repeatingtrisuifated/sulfonateddisaccharideunit (a+-iduonic acid 2-sulfate [ 1+4] 2deoxy-2-suifamlno-a+glucose &sulfate) in
heparin structure that appears to be critical for membrane electrode’s response.
01
10
S A
-l01 -20
1
U
-30
00
-
~
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ACT,Sec Figure 4. Potentiometricresponse signal(A€) plotted against the blood clotting time (ACT) for human whole blood samples: (A) blood samples of patients administered porcine Intestine mucosa heparin and (6)
blood samples of patients given beef lung heparin; (A) before administering heparin (baseline, 0 mV); (0)after systemic anticoag ulation with heparin; (0)after administering protamine. The average normal blood clotting time is 120 s.
minel.23 The fact that the polymer membrane electrode appears to respond to different heparins in proportion to their chemicalbinding activity suggeststhat the sensor could be used to monitor heparin levels in physiological samples. The response time of the membrane electrode toward heparin at different concentrations and of various molecular weighta was examined. The response time appears to be concentration dependent, becomingshorter at higher heparin concentrations. The t96 values, which represent the time required to achieve 95 % of the potential change to the steady state, were determined to be 7.0,4.0,3.5,2.7, and 1.5 min for heparin at concentrations of 0.5,0.8, 1.0, 2.0, and 4.0 units/ mL, respectively. On the other hand, the response time of the membrane electrode is independent on the size of heparin and remains constant (3.45 f 0.24 min) for heparins (6.0 pg/ mL) with different molecular weights ranging from 2500 to 25 000. Whole Blood Measurements. To examine this prospect, citrated blood samples were obtained from patients undergoing open heart surgery. Potentiometric measurements were made on three samples obtained during the course of each surgery: (1)before administering heparin; (2) after initiating heparin therapy; and (3)after administeringprotamine sulfate to neutralize the heparin. The electrode results are compared with the activated clotting time (ACT) measurements in Figure 4A,B. The data in Figure 4A are for patients given porcine heparin, while those in Figure 4B are for patients treated with beef lung heparin. The choice of heparin used for a given procedure is primarily determined by the surgeon’s
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 15, AUGUST 1, 1993
personal preference. As shown in both figures, a significant negative potential change is observed (compared to the baseline signal; normalized to 0 mV for the initial blood samples (no heparin added)) when the membrane electrode is placed in the heparinized blood samples. At the same time, the clotting time values for blood drawn at the same time are elongated, as expected. After neutralization with protamine, the electrode signals return to near-baseline values as did the ACT values. These data clearly show that the heparin sensor exhibits a measurable signal in whole blood that correlates with whether the administered heparin has been fully neutralized by protamine. Although for all samples tested in this study, the clotting times also correlated reasonably well with the potentiometric signals, this may not always be the case. Indeed, there are often situations where blood coagulation times do not return to normal baseline values after the addition of appropriate levels of protamine. Since at present, the clinician cannot detect heparin level directly (only via clotting time measurement), more protamine is usually administered, even though all the heparin may already be fully titrated. Because excess amount of protamine itself may cause adverse effects, and since delays in properly diagnosing the route cause of elongated clotting times will likely occur when more than the required level of protamine is administered (due to protamine’s own anticoagulant activity30), it would be advantageous clinically to use the proposed electrochemical heparin sensor to determine whether or not all the heparin has been fully titrated by protamine at the termination of any procedure in which the patient is systemically heparinized. That is, if the anionic response for blood samples is still negative compared to the baseline voltage value, this would be evidence that heparin is still present. If the membrane potential in blood returns to a value close to the baseline, and ACT values are still longer than baseline values, it is likely that the patient’s hemostatic status has been partially impaired by the extracorporeal procedure (e.g., insufficient platelets, etc.), and there is no need, and actually no use, to add more protamine sulfate in an attempt to reduce clotting times. At the same time, measurements with the heparin sensor may also be valuable in predicting the amount of protamine required to terminate heparin therapy. At present, the amount of protamine administered is calculated based on the total amount of heparin added during the entire extracorporeal procedure. However, heparin is continuously metabolized in the body, and the amount present at any given time is unknown. By measuring the potentiometric response of the heparin sensor in a given blood sample, it may be possible to calculate what dose of protamine will be required to neutralize all of the heparin detected at a given point in time. (30) Hougie, C. Proc. SOC.Exp. Biol. Med. 1958, 98,130.
CONCLUSIONS In conclusion, we have documented that the specific formulations of polymeric membranes doped with quaternary ammonium heparin ion pairing reagents is critical to the overall potentiometric anion response of such membranes to heparin. This response is likely due to the simultaneous interaction of the many sulfate groups on the heparin polysaccharide chain with tridodecylmethylammonium groups (preferred ion pairing reagent) doped within the membrane. This interaction appears to be quite strong, based on the magnitude of response to very low levels of heparin in the presence of high levels of chloride and citrate anions. It has been shown that the sensor can serve as a useful tool for quantitatively monitoring the interaction of heparin with its antagonists via simple potentiometric titrations. More importantly, we have further demonstrated that the heparin sensor can be used directly in whole blood to provide valuable information about the status of heparin therapy. At present, the main limitation of the heparin-sensing polymeric membrane is that heparin’s interaction with the surface is not completely reversible without an additional rinsing step. When transferring the sensor from a solution (including blood) that contains high levels of heparin to one with no heparin, it is necessary to dissociate the heparin by first rinsing the sensing membrane with 1-2 M NaCl solution, in order to obtain the original and reproducible baseline potential values. Although this prevents application of the membrane system as a continuous real-time monitor of heparin, it is likely that the electrode could be used in a simple flow-througharrangement where automatic rinse capabilities could be incorporated for continuous regeneration of the sensor. Alternately, because of the inexpensive nature of the membrane technology employed,disposablesingle-usedevices could also be developed for rapid whole blood measurements. Efforts in both of these directions are currently in progress, as are experiments aimed at gaining more insight into the exact mechanism responsible for potentiometric response to heparin.
ACKNOWLEDGMENT We greatfully acknowledge Dr. V. Shanberge from Beaumont Hospital (Royal Oak, MI) for providing the whole blood samples, and Dr.R. Linhardt (Collegeof Pharmacy, University of Iowa) for providing the tetra- and hexasaccharide heparin fragments. This work was partially supported by grants from the National Institutes of Health, HL 38353 and GM-28882, and the College of Pharmacy Upjohn Research Award. RECEIVED for review January 22,1993. Accepted April 13, 1993.
