Heat inhibition of in vitro lipolysis and 14C ibuprofen protein binding in plasma from heparinized uraemic subjects

Protein binding determination in post heparin plasma samples is complicated by the continued post heparin lipase activity, in vitro, during the binding analysis. The decomposition of lipoproteins and accumulation of nonesterified fatty acids (NEFA) results in artifically elevated free fractions of many drugs. This artefact is particularly accentuated in haemodialysis patients who are frequently hypertriglyceridaemic and receive large doses of heparin. Rapid heat treatment (60 degrees for 15 min) of plasma from heparinized uraemic subjects is shown to inhibit the in vitro lipolysis occurring during 2 hours of equilibrium dialysis at 37 degrees (ED). Mean NEFA concentrations in heat treated plasma after ED (means = 400 +/- 141 mumol/L) were not different (p greater than 0.05, n = 9) from the baseline values in fresh plasma (means 351 +/- 117 mumol/L) but were considerably less (p less than 0.005) than NEFA levels in untreated plasma after ED (means = 1025 +/- 523 mumol/L). The degree of in vitro lipolysis inhibition (92 +/- 6.6%) was very much greater than using the chemical inhibitors phenyl methyl sulphonyl fluoride, EDTA, Triton X100 or protamine sulphate. Heat treatment at 60 degrees for 15 min increased the percentage of free 14C ibuprofen in 3.5% isolated human serum albumin from 0.34% to 0.62%. Reduced binding as a result of heat treatment was not observed however in whole plasma. The percentage free ibuprofen in heat treated, whole plasma from both heparinized and non heparinized subjects (means = 1.22 +/- 0.19; n = 29) was not different (p greater than 0.05) from the percentage free determined in plasma from a non heparinized group (means = 1.16 +/- 0.23; n = 15). In contrast the % free ibuprofen in untreated plasma from heparinized subjects was markedly higher (means = 1.56 +/- 0.41; n = 24; p less than 0.05). There was a strong correlation between % free ibuprofen and plasma NEFA concentration (r = 0.8; p less than 0.005; n = 68). The heat treatment of plasma for 15 min at 60 degrees is proposed as an effective means of controlling heparin induced lipolysis in vitro and may be valuable in overcoming the post heparin binding artefact.     

Paclitaxel-HSA interaction. Binding sites on HSA molecule

Paclitaxel (trade name Taxol) is one of the world's most effective anticancer drugs. It is used to treat several cancers including tumours of the breast, ovary and lung. In the present work the interaction of paclitaxel with human serum albumin (HSA) in aqueous solution at physiological pH has been investigated through CD, fluorescence spectroscopy and by the antibody precipitate test. Binding of paclitaxel to albumin impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or bilirubin. The paclitaxel-HSA interaction causes the conformational changes with the loss of helical stability of protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the paclitaxel-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin binding site, which includes Trp 214, and high affinity bilirubin binding site located in subdomain IIA.     

Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparin interacts with a selenoprotein in human plasma

The major selenium-containing peak at gel filtration of human serum on Sephadex G-150 had a Kav of 0.19, but at chromatography of heparinized plasma this peak had a Kav of 0.01, indicating an interaction between heparin and a major selenium-containing protein. The addition of protamine abolished the effect of heparin. The heparin-interacting protein was not identical to glutathione peroxidase. The data explain some previous discrepancies on selenium distribution in plasma due to the use of different anticoagulants.     

Stereoselective effect of warfarin and bilirubin on the binding of 5-(o-chlorophenyl)-1,3-dihydro-3-methyl-7-nitro-2H-1,4-benzodiazepin-2- one enantiomers to human serum albumin

The binding of the title benzodiazepine enantiomers and its modulation by warfarin and bilirubin were studied by chromatography on human serum albumin (HSA) immobilized on Sepharose 4B, and also by a combination of ultrafiltration and circular dichroism (UF-CD) methods. In the absence of warfarin and bilirubin the binding of the benzodiazepine was not stereoselective. (S)-Benzodiazepine and (S)-warfarin mutually increased the binding of each other, while the binding of (R)-benzodiazepine was preferentially enhanced on HSA saturated with bilirubin.     

Crystal structure analysis of warfarin binding to human serum albumin: anatomy of drug site I

Human serum albumin (HSA) is an abundant transport protein found in plasma that binds a wide variety of drugs in two primary binding sites (I and II) and can have a significant impact on their pharmacokinetics. We have determined the crystal structures at 2.5 A-resolution of HSA-myristate complexed with the R-(+) and S-(-) enantiomers of warfarin, a widely used anticoagulant that binds to the protein with high affinity. The structures confirm that warfarin binds to drug site I (in subdomain IIA) in the presence of fatty acids and reveal the molecular details of the protein-drug interaction. The two enantiomers of warfarin adopt very similar conformations when bound to the protein and make many of the same specific contacts with amino acid side chains at the binding site, thus accounting for the relative lack of stereospecificity of the HSA-warfarin interaction. The conformation of the warfarin binding pocket is significantly altered upon binding of fatty acids, and this can explain the observed enhancement of warfarin binding to HSA at low levels of fatty acid.     

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Impact of Potential Blockers on Ru(III) Complex Binding to Human Serum Albumin

The effects of aspirin, vitamin B2 and warfarin as potential blockers of the ruthenium binding sites in HSA were investigated through UV/visible, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy ICP(AES). The studies on the interactions of several biologically relevant molecules with HSA have shown that drugs like aspirin or warfarin may strongly influence the interaction of serum protein with anticancer drugs. It can derive from the influence of the drug on protein conformation or binding close to binding site of anticancer drug. Aspirin, vitB2 and warfarin bind to IIA subdomain leading to partial blocking of the ruthenium binding site in HSA.     

Proton nuclear magnetic resonance study on the multimode interactions of human serum albumin with drug molecules

The interactions of human serum albumin (HSA) with a number of ligands (mostly drugs) were examined by proton nuclear magnetic resonance spectroscopy. Ligand presence-absence difference spectra of HSA solutions were measured. Nonspecifically bound drugs such as tiaramide showed difference spectrum patterns which were similar to the spectra of the drugs themselves but were broadened as to the line-widths of signals. Thus, the difference spectra of these drugs reflect only the changes in the surroundings of the drug molecules, that is, between the bound and free states. In contrast, specifically bound drugs like ibuprofen and warfarin showed difference spectra in which signals from the HSA molecule only were observed. Furthermore, according to the characteristic peaks in these difference spectrum patterns, specifically bound drugs may be classified into several groups; the drugs in the first group bind to the ibuprofen binding site, those in the second group to the warfarin binding site, and those in the third group to sites other than the warfarin and ibuprofen sites. These findings suggest that the specific binding of drugs to HSA brings about a conformational change of this protein which is specifically correlated to the binding site.     

A comparative study of the binding of l-tryptophan and bilirubin by plasma proteins

The binding of l-tryptophan and bilirubin by plasma proteins from a variety of animals has been studied. Bilirubin was bound by plasma protein(s) from all animals investigated. l-Tryptophan, on the other hand, was bound only by plasma protein(s) from warm-blooded animals. Our results in vertebrates are consistent with serum albumin being the binding protein concerned.     

Effect of holding time and temperature of bovine whole blood on concentration of progesterone, estradiol-17beta and estrone in plasma and serum samples

Bovine jugular venous blood was collected, with and without heparin, and aliquoted into 140 12-ml tubes. Four subsamples (two heparinized and two coagulated) were centrifuged immediately (time zero) and plasma or serum was aspirated and stored at -20 degrees C. One-half of the remaining subsamples were stored at 4 degrees C and the other one-half at 25 degrees C (room temperature). At 1-h intervals (0 to 24 h), 6-h intervals (24 to 72 h) and at 96 and 120 h, four subsamples (heparinized and coagulated at both 4 degrees C and 25 degrees C) were centrifuged, plasma or serum was aspirated and stored at -20 degrees C. Whole blood incubation for 1 h at 25 degrees C reduced mean plasma and serum progesterone (P(4)) concentration (P<0.05). Similarly, whole blood incubation at 4 degrees C for 2 and 3 h, respectively, reduced mean plasma and serum P(4) concentration (P<0.05). No difference was found in mean P(4) concentration between plasma and serum samples harvested from whole blood incubated at 4 degrees C or 25 degrees C. Concentration of estradiol-17beta (E(2)) and estrone (E(1)) fluctuated over time, irrespective of holding temperature. There was a blood type, heparinized or coagulated, by time interaction (P<0.01) for both E(2) and E(1) concentrations It was concluded that incubation time and temperature between collection and centrifugation of bovine blood samples influenced the assayable P(4) concentration in both plasma and serum. In contrast, incubation temperature had no effect on assayable E(2) and E(1) concentrations, but assayable E(2) and E(1) over time were differentially affected, depending on whether plasma or serum was assayed.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

Role of apolipoprotein D in the transport of bilirubin in plasma

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.     

Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q.

Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Cys34-Cysteinylated Human Serum Albumin Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.     

Preparation and characterization of heparinized multi-walled carbon nanotubes

The characteristics of heparinized multiwalled carbon nanotubes (MWNTs) were investigated in terms of the activated partial thromboplastin time (APTT) to verify the heparin activity, a carbazole assay was done to measure the content of the immobilized heparin, and the octanol-water partition coefficient was assessed to determine the lipophilicity. Two heparin-immobilized MWNTs were prepared to evaluate their differences. The first preparation method involved polymer-coated MWNTs with heparin indirectly center-point-attached. In the second approach, heparin was directly end-point-attached through its reducing end onto acid-treated MWNTs. The blood compatibility of MWNTs to which heparin was end-point-attached through its reducing end was greatly enhanced compared to that of the MWNTs onto which heparin was center-point-attached. The APTT and carbazole assay results demonstrated that heparinized MWNTs prepared through end-point attachment result in prolonged plasma-based anticoagulant activity. The blood compatibility of MWNTs heparinized by end-point attachment was not decreased up to the fourth pasteurization. Heparinized MWNTs were also studied using octanol-water partition, which should be useful for exploring heparinized MWNTs as drug carriers including delivery systems. The results of octanol/water partition on the design of heparinized MWNTs prepared by end-point attachment with a specific binding can facilitate the design of drug delivery carriers with high blood compatibility.     

Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.