Point-of-care fibrinolytic tests: the other side of blood coagulation.

Point-of-care (POC) coagulation tests with paramagnetic iron oxide particles have provided alternatives to testing previously done only in the laboratory. With this technology, POC fibrinolytic tests have followed quietly the trail blazed by POC clotting tests and have found specific applications. These include rapid verification of in vivo thrombolytic drug action by in vitro testing with subsequent quantitative drug monitoring of the systemic lytic state, and also the determination of in vitro thrombolytic drug response before the drug is actually administered, to individualize therapy by selection of the most appropriate drug. Other applications include POC coagulation factor assays associated with fibrinolysis, and most recently the POC screening of patients with fibrinolytic defects. In this latter application, plasma from cardiac catheterization (n = 19) and venous thrombosis (n = 47) patient groups were tested. Controls consisted of two independent donor pools (n = 10, n = 21) as negatives and two plasma samples with known genetic defects in the fibrinogen molecule (A alpha 554 Arg-->Cys) as positives.

The significance of non-visualization of the fetal bladder during an ultrasound examination to evaluate second-trimester oligohydramnios.

Our objective was to determine the pregnancy outcome after targeted sonography performed for the evaluation of second-trimester oligohydramnios fails to visualize the fetal bladder. A retrospective review identified patients in 1990-94 who were referred for targeted sonography from 16 to 24 weeks to evaluate oligohydramnios. The ultrasound records and photographs from the initial examination were reviewed and compared to later pregnancy outcome data obtained by reviewing delivery and neonatal records, neonatal renal ultrasound reports or autopsy information. Complete outcome data were available in 98 patients. The fetal bladder was not visualized in 29 patients (30%). Post-delivery analysis of the fetal urinary tract was performed in 25 patients either by autopsy or by neonatal renal ultrasound scanning. A severe anatomic malformation of the urinary system was present and had been detected by targeted sonography in 23 of 25 cases (92%). The remaining two fetuses were found to have anatomically normal urinary tracts at autopsy, but other pathological findings suggested that a functional derangement of the urinary system had led to non-visualization of the bladder at ultrasound examination. Non-visualization of the fetal bladder during targeted sonography to evaluate oligohydramnios indicates a severe anatomic or functional problem of the fetal urinary tract. In our study, this ultrasound finding was associated with universal fetal or neonatal death.

Selective inhibitors of monoamine oxidase. 2. Arylamide SAR.

Monoamine oxidase (MAO) exists in two forms distinguishable by substrate specificity. Inhibition of MAO A is believed to be responsible for the antidepressant activity of MAO inhibitors. A group of N-arylacetamides are highly specific inhibitors of MAO A, some with IC50 values in the 10-100 nM range. The requirements for high activity and specificity include a nearly linear tricyclic aromatic portion but a larger and a smaller central ring component. The amide group, which is best acetamido, is optimally placed para to the smaller central group. The size and shape of the aromatic moiety appear to be the major influence on activity and specificity for MAO A.

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.

Dual-enzyme cascade--an amplified method for the detection of alkaline phosphatase.

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.

The development of a novel immunoassay amplification system and its use in viral detection.

A novel amplification system has been developed for the detection of free or antibody-conjugated alkaline phosphatase. The amplification system provides a 100 fold enhancement in the detection of the enzyme, compared to direct detection with chromogenic substrates. The key to the amplification system is the dephosphorylation of a potent phosphorylated inhibitor, and the visualization of this inhibitor using a second, indicator, reaction. This system is shown to provide increased sensitivity for immunoassays detecting either herpes simplex virus or respiratory syncytial virus in clinical samples. In addition, this general concept for amplification may be applicable to a variety of other hydrolytic enzymes, and is demonstrated for the enhanced detection of beta-galactosidase.

Melanocytotoxicity and the mechanism of activation of gamma-L-glutaminyl-4-hydroxybenzene.

gamma-L-Glutaminyl-4-hydroxybenzene is converted by the tyrosinase of the common mushroom, Agaricus bisporus, to the toxic, dormancy-inducing metabolite 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one. Hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene by mammalian tyrosinase was monitored by determining tritium water release from gamma-L-glutaminyl-[3,5-(3)H[4-hydroxybenzene and occurred at only 25% of the rate found with tyrosine. The dihydroxy product of the hydroxylation reaction, gamma-L-glutaminyl-3,4-dihydroxybenzene, was not oxidized by the mammalian enzyme. Therefore, oxidation of gamma-L-glutaminyl-4-hydroxybenzene to sulfhydryl-reactive quinones by mammalian tyrosinase is an unlikely explanation for the hair depigmentation and inhibition of melanocarcinoma growth observed following administration of this compound. Cleavage of gamma-L-glutaminyl-4-hydroxybenzene by gamma-glutamyl transpeptidase releasing p-aminophenol was demonstrated. p-Aminophenol was an active depigmenting and melanocytotoxic compound. N2-Methyl-gamma-L-glutaminyl-4-hydroxybenzene was synthesized, differing from gamma-L-glutaminyl-4-hydroxybenzene only by the presence of a methylated amide linkage. This chemical modification resulted in a compound resistant to cleavage by gamma-glutamyl transpeptidase and lacking in melanocytotoxic activity. gamma-Glutamyl transpeptidase cleavage is proposed as the route for transformation of gamma-L-glutaminyl-4-hydroxybenzene into an active inhibitor of melanocytes.

The role of gamma-glutamyl transpeptidase in the nephrotoxicity of an Agaricus bisporus metabolite.

The mushroom metabolite gamma-L-glutaminyl-3,4-dihydroxybenzene (GDHB) was found to have an LD50 of 100 to 200 mg/kg in neonatal C57Bl/6J mice. Adult mice given 200 mg/kg GDHB showed histopathologic evidence of proximal convoluted tubular injury as early as 2 hours after injection, which progressed by 24 hours to profound acute tubular necrosis. Focal acinar epithelial cell necrosis in the pancreas was also observed. The time course and location of the injury suggested that appearance of the ultimate toxic metabolite could be due to cleavage of GDHB by gamma-glutamyl transpeptidase (GGTP). The reaction in vitro of GDHB with crude porcine GGTP resulted in the release of 4-amino-catechol which air oxidized to 2-hydroxy--4-iminoquinone (HIQ), a known sulfhydryl reagent and cytotoxic compound. Synthesis of N2-methyl-gamma-glutaminyl-3,4-dihydroxybenzene (MeGDHB) provided a compound whose oxidized derivatives, when compared with those of GDHB, had similar half-wave potentials and visible absorption maxima. MeGDHB was resistant to cleavage by GGTP and was without apparent toxicitiy at 2-3 times the LD50 of GDHB. Therefore, cleavage by GGTP, an enzymatic transformation accessible to GDHB but unavailable to MeGDHB, is proposed as the mechanism of activation of the mushroom metabolite. The following pathogenic sequence is indicated: 1) release of 4-aminocatechol from GDHB by the action of GGTP and 2) irreversible injury resulting both from the generation of free radicals by the autoxidation of 4-aminocatechol and from the reaction of HIQ with cellular nucleophils, particularly sulfhydryl groups.

The metabolic pathway catalyzed by the tyrosinase of Agaricus bisporus.

N-t-Butyloxycarbonyl-gamma-L-glutaminyl-2-bromo-4-hydroxybenzene alpha-benzyl ester was synthesized as a precursor to gamma-L-glutaminyl-4-hydroxy[2-3H]benzene. With this labeled compound and the previously synthesized gamma-L-glutaminyl-4-hydroxy[3,5-3H]benzene, the stoichiometry of ring substitution was determined for the tyrosinase-catalyzed metabolic pathway of Agaricus bisporus. In this pathway, gamma-L-glutaminyl-4-hydroxybenzene is hydroxylated to gamma-L-glutaminyl-3,4-dihydroxybenzene which is oxidized to gamma-L-glutaminyl-3,4-benzoquinone and a compound of previously unknown structure, "490." The results indicated that the "490" quinone was derived from gamma-L-glutaminyl-3,4-benzoquinone without further ring substitution. A base-catalyzed, nonenzymatic reaction of gamma-L-glutaminyl-3,4-benzoquinone was observed which yielded a compound with a 490 nm chromophore. gamma-Glutamyl transpeptidase cleavage of gamma-L-glutaminyl-3,4-dihydroxybenzene led to the release of 4-aminocatechol which air-oxidized to a compound with identical spectral properties to "490." The structure of "490" was thus determined to be 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one(2-hydroxy-4-iminoquinone). The tyrosinase-catalyzed hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene was found to be optimal at pH 8.0, while the enzymatic oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene was optimal at pH 6.0.

Synthesis of gamma-L-glutaminyl-[3,5-3H]4-hydroxybenzene and the study of reactions catalyzed by the tyrosinase of Agaricus bisporus.

gamma-L-Glutaminyl-[3,5-3H]4-hydroxybenzene was synthesized in order to study the kinetics of its hydroxylation by tyrosinase purified from Agaricus bisporus and to explore its role in the induction of the dormant state in the spores of this species. It was found to be unique among the monophenolic substrates for tyrosinase in that the lag period for the hydroxylation reaction decreased with increasing substrate concentration. Unlike previously studied compounds, this phenol appeared to function as an electron donor, allowing it to act as its own co-substrate in the hydroxylation reaction. Its catechol product, gamma-L-glutaminyl-3,4-dihydroxybenzene, was found to be a superior co-substrate, yielding its electrons more readily (oxidation peak potential +0.18 V as compared with +0.65 V for the phenol). In situ periodate oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone confirmed the co-substrate role of the catechol in the hydroxylation reaction. The tyrosinase-mediated oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone occurred with an apparent Km = 1.54 mM and Vmax = 0.36 mmol/min/mg of enzyme. gamma-L-Glutaminyl-4-hydroxybenzene acted as an inhibitor of the oxidation reaction.