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Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays

Division of Clinical Chemistry, Department of Biomedicine and Surgery, Linköping University, S-581 85 Linköping, Sweden.
^a Author for correspondence. Fax 46 13 223240; e-mail tony.gojceta{at}mbox200.swipnet.se

	Abstract

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Background: Ca²⁺ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca²⁺ activity at ~1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca²⁺ buffering improves diagnostic efficacy in global diagnostic coagulation tests.

Methods: Buffering was investigated by mixing CaCl₂ and 11 candidate compounds and determining Ca²⁺ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca²⁺ activity to ~1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.

Results: Two suitable Ca²⁺ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca²⁺ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca²⁺ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P <0.005) for the original APTT.

Conclusions: Isocitrate can be used to buffer Ca²⁺ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca²⁺ activity shows signs of improved diagnostic efficacy.© 1999 American Association for Clinical Chemistry

	Introduction

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It is well known that the activity of the calcium ion, Ca²⁺, above a certain threshold value is necessary for coagulation of mammalian biological fluids such as blood, blood plasma, and synovial fluid (1)(2)(3)(4)(5). Less generally recognized is the fact that coagulation is inhibited by increased Ca²⁺ activity (2)(3)(4)(6). Thus, the coagulation reactions that occur in biological fluids require Ca²⁺ activity within a certain range; this range is ~0.5–20 mmol/L. The physiological Ca²⁺ activity of biological fluids is ~1.3 mmol/L. A motivation for the present study was the belief that the exact Ca²⁺ activity within the range could be an important factor for the diagnostic performance of laboratory coagulation tests. The starting point for our investigation was the assumption that the physiological activity, 1.3 mmol/L, is the diagnostically most relevant condition. To allow precise regulation of Ca²⁺ activity at physiological concentrations, the present study explores the possibility of buffering Ca²⁺ at such concentrations. The main goals of the study were as follows: (a) to identify substances that buffer Ca²⁺ activity in the physiological range, (b) to study the effects of the Ca²⁺-buffering substances on coagulation reactions, (c) to select the best buffer and design an activated partial thromboplastin time (APTT) procedure with final Ca²⁺ buffered to near-physiological concentrations, and (d) to investigate whether such an APTT procedure would show signs of improved diagnostic efficacy in mild bleeding disorders. The fourth goal of the study was inspired by reports that some patients with mild clinical bleeding symptoms have APTT values within the reference interval (5)(7)(8).

	Materials and Methods

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chemicals
Trisodium DL-isocitrate (cat. no. I-1252), phthalic acid (cat. no. P-2944), 2,6-diaminopurine (hemisulfate salt; cat. no. D-3289), trans-aconitic acid (cat. no. A-7376), tricarballylic acid (cat. no. T-9251), adenine (cat. no. A-8626), and HEPES (cat. no. H-3375) were from Sigma Chemical Co. Methylmalonic acid (cat. no. 17503 43) was from Fluka AG. Dimethylmalonic acid (cat. no. D16 800-9) was from Aldrich-Chemie. Iminodiacetate (cat. no. I 120-0) was from Ega-Chemie KG. Oxalic acid (cat. no. 495) was from Merck. Trisodium citrate (cat. no. 32320) was from Riedel-de Haën.

patients
Patient plasmas from which all identity-revealing labels had been removed were obtained from the routine clinical chemistry laboratory at University Hospital, Linköping. Patient plasma was obtained by centrifugation for 20 min at 2500g after collecting 9 volumes of blood into 1 volume of 0.13 mol/L citrate.

procedures
APTT and prothrombin complex activity assays were performed in compliance with manufacturers' recommendations, using the following reagents: PTT Automate from Diagnostica Stago, and prothrombin complex reagent GHI 129 from Global Hemostasis Institute AB. The Ca²⁺ concentration and pH were determined potentiometrically with an ICA 2 Ionized Calcium Analyzer (Radiometer). Coagulation assays were performed with nephelometric clot detection, using an ACL 300R from Instrumentation Laboratories. The Ca²⁺ affinity, defined as the ligand complexation strength or chelating strength, of various water-soluble organic compounds was determined at ligand concentrations of 6–50 mmol/L. The ligand was dissolved in 20 mmol/L HEPES to a concentration of ~100 mmol/L and neutralized to pH 7.3 with NaOH or HCl. One volume of 100 mmol/L ligand solution was mixed with one volume of 50 mmol/L CaCl₂ and further diluted with 150 mmol/L NaCl to obtain pH-neutral solutions with one-half the stoichiometric amount of Ca²⁺ relative to the ligand concentration. The Ca²⁺ activity and pH were determined at 37 °C, and the apparent dissociation constant was calculated.

The Ca²⁺-buffering capacities of citrate and isocitrate were investigated in a fluid composed of one part thromboplastin, two parts plasma depleted of vitamin K-dependent coagulation factors, and one part CaCl₂ buffer mixed with two parts normal human plasma, prediluted 1:7 [the final reaction mixture of a prothrombin complex activity assay (9)]. The CaCl₂ buffer contained 30–250 mmol/L CaCl₂ and 0, 30, 60, or 120 mmol/L citrate or isocitrate. This yielded final additions of 0, 5, 10, and 20 mmol/L citrate or isocitrate. The Ca²⁺-buffering capacity was calculated, between adjacent data points, as the difference in added calcium divided by the difference in the final Ca²⁺ activity. For each point at each of the three added concentrations, the relative increases in Ca²⁺-buffering capacity per each mmol/L CaCl₂ addition were calculated. The mean and SD for each triad of determinations were calculated. An APTT procedure, PTT Automate, was modified to contain final concentrations of 10 mmol/L isocitrate and 11.7 mmol/L CaCl₂. In comparison, the standard APTT procedure contained a final concentration of 8.3 mmol/L CaCl₂.

	Results

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A search for water-soluble ligands that could buffer Ca²⁺ activity in the physiological range at ~1.3 mmol/L was conducted with the emphasis that the ligand must form a soluble Ca²⁺ complex and display an apparent dissociation constant of 0.2–5 mmol/L at ligand concentrations of 5–50 mmol/L. The screening for ligands with desired properties included laboratory examination of methylmalonate, oxalate, iminodiacetate, dimethylmalonate, phthalate, diaminopurine, adenine, trans-aconitate, tricarballylate, citrate, and isocitrate. Apart from oxalate, which precipitated in the presence of Ca²⁺, all ligands except citrate and isocitrate showed apparent dissociation constants above 10 mmol/L at ligand concentrations of 5–50 mmol/L. Citrate exhibited apparent dissociation constants of 0.45–0.57 mmol/L, and isocitrate exhibited apparent dissociation constants of 3.9–4.7 mmol/L. These two ligands were further investigated for use as Ca²⁺ buffers in coagulation assays.

An initial investigation was performed to determine whether citrate and isocitrate had an effect on coagulation reactions not related to a reduction of Ca²⁺ activity. Coagulation time was determined in a fluid composed of blood plasma, thromboplastin, various concentrations of CaCl₂, and 0, 5, 10, or 20 mmol/L citrate or isocitrate. Citrate and isocitrate inhibited coagulation reactions at all Ca²⁺ activity values in the coagulation-permissive range, as shown in Figs. 1 and 2. On a molar basis, this inhibitory effect was about twice as large for citrate as for isocitrate, which was readily demonstrated by plotting the coagulation time at any Ca²⁺ activity against the ligand concentration. This inhibition limited the concentration of citrate and isocitrate that could be used for buffering Ca²⁺ in a coagulation test. At concentrations >20 mmol/L, the inhibitory effect was predominant. In addition, it appeared that a smaller reduction in Ca²⁺ activity was required to achieve anticoagulation at increased concentrations of citrate or isocitrate, which may indicate that sufficiently increased concentrations of citrate or isocitrate may block coagulation of biological fluids at physiological Ca²⁺ activities.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of added citrate on coagulation time of a fluid composed of plasma, thromboplastin, and various concentrations of CaCl2. •, no added citrate; , 5 mmol/L added citrate; , 10 mmol/L added citrate; , 20 mmol/L added citrate. Ca2+ activity was measured potentiometrically.

The data in Figs. 1 and 2 also allowed an estimate of Ca²⁺-buffering capacity at various concentrations of Ca²⁺ activity because the difference in added CaCl₂ and the difference in Ca²⁺ activity between any two experimental points were known. Using adjacent data points, the Ca²⁺-buffering capacity of the fluid with 0, 5, 10, and 20 mmol/L citrate or isocitrate was estimated for Ca²⁺ activity in the range 1–5 mmol/L. The results are shown in the insets of Fig. 3 . The increased Ca²⁺-buffering capacity was assumed to be proportional to the ligand concentration, and the data at 5 and 20 mmol/L were used in estimating the increased Ca²⁺-buffering capacity at 10 mmol/L. The mean increased Ca²⁺-buffering capacity and SD from the three estimates for both citrate and isocitrate are shown in Fig. 3 . Both citrate and isocitrate significantly increased the Ca²⁺-buffering capacity of the solutions in the Ca²⁺ activity range studied (P <0.0007 and P <0.0009, respectively, Wilcoxon matched-pairs test). Isocitrate tended to give a greater increase than citrate (P <0.09), on average, 32% greater. This additional increase notwithstanding, isocitrate was a better Ca²⁺ buffer for coagulation assays because it could be present at twice the concentration without causing excessive inhibition. Thus, compared with citrate, isocitrate yielded at least twice as much increased Ca²⁺-buffering capacity in the Ca²⁺ activity range 1–5 mmol/L.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effects of added isocitrate on coagulation time of a fluid composed of plasma, thromboplastin, and various amounts of CaCl2. •, no added isocitrate; , 5 mmol/L added isocitrate; , 10 mmol/L added isocitrate; , 20 mmol/L added isocitrate. Ca2+ activity was determined potentiometrically.

View larger version (21K): [in this window] [in a new window]   Figure 3. Ca2+-buffering capacity of citrate and isocitrate. Left inset, Ca2+-buffering capacity in a fluid composed of plasma, thromboplastin, CaCl2, and added citrate; right inset, Ca2+-buffering capacity in a fluid composed of plasma, thromboplastin, CaCl2, and added isocitrate. •, no added citrate or isocitrate; , 5 mmol/L citrate or isocitrate; , 10 mmol/L citrate or isocitrate; , 20 mmol/L citrate or isocitrate. The data are from experiments displayed in Figs. 1 and 2 , and the Ca2+-buffering capacity was calculated as described in Materials and Methods. Main panel, estimated increase in Ca2+-buffering capacity caused by addition of 10 mmol/L citrate (•) or isocitrate (). The increases are expressed as percentages.

Isocitrate was chosen to modify an APTT procedure with the aim to adjust the Ca²⁺ activity to near-physiological concentrations. As described in Materials and Methods, the APTT procedure was modified by the addition of 30 mmol/L isocitrate to the CaCl₂ solution, yielding a final isocitrate concentration of 10 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with the modified and the original APTT procedures. The mean (± SD) results for the 22 healthy individuals were 46.4 ± 4.6 s for the modified APTT and 32.5 s for the original APTT, allowing respective reference intervals (within 2 SD of the mean) of 37.2–55.6 s and 26.5–38.5 s to be estimated. The results for the 120 patients showed wide variation: 35–169 s for the modified APTT and 27–117 s for the original APTT. The deviations from the mean of the gaussian distribution expressed in corresponding SD units were 4.3 ± 5.7 for the modified APTT and 3.6 ± 5.0 for the original APTT; these deviations were statistically highly significant (P <0.005, according to the Wilcoxon matched-pairs test). The final Ca²⁺ activities of the procedures were 1.60 ± 0.07 mmol/L and 2.73 ± 0.20 mmol/L for the modified and the original APTT procedures, respectively. The pH values in the final reaction mixtures of the two procedures were nearly identical, both being 7.33–7.40.

Fig. 4 A is a correlation diagram displaying the analytical results with the two APTT procedures expressed as the deviation in SD units from the mean for 120 patients. Fig. 4A includes two lines, drawn parallel to the x- and y-axes, at the upper reference limits. Patients with abnormally prolonged APTT with the original procedure are to the right of the perpendicular line, and patients with abnormally prolonged APTT according to the modified procedure are above the horizontal line. Inspection of Fig. 4B , an enlargement of Fig. 4A , indicates that 14 patients had abnormal APTT values with the modified procedure and values within the reference interval with the original procedure. The reverse was true for five other patients. This difference, according to binomial distribution theory, was borderline significant (P <0.07, McNemar test) (10).

View larger version (20K): [in this window] [in a new window]   Figure 4. APTT. (A), in the original APTT procedure, the reaction was recalcified by the addition of 25 mmol/L CaCl2; in the modified procedure, the reaction was recalcified by the addition of a mixture of 35 mmol/L CaCl2 and 30 mmol/L isocitrate. The experimental points represent 120 patient plasmas expressed as multiples of the SD from the gaussian mean for the original procedure and the modified procedure, respectively. The intersecting lines denote the upper limits of the reference intervals, defined as the gaussian mean ± 2 SD, calculated from 22 healthy individuals. (B), enlargement of Fig. 4A showing the region near the upper limits of the reference intervals.

	Discussion

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This study investigated 11 Ca²⁺ ligands for their utility as buffers for Ca²⁺ activity in the physiological range for possible application in laboratory diagnostic coagulation procedures. Only two of the ligands, citrate and isocitrate, formed Ca²⁺ complexes with apparent dissociation constants between 0.5 and 5 mmol/L and thus were suitable as Ca²⁺-buffering agents in the physiological Ca²⁺ activity range. Citrate and isocitrate were further investigated regarding their effect on coagulation procedures and their ability to increase Ca²⁺-buffering capacity in the final reaction mixture. The study provided two reasons to prefer isocitrate to citrate: isocitrate was less inhibitory in coagulation procedures and hence could be used at higher concentrations, and isocitrate was more efficient in increasing the Ca²⁺-buffering capacity of coagulation procedures.

The study clearly demonstrated that citrate and isocitrate have an inhibitory effect on coagulation reactions that is independent of Ca²⁺ activity. Citrate and isocitrate thus may prevent coagulation of a biological fluid with Ca²⁺ activity retained at physiological concentrations.

A commonly used APTT procedure was modified by the addition of isocitrate to a final concentration of 10 mmol/L. Analysis of 22 healthy individuals established reference intervals for the two procedures. Analysis of 120 randomly selected hospital laboratory patient plasma samples with both the modified and the original APTT procedures revealed intriguing aspects related to diagnostic power. In the modified APTT, the patient samples were abnormal both to a higher degree (P <0.005) and to a higher frequency (P <0.07). These results are interesting in the light of known discrepancies between clinical and laboratory findings. A good fraction of patients that display mild clinical bleeding may test normal with APTT (5)(7)(8). The present study indicates that APTT procedures in which the Ca²⁺ activity is in the near-physiological range may have a greater diagnostic power than those with higher Ca²⁺ activity.

It is not immediately obvious why the widely used commercial APTT procedure, referred to above as the original APTT, displayed a Ca²⁺ activity of 2.7 mmol/L and not the physiological activity of 1.3 mmol/L. APTT is considered a global coagulation procedure with conditions that are, as far as practically possible, physiological at 37 °C, pH 7.3, and an ionic strength 150 mmol/L. One would then expect that the Ca²⁺ activity should be nearly 1.3 mmol/L, which is clearly not the case. It has been reported that APTT becomes more sensitive to heparin at higher Ca²⁺ activities (11). The rational for the hyperphysiological Ca²⁺ activity could be that the manufacturer has aimed for high heparin sensitivity and has been unaware of the risk for reduced diagnostic power in identifying bleeding conditions. Another possible explanation is that the APTT procedure has a built-in safety margin to hypophysiological Ca²⁺ activities because these are generally recognized as being detrimental for coagulation reactions.

In conclusion, the present study indicates a possibility to increase the diagnostic power of global coagulation assays in mild hemophilia. This increased power may be obtained by buffering the final Ca²⁺ activity to near physiological concentrations. The study also points to the possibility of more gentle anticoagulation of biological fluids in which the final Ca²⁺ activity is only moderately reduced. The study identifies isocitrate as a substance that can function as a Ca²⁺ buffer in coagulation assays and as an anticoagulant in biological fluids.

	Acknowledgments

 
We thank the staff of the Laboratory of Clinical Chemistry at the University Hospital, Linköping, Sweden, for generous cooperation. We also thank Global Hemostasis Institute AB, Linköping, Sweden, for supporting the study.

	References

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