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PubMed1993

Characterization of the antiplasmin activity of human thrombospondin-1 in solution.

These studies demonstrate relatively rapid association of plasmin with thrombospondin and the effects of this interaction on plasmin activity towards D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) and the proteinase inhibitors alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M). Binding of plasmin to thrombospondin reached an apparent reversible equilibrium within 3 min at 22 degrees C. The amidase activity of bound plasmin was inhibited. An analysis of S-2251 hydrolysis indicated that thrombospondin is a linear mixed-type plasmin inhibitor. The dissociation constant (KD) for the binding of plasmin to thrombospondin was 0.5 microM, assuming one plasmin binding site per thrombospondin homotrimer. Plasmin and miniplasmin slowly cleaved thrombospondin, yielding products which were comparable with those generated by other proteinases. Tranexamic acid inhibited the digestion of thrombospondin by plasmin and miniplasmin, suggesting an important role for the kringle-5 domain in this process. When plasmin was incubated first with thrombospondin and then with alpha 2AP, plasmin that was apparently bound to thrombospondin reacted with alpha 2AP at a decreased rate; however, within 20 min, all of the plasmin was recovered in complex with alpha 2AP. Similar results were obtained with alpha 2M. Transfer of plasmin from thrombospondin to alpha 2AP or alpha 2M probably required plasmin-thrombospondin-complex dissociation. A low level of reaction of alpha 2AP with thrombospondin-associated plasmin could not be ruled out. These results demonstrate that the activity of plasmin, when bound to thrombospondin, is greatly diminished or eliminated. The plasmin-thrombospondin complex, which is formed within 3 min, is fully reversible and the associated plasmin is in a latent form protected from proteinase inhibitors. Therefore, thrombospondin may regulate plasmin activity in a manner which is distinct from conventional proteinase inhibitors and other extracellular-matrix proteins.

Amidohydrolases↗
PubMed1994

Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate.

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.

Adenosine Triphosphate↗
PubMed1994

Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.

Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.

Bacillus↗
PubMed1994

Binding of quinomycin antibiotic UK-65,662 to DNA: 1H-n.m.r. studies of drug-induced changes in DNA conformation in complexes with d(ACGT)2 and d(GACGTC)2.

Quinomycin antibiotic UK-65,662 binds selectively to the 5'-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5'-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.

Anti-Bacterial Agents↗
PubMed1997

Allosteric modulation of the activity of thrombin.

Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu192-->Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192-->Gln mutant. A peptide based on the sequence of hirudin (rhir52 65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192-->Gln mutant; a 5-fold decrease in the K(m) value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52-65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both alpha-thrombin and the Glu192-->Gln mutant. However, the increases in the ki values were greater with the Glu192-->Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable D-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 site of thrombin.

Allosteric Regulation↗
PubMed1991

Heparin and heparan sulphate are inhibitors of human leucocyte elastase.

1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(L-alanine)3-p-nitroanilide (50% inhibition of 250 microliters of 10 micrograms of human leucocyte elastase/ml was obtained with 80 microliters of 2.8 micrograms of heparin/ml and 8 micrograms of heparan sulphate/ml). Less significant inhibition at the same concentrations was seen with the other glycosaminoglycans tested: hyaluronic acid and chondroitin sulphates A-C. 2. Heparin and heparan sulphate also strongly inhibited human leucocyte elastase activity towards insoluble human lung elastin, as determined by an e.l.i.s.a. for soluble elastin-derived peptides released by elastolytic activity on the elastin. This inhibition was shown not to be due to a direct interference of the glycosaminoglycans in the e.l.i.s.a. nor to the inhibition causing a change in the size of the elastin-derived peptides. However, unlike the chromogenic assay with n-succinyl-(L-alanine)3-p-nitroanilide as substrate, where heparin was the more effective inhibitor, in this assay system heparan sulphate was the more effective inhibitor (50% inhibition of 100 microliters of 50 ng of human leucocyte elastase/ml was obtained with 100 microliters of 4.5 micrograms of heparin/ml and 0.8 microgram of heparan sulphate/ml). These results suggest that heparin and heparan sulphate, as components of cellular and basement membranes, are likely to have a role in protecting structural proteins, such as elastin, from the proteolytic activity of human leucocyte elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed2002

Oral yeast carriage in patients with advanced cancer.

The aim of this study was to investigate oral yeast carriage amongst patients with advanced cancer. Oral rinse samples were obtained from 120 subjects. Yeasts were isolated using Sabouraud's dextrose agar and CHROMagar Candida, and were identified using a combination of the API 20 C AUX yeast identification system, species-specific PCR and 26S rDNA gene sequencing. Oral yeast carriage was present in 66% of subjects. The frequency of isolation of individual species was: Candida albicans, 46%; Candida glabrata, 18%; Candida dubliniensis, 5%; others, < 5%. The increasing isolation of non-Candida albicans species is clinically important, since these species are often more resistant to antifungal drugs. Oral yeast carriage was associated with denture wearing (P = 0.006), and low stimulated whole salivary flow rate (P = 0.009). Identification of these risk factors offers new strategies for the prevention of oral candidosis in this group of patients.

Adult↗
PubMed2002

Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus-infected (HIV+) subjects.

Candida dubliniensis, an emerging oral pathogen, phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The aim of the present study was to evaluate the usefulness of two phenotypic methods, growth at 45 degrees C and 2,3,5-triphenyltetrazolium chloride (TTC) reduction, for confirming presumptive identification of C. dubliniensis and C. albicans by colony color on CHROMagar Candida (CAC) medium. A combination of these methods was used to establish the prevalence of oral C. dubliniensis in an Italian population of 45 human immunodeficiency virus (HIV)-infected subjects. Twenty-two samples (48.9%) were positive for yeasts on CAC medium producing a total of 37 fungal isolates. The colony color and 45 degrees C growth ability test correctly identified all C. dubliniensis and C. albicans isolates (5/37, 13.5%, and 16/37, 43.2%, respectively), while assessment of TTC reduction misidentified one C. albicans isolate. The isolation rate of C. dubliniensis was 11.1% (5/45 patients). All of the C. dubliniensis isolates were highly susceptible to fluconazole (MIC = 0.5 microg/ml). The combination of CAC medium screening with growth at 45 degrees C and TTC reduction tests may represent a simple, reliable and inexpensive identification protocol for C. dubliniensis.

Adult↗
PubMed2003

Rapid and unequivocal differentiation of Candida dubliniensis from other Candida species using species-specific DNA probes: comparison with phenotypic identification methods.

Candida dubliniensis is a recently described opportunistic pathogen which shares many phenotypic characteristics with Candida albicans but which has been reported to rapidly acquire resistance to azole antifungal drugs. Therefore, differentiation of C. dubliniensis from C. albicans becomes important to better understand the clinical significance and epidemiologic role of C. dubliniensis in candidiasis. We compared phenotypic methods for the differentiation of C. dubliniensis from C. albicans (i.e. the ability to grow at elevated temperatures, colony color on CHROMagar Candida medium, and carbohydrate assimilation patterns) to amplify the results of a polymerase chain reaction (PCR) assay using universal fungal primers to the internal transcribed spacer 2 (ITS2) region of rDNA and species-specific DNA probes in an enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 rDNA region was also conducted. The C. dubliniensis ITS2 probe correctly identified all C. dubliniensis isolates without cross-reaction with any other Candida species tested (mean A(650 nm) +/- SE, C. dubliniensis probe with C. dubliniensis DNA, 0.372 +/- 0.01, n = 22; C. dubliniensis probe with other Candida species DNA, 0.001 +/- 0.02 n = 16, P < 0.001). All other Candida species tested (C. albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) were also correctly identified by the PCR-EIA without any detectable cross-reactions among species. Phenotypically, C. dubliniensis isolates demonstrated an increased sensitivity to heat compared to C. albicans isolates. At 42 degrees C, only 50% of C. dubliniensis isolates grew compared to 73% of C. albicans isolates and, at 45 degrees C, 91% of C. dubliniensis isolates failed to grow compared to 64% of C. albicans isolates. C. albicans was more likely to demonstrate a dark green or blue green colony color on CHROMagar Candida medium obtained from Becton Dickinson (i.e. 100% of C. albicans isolates were dark green or blue green versus 64% of C. dubliniensis isolates) whereas no difference in the percentage of C. albicans or C. dubliniensis isolates producing dark green or blue green colony color was detected using CHROMagar Candida medium from Hardy Diagnostics (82% for both species). The API 20C AUX carbohydrate assimilation system incorrectly identified C. dubliniensis as C. albicans in all but three cases: remaining isolates were misidentified as C. albicans/C. tropicalis, C. tropicalis/C. albicans, and Candida lusitaniae/C. albicans. In all, 82% of C. albicans isolates and 100% of C. dubliniensis isolates assimilated trehalose; the latter finding was opposite to that reported for C. dubliniensis in the API 20C AUX profile index. Xylose and alpha-methyl-D-glucoside assimilation, respectively, were negative for 100 and 95% of C. dubliniensis isolates and positive for 100 and 91% of C. albicans isolates, confirming earlier reports that assimilation results for xylose and alpha-methyl-D-glucoside may be helpful in the discrimination of these two species. However, conventional phenotypic species identification tests required days for completion, whereas the PCR-EIA could be completed in a matter of hours. In addition, identification of Candida species by ITS1 rDNA sequencing gave 100% correspondence to the results obtained by the PCR-EIA, confirming the specificity of the PCR-EIA method. These data indicate that although a combination of phenotypic methods may help differentiate C. dubliniensis from C. albicans to some extent, the PCR-EIA can provide a simple, rapid, and unequivocal identification of the most medically important Candida species in a single test.

Candida↗
PubMed2002

Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies.

We have investigated the possibility that differences in the profile of factor VIII (FVIII) activation, by thrombin, may help to explain the one-stage/chromogenic potency discrepancies in two therapeutic concentrates. A Method M concentrate and a recombinant B-domain-deleted (B-DD) concentrate were found to have one-stage/chromogenic ratios of approximately 1.15 and 0.70, respectively, relative to the World Health Organization (WHO) 6th International Standard (IS) FVIII concentrate, whether pre-diluted in FVIII-deficient plasma or buffer (+/- von Willebrand factor, VWF). The activation of FVIII, by thrombin, was followed in a buffer medium (+/- VWF) and all three concentrates showed similar times to reach peak FVIII coagulation (FVIII:C) activity. However, despite the use of equivalent amounts of FVIII:C for all three concentrates, the B-DD concentrate reached a higher peak level and maintained higher FVIII:C compared with the WHO 6th IS throughout the incubation period, whereas the Method M concentrate reached a lower peak level and maintained lower FVIII:C throughout the incubation period. We propose that the higher levels of FVIII:C found with the B-DD concentrate and the lower levels with the Method M concentrate, following activation, may be reflected in the potencies obtained by the chromogenic method and may be consistent with one-stage/chromogenic ratios of < 1.0 and > 1.0 respectively.

Blood Coagulation Tests↗
PubMed2003

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique.

This study assessed the inter-laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio approximately 2.0 and approximately 4.0] and a further two subjects receiving treatment with unfractionated heparin (anti-Xa 0.07 i.u./ml and 0.31 i.u./ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0.96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL-300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.

Anticoagulants↗
PubMed2004

High plasma levels of factor VIII and risk of recurrence of venous thromboembolism.

The aim of this study was to evaluate the relationship between factor VIII (FVIII) levels, measured by chromogenic and clotting assays, and risk of venous thromboembolism (VTE) recurrence. A total of 564 patients underwent clinical follow-up after oral anticoagulant withdrawal (total follow-up = 924.4 years). Recurrent VTE developed in 39 of 309 (12.6%) patients with a first idiopathic VTE and in 14 of 255 (5.5%) patients whose first event was secondary. In patients with a first idiopathic VTE, the risk of recurrence was more than fivefold higher in patients with FVIII levels exceeding the 90th percentile [chromogenic FVIII: relative risk (RR) 5.43 (95% CI 1.76-16.8); clotting FVIII: RR 6.21 (95% CI 1.57-24.5)] after adjustment for all possible confounding variables. In patients with a first secondary VTE, the risk of recurrence was slightly higher in patients with high FVIII levels [chromogenic FVIII: RR 2.62 (95% CI 0.34-19.9); clotting FVIII: RR 1.74 (95% CI 0.25-12.1)], but, given the low number of recurrences, the 95% CI were very large. In conclusion, this study shows that high FVIII levels are associated with increased risk of VTE recurrence in patients with a first idiopathic VTE. Although the measurement of FVIII levels by a specific chromogenic assay might, in principle, be preferred to avoid the risk of aspecific clotting effects, no significant differences in results obtained by chromogenic or clotting methods were found.

Adolescent↗
PubMed2001

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin.

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.

Animals↗
PubMed1999

Three different chromogenic methods do not give equivalent anti-Xa levels for patients on therapeutic low molecular weight heparin (dalteparin) or unfractionated heparin.

In this study we compare three chromogenic methods (IL-Heparin, Stachrom Heparin and Heparin Sigma) on two different instruments (ACL300+ and AMAX CS190) for patients on dalteparin (n = 41), a low molecular weight heparin or unfractionated heparin (n = 50). For dalteparin the mean anti-Xa levels for IL-Heparin, Stachrom Heparin and Heparin Sigma were 0.27, 0.30 and 0.21 U/ml, respectively, while for heparin they were 0.52, 0.55 and 0.41 U/ml, respectively. To test for instrument specific effects, IL-Heparin and Stachrom Heparin were repeated on both instruments on 42 patients receiving unfractionated heparin. For IL-Heparin the mean anti-Xa levels on the AMAX CS190 and ACL300+ were 0.51 and 0.59 U/ml, respectively, while for Stachrom Heparin they were 0.55 and 0.67 anti-Xa U/ml. We conclude that different chromogenic anti-Xa methods do not give equivalent anti-Xa levels for the same samples. Moreover, the differences are clinically significant. This is not explained entirely by instrumentation effects. Recommended therapeutic ranges may need to be method and instrument specific.

Analysis of Variance↗
PubMed1996

Detection of gram-negative bacteraemia in early sepsis by a quantitative chromogenic and kinetic endotoxin assay. The Study Group.

A kinetic chromogenic limulus test was carried out in order to investigate the possibility of a sensitive and specific detection of circulating endotoxin during the first 24 h of septic shock or severe sepsis in 76 patients. Two commercial kits, Whittaeker (W) and Chromogenix (C), were used. Blood culture was taken as a reference. At 1:10 plasma dilution (a currently used dilution in the end point limulus test) abnormal reaction kinetics were found in 13% and 41% of tests, for C and W respectively (P = 0.0008), resulting in unreliable results. Retesting plasma at a greater dilution, until the reaction kinetic was identical to calibration curve control values, gave similar results between the two kits and a better accuracy. Beyond a 0.5 EU mL-1 endotoxin level, the probability of Gram-negative bacteraemia was high (sensitivity = 0.53 and 0.47; specificity = 0.95 and 0.93 for C and W respectively). This kinetic limulus amoebocyte lysate (LAL) test may be useful in therapeutic decisions for treatment of endotoxaemia.

Aged↗
PubMed2002

Single-dose pharmacokinetics of porcine factor VIII (Hyate C).

The objective was to establish the pharmacokinetic properties of porcine factor VIII (pFVIII) at three different doses in a single patient. The patient, born in 1952, had severe haemophilia A and developed an inhibitor to human FVIII (hFVIII) in 1966 aged 14 years. He was first treated with pFVIII in 1980. Apart from a short period of treatment with hFVIII in 1998 which resulted in the reappearance of the inhibitor, pFVIII has been constantly used since 1984. No inhibitor against human or porcine FVIII had been recordable over the 2-year period prior to the study. Three separate pharmacokinetic studies were performed using a washout period of 72 h and doses of 10 U kg (-1), 25 U kg (-1), or 50 U kg (-1), respectively, with sampling at preinfusion, then at 15 and 30 min and 1, 3, 6, 9, 12 and 24 h postinfusion. The FVIII levels were measured using both plasma derived one stage APTT based assay and chromogenic assay. The results were computed using a model-independent analysis and a model-dependent analysis. The respective clearances (mL h(-1)kg(-1)) at doses 50 U, 25 U or 10 U kg (-1) were 1.32, 1.33 1.8 (bioassay) and 2.54, 2.93, 9.43 (chromogenic). The respective half-lives (h) at doses 50 U, 25 U, and 10 U kg (-1) were 23.71, 16.54, 25.17 (bioassay) and 15.71, 17.39, and 10.66 (chromogenic). The respective recoveries (u dL(-1)/u kg(-1)) at doses 50 U, 25 U, and 10 U kg (-1) were 2.32, 2.44, 2.7 (bioassay) and 1.42, 1.16 and 0.9 (chromogenic). It was found that the two compartment model best fitted the curves of the bioassay and a one compartmental model best fitted the curves of the chromogenic assay. The pharmacokinetic studies are the first to be performed at different dose levels and using different assay methods for pFVIII. Using the bioassay, they show a long half-life and high recovery compared to hFVIII. The differences between the bioassay and the chromogenic assay reflect their different biological basis and are of relevance when potency labelling is performed using chromogenic assay (European Pharmacopeia).

Animals↗
PubMed2002

A blinded in vitro study with Refacto mock plasma samples: similar FVIII results between the chromogenic assay and a one-stage assay when using a higher cephalin dilution.

Assay of factor VIII (FVIII) in patient samples is routinely carried out using the one-stage assay rather than the chromogenic substrate assay. The introduction of new FVIII preparations for the treatment of haemophilia A, including immunopurified FVIII and particularly, recombinant FVIII (rFVIII) concentrates, has led to discrepancies between the results obtained with the two assays. In patients treated with rFVIII concentrates, FVIII levels measured with the one-stage assay can be 20-50% lower than those measured with the chromogenic assay. In this study, the one-stage assay was performed with cephalin dilutions higher than those recommended by the manufacturer. B-domain-deleted recombinant FVIII, Refacto, was diluted to eight different concentrations, ranging from 1-100 IU dL(-1), in FVIII-deficient plasma and the FVIII activity of the eight solutions was determined by the chromogenic method in a central laboratory. Aliquots were then assayed by the one-stage method in the four participating laboratories, using different dilutions of CK-Prest. When CK-Prest was reconstituted according to the manufacturer's recommendations (dilution 1 : 1), the difference between the one-stage and chromogenic methods was close to 30%. CK-Prest cephalin dilutions of 1 : 5 and 1 : 8 gave very similar results with the two methods, without increasing the interlaboratory coefficient of variation. These findings confirm the influence of phospholipids on the one-stage assay, particularly the importance of using a phospholipid concentration close to the physiological value in platelets. This modified one-stage method may therefore offer an alternative to the use of a concentrate-specific standard.

Chromogenic Compounds↗