Indomethacin improves locomotor deficit and reduces brain concentrations of neuroinhibitory steroids in rats following portacaval anastomosis.

Hepatic encephalopathy (HE) is a neuropsychiatric complication of both acute and chronic liver failure characterized by progressive neuronal inhibition. Some neurosteroids are potent positive allosteric modulators of the gamma-aminobutyric acid (GABA)-A receptor complex, and 'increased GABAergic tone' has been proposed to explain the neuroinhibition characteristics of HE. Brain levels of the neurosteroids pregnenolone, allopregnanolone and tetrahydrodesoxycorticosterone (THDOC) and the functional status of the GABA-A receptor complex were assessed in rats following portacaval anastomosis (PCA). Effects of indomethacin, an inhibitor of the 3alpha-hydroxysteroid dehydrogenase enzyme involved in neurosteroid synthesis, on PCA rat locomotor activity and brain neurosteroid levels were also assessed. Significant increases of the neurosteroid pregnenolone (2.6-fold), allopregnanolone (1.7-fold) and THDOC (4.7-fold) were observed in brains of PCA rats. Brain levels of these neurosteroids were in the nanomolar range, sufficient to exert positive allosteric modulatory effects at the GABA-A receptor. Indomethacin (0.1-5 mg kg(-1)) ameliorated dose-dependently the locomotor deficit of PCA rats and concomitantly normalized brain levels of allopregnanolone and THDOC. Increased brain levels of neurosteroids with positive allosteric modulatory actions at the neuronal GABA-A receptor offer a cogent explanation for the notion of 'increased GABAergic tone' in HE. Pharmacological approaches using agents that either reduce neurosteroid synthesis or modulate the neurosteroid site on GABA-A receptor could offer new therapeutic tools for the management and treatment of HE.

Microbial and reducing agents catalyze the rearrangement of taxanes.

5alpha, 7beta, 9alpha, 10beta, 13alpha-Pentahydroxy-4(20),11(12)-taxadiene derivative 1 was converted to two unprecedented 1(15-->11)abeo-taxanes and a taxane derivative with a C10-C11 double bond by Absidia coerula ATCC 10738a. A similar compound was obtained from treatment with zinc of a triacetoxy-4(20),11(12)-taxadiene derivative.

Initial catabolic steps of isoleucine, the R-pathway and the origin of alloisoleucine.

The initial catabolic steps of isoleucine by mammals has been misunderstood and misapprehended in the scientific literature for many years. The suggestion that the interconversion of isoleucine and alloisoleucine occurs through the keto-enol racemization of their respective transaminated alpha-keto acids was first tentatively advanced by Alton Meister in the early 1950s, and accepted without hard confirming evidence by many authors. It will be shown in this brief review that isoleucine is converted to alloisoleucine with conservation of a 15N label denying the intermediacy of the alpha-keto acids, and that alloisoleucine arises as an unavoidable consequence of isoleucine transamination.

In vivo variability of TMA oxidation is partially mediated by polymorphisms of the FMO3 gene.

Trimethylaminuria (TMAU) results from an accumulation of an excessive amount of unoxidized trimethylamine that is excreted in urine and body secretions. Mutations of the flavin-containing monooxygenase 3 (FMO3) gene (a hepatic phase I drug-metabolizing enzyme) account for the severe recessively encoded form of this condition. We have previously described a number of FMO3 polymorphisms which in vitro exhibit reduced substrate affinity for several FMO substrates. Here we show that three prevalent polymorphisms (E158K, V257M, and E308G) inherited in particular combinations confer a slight decrease in TMA oxidation under normal physiological conditions, which may be clinically "silent." With the use of substrate loading or with the interaction of other known modulators of FMO3 activity such as hormonal influences, these genotypes may predispose to mild TMAU.

Kinetics and mechanisms of hepatic acute phase response to subtotal partial hepatectomy and cultural impact on environmental hepatic end-stage liver injury in the homeless.

Intoxication and liver damage induced by carbon tetrachloride (CCl(4)), aflatoxin B1, diabetes, and subtotal partial hepatectomy (PH(90)) in rats in which approximately 90% of the total hepatic tissue mass is surgically removed produces an acute-phase response (APR) whose initial stage prior to regression closely mimics the APRs associated with the life-threatening hepatic failure seen in the homeless. Rats treated by PH(90)were either healthy, CCl(4)-intoxicated, diabetic, or alflatoxin B1 (AFB1) intoxicated to the point of 75% liver insufficiency. It is well documented that high rates of mortality following PH(90)in aseptic rats could be minimized by supplementing drinking water with 20% glucose, organic components of L-15 medium and housing animals in cages maintained at 33-35;C. Aseptic rats showed a mild 20-30% decrease in APR proteins during the first 4-5 days following PH(90), while a maximal APR was noted 9-12 days post PH(90)and lasted for ~30 days when it returned to values close to those of healthy controls. This delay in hepatic APR of the remnant caudate lobe favoured replacement of lost basophilic clumps and ribosomes. The newly synthesized ribosomes of the nascent hepatocytes quantitatively maintained the APR signals of the injured caudate hepatocytes, and biosynthesized and released a typical spectrum of APR proteins. We suggest that massively injured liver has decoded an already stored and irreversible DNA-biochemical sequence of events in which priority is given to recovery of lost tissues by delaying an APR response to injury. In PH(90)of diabetic and CCl(4)-intoxicated rats, the hepatic dual functions of regeneration and APR processes associated with intoxication-initiated catabolic signals, created a heavy metabolic burden on the remnant caudate lobe leading to higher rates of mortality. APR of healthy rats to AFB1 parallels that of alpha-amanitin-induced intoxication. Similarly, within shorter time scale proportional to the severity of surgery, livers undergoing 75% partially hepatectomy (PH(75)) delayed both the onset and regression of APR. We are therefore led to believe that approaches other than liver transplantation should be considered as viable alternatives in the treatment of various acute and chronic liver diseases to avoid rejection and retransplantation. Scarcity of cadaveric liver has forced the medical community to investigate xenotransplantation with its unknown risks. Concomitantly, it is suggested that in view of the incalculable risks of indifference, the homeless must receive much improved medical care as we have found that two-dimensional immunoelectrophoretic assay of their serum is indicative of acute and chronic liver injury. The scientific and moral interrelationships of related matters are illuminated.

Biotransformation of a 4(20),11(12)-taxadiene derivative.

A 4(20),11(12)-taxadiene derivative was converted to hydroxylated derivatives by Cunninghamella elegans AS3.2033 and Cunninghamella elegans var chibaensis ATCC 20230. Both microorganisms led to C-1 hydroxylations and conversion to a C-15-hydroxylated abeo-taxane. Additional products from the two fungi differed: a C-14 oxidation and a trans-cis isomerization of the cinnamoyl for one and an unprecedented hydroxylation at C-17 for the other.

New taxane analogues from the needles of Taxus canadensis.

Eight minor taxanes have been identified for the first time in Taxus canadensis needles. Four of these metabolites are new taxane analogues: 7-acetyl-10-deacetyltaxol (1), 2'-acetyl-7-epi-cephalomannine (2), 10-deacetyl-10-oxo-7-epi-cephalomannine (3), and 10-acetylglycollylbaccatin VI (4).

Parenteral vitamin B12 reduces hyperhomocysteinemia in end-stage renal disease.

OBJECTIVE: The authors found considerably lower plasma total homocysteine (tHcy) concentrations in patients with end-stage renal disease (ESRD) on maintenance hemodialysis, who routinely received high-dose parenteral vitamin B12, than in comparable patients receiving much higher doses of folic acid but only replacement-dose oral vitamin B12. They therefore sought prospective evidence that high-dose parenterally administered vitamin B12 may partially ameliorate renal failure-associated hyperhomocysteinemia. DESIGN: Open phase 2 clinical trial. SETTING: Outpatient hemodialysis unit. PATIENTS: Fourteen clinically stable patients on maintenance hemodialysis with normal baseline serum vitamin B12 concentrations. INTERVENTION: Three parenteral injections of 1 mg vitamin B12 given at 4-week intervals. OUTCOME MEASURES: Plasma tHcy and serum vitamin B12 concentrations were measured before, during and 7 months after the termination of vitamin B12 therapy. RESULTS: The mean (and standard error) baseline plasma tHcy was 26.5 (1.8) micromol/L. The plasma tHcy value fell successively after each vitamin injection to reach a value of 23.6 (1.6) micromol/L 1 month after the final injection (p < 0.05), while the serum vitamin B12 concentration increased from 471 (42) pmol/L to 890 (74) pmol/L (p < 0.05). Seven months after the final injection, the serum B12 concentration had fallen and tHcy had risen to near their original values. CONCLUSIONS: Three monthly vitamin B12 injections modestly but distinctly reduced tHcy concentrations in hemodialysis patients whose prior vitamin B12 status was normal. Randomized placebo-controlled clinical trials of longer duration and using larger or more frequent parenteral doses are indicated to determine whether administration of this safe and inexpensive vitamin can improve hyperhomocysteinemia in ESRD.

Measurement of succinylcholine concentration in human plasma by electrospray tandem mass spectrometry.

An electrospray mass spectrometric method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine (SUX) is described. An extraction method compatible with direct infusion inlet was developed and leads to an analysis cycle time of 7--8 min instead of 25 min that would be required for HPLC inlet. SUX was extracted from human plasma on C1 solid-phase cartridges and was analyzed using positive ion electrospray tandem mass spectrometry (ESI-MS/MS). SUX plasma concentrations were determined by a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide (SUXd6) as the internal standard. The calibration curve was prepared using the ratio of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of SUX and SUXd6 in plasma. Calibration curves for the quantification were linear from 25 to 4000 ng/ml. For intraday precision, CV were < or =6% and accuracy ranged from 98 to 103%. For the interday precision, CV were < or =10% and accuracy ranged from 90 to 102%. This method is specific, sensitive, reproducible, and practical in a clinical setting.

Plasma reduced homocysteine concentrations are increased in end-stage renal disease.

BACKGROUND: Plasma total homocysteine (tHcy) concentrations> 15 micromol/L are associated with an increased risk of cardiovascular disease. This is especially the case in end-stage renal disease (ESRD), in which tHcy concentrations commonly range between 20 and 30 micromol/L. Adverse vascular or prothrombotic effects associated with hyperhomocysteinemia are assumed to be mediated by the free sulfhydryl (reduced) form of the molecule (rHcy), but data based on fluorescence high-pressure liquid chromatography (HPLC) indicate that rHcy concentrations are not increased in ESRD despite two- to threefold elevations in tHcy. METHODS: We developed a sensitive method for measuring plasma rHcy concentrations in which freshly drawn blood is incubated with sodium iodoacetate, and the resulting S-carboxymethylhomocysteine is analyzed by gas chromatography mass spectrometry. RESULTS: Unlike with the earlier methodology, we found plasma rHcy concentrations two to four times higher than normal in ESRD. These concentrations were lowered by hemodialysis and were proportional to plasma tHcy over the range of tHcy concentrations that has been associated with increased cardiovascular risk (r2 = 0.39, P < 0.0001). CONCLUSIONS: These results support the hypothesis that homocysteine could directly mediate vascular disease through mechanisms related to the reactivity of its free sulfhydryl group. It remains to be determined how much of the variability between plasma tHcy and rHcy is due to analytical variation and how much is due to biologic factors that separately influence concentrations of the disease marker, tHcy, and its presumed mediator, rHcy.

New taxanes from the needles of Taxus canadensis.

Nine minor taxanes were identified for the first time in the Canadian yew needles. Four of these metabolites are new: 5-epi-cinnamoylcanadensene (1), 2,9,10, 13-tetraacetoxy-20-cinnamoyloxy-taxa-4(5),11(12)-diene (2), 2'-acetyl-epi-taxol (3), and 9-deacetyltaxinine E (4).

Taxus canadensis abundant taxane: conversion to paclitaxel and rearrangements.

An efficient conversion of Taxus canadensis abundant taxane, 9-dihydro-13-acetylbaccatin III to baccatin III is described. Since the synthesis of paclitaxel from baccatin III has been reported, this work can be used for additional supply of this powerful anticancer drug. In addition, new taxanes derived from skeletal rearrangements originating from oxidation reduction reactions of the Canadian yew major taxane, are reported.

Taxoids from the needles of the Canadian yew.

Systematic characterization of the taxoids in the needles of Taxus canadensis led to the discovery of seven taxanes along with three known congeners. Their structures were rigorously established by spectroscopic methods as 15-benzoyl-10-deacetyl-2-debenzoyl-10-dehydro-abeo-baccat in III; 15-benzoyl-2-debenzoyl-7, 9-dideacetyl-abeo-baccatin VI; N-acetyl-N-debenzoyltaxol; 7,9,13-trideacetylbaccatin VI; 10-deacetyl-10-glycolylbaccatin IV; 1 beta-hydroxy-10-deacetyl-10-glycolylbaccatin I; and 7-deacetyltaxuspine L. These taxanes, specific to the Canadian yew, were co-isolated with taxacustin, taxagifine and 2-deacetyl-7,10-diacetyl-5-deaminoacyl taxine A previously found in Taxus cuspidata, baccata, and yunnanensis, respectively.

Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia.

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development.

Plasma kinetics of apoC-III and apoE in normolipidemic and hypertriglyceridemic subjects.

Apolipoprotein (apo) C-III and apoE play a central role in controlling the plasma metabolism of triglyceride-rich lipoproteins (TRL). We have investigated the plasma kinetics of total, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) apoC-III and apoE in normolipidemic (NL) (n = 5), hypertriglyceridemic (HTG, n = 5), and Type III hyperlipoproteinemic (n = 2) individuals. Apolipoprotein kinetics were investigated using a primed constant (12 h) infusion of deuterium-labeled leucine. HTG and Type III patients had reduced rates of VLDL apoB-100 catabolism and no evidence of VLDL apoB-100 overproduction. Elevated (3- to 12-fold) total plasma and VLDL apoC-III levels in HTG and Type III patients, although associated with reduced apoC-III catabolism (i.e., increased residence times (RTs)), were mainly due to increased apoC-III production (plasma apoC-III transport rates (TRs, mean +/- SEM): (NL) 2.05 +/- 0.22 (HTG) 4.90 +/- 0.81 (P < 0.01), and (Type III) 8.78 mg. kg(-)(1). d(-)(1); VLDL apoC-III TRs: (NL) 1.35 +/- 0. 23 (HTG) 5.35 +/- 0.85 (P < 0.01), and (Type III) 7.40 mg. kg(-)(1). d(-)(1)). Elevated total plasma and VLDL apoE levels in HTG (2- and 6-fold, respectively) and in Type III (9- and 43-fold) patients were associated with increased VLDL apoE RTs (0.21 +/- 0.02, 0.46 +/- 0. 05 (P < 0.01), and 1.21 days, NL vs. HTG vs. Type III, respectively), as well as significantly increased apoE TRs (plasma: (NL) 2.94 +/- 0.78 (HTG) 5.80 +/- 0.59 (P < 0.01) and (Type III) 11.80 mg. kg(-)(1). d(-)(1); VLDL: (NL) 1.59 +/- 0.18 (HTG) 4.52 +/- 0.61 (P < 0.01) and (Type III) 11.95 mg. kg(-)(1). d(-)(1)). These results demonstrate that hypertriglyceridemic patients, having reduced VLDL apoB-100 catabolism (including patients with type III hyperlipoproteinemia) are characterized by overproduction of plasma and VLDL apoC-III and apoE.

Charged residues in surface-located loops influence voltage gating of porin from Haemophilus influenzae type b.

Porin of Haemophilus influenzae type b (341 amino acids; M(r) 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24 +/- 0.41 vs. 0.85 +/- 0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50-55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89-91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.

Measurement of trimethylamine and trimethylamine N-oxide independently in urine by fast atom bombardment mass spectrometry.

We report a method based upon fast atom bombardment mass spectrometry (FAB-MS) and stable isotope dilution techniques for the measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAOx). TMA is extracted from urine that was spiked with (15)N-labeled TMA. The extracted TMA isotopomers are quaternized with trideuteromethyl iodide and analyzed in FAB-MS with hexaethylene glycol as matrix. TMAOx is measured by evaporation of another sample of the urine spiked with (15)N-labeled TMAOx on the FAB probe and analyzed as for the TMA. The method allows the ready and simple distinguishing of controls and patients with TMAuria, and is useful in monitoring patients with the disorder. We give examples of its use in determining normal control ranges for these metabolites and in evaluating patients.

Biosynthesis of trichothecenes and apotrichothecenes.

Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.