Altered gene and protein expression by Nm23-H1 in metastasis suppression

The aim of our work was to study (1) the antioxidant properties of lipoic acid (LA) and its reduced metabolite dihydrolipoic acid (DHLA) formed by reduction of LA and (2) the effects of treatment with LA and DHLA on (a) K(+) efflux from human red blood cells and (b) post-ischemic recovery and oxidative stress in isolated perfused rat hearts challenged with an ischemia-reperfusion (IR) sequence. In vitro, we used xanthine and xanthine oxidase to generate superoxide anion, which is not directly measurable by electron paramagnetic resonance (EPR), but specifically oxidizes the spin probe CPH into an EPR-detectable long lasting CP(*) nitroxide radical. While 5 mM of LA was ineffective in reducing the kinetics of CP(*) nitroxide formation, DHLA was shown to lessen this rate in a dose-dependent manner and at 30 mM was even more efficient than 300 UI/ml SOD. These results are in agreement with the fact that DHLA is able to directly scavenge superoxide anion. Red cells are a good model to investigate oxidative damage in biological membranes; hence, we used a suspension of erythrocytes incubated with 2,2(')-azobis(2-amidinopropane) hydrochloride (AAPH) which generates in vitro free radicals. DHLA provided more effective protection of red cells membranes than LA; DHLA was comparable to Trolox for its antioxidant potency. In vivo, treatment of rats (50 mg/kg/day i.p. for 7 days) with LA induced a slight increase in coronary flow (CF) in isolated perfused hearts, after 30 min of global total ischemia. This effect was not associated with an improvement in contractile function and reduction of myocardial oxidative stress. In conclusion, because of their ability to scavenge free radicals, LA and to an even greater degree DHLA were able to protect the membranes of red blood cells. This finding suggests that LA and DHLA might be useful in the treatment of diseases associated with oxidative stress such as diabetes.     

Dihydrolipoic acid lowers the redox activity of transition metal ions but does not remove them from the active site of enzymes

Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelate transition metal ions and, hence, mitigate iron- and copper-mediated oxidative stress in biological systems. However, it remains unclear whether LA and DHLA chelate transition metal ions in a redox-inactive form, and whether they remove metal ions from the active site of enzymes. Therefore, we investigated the effects of LA and DHLA on iron- or copper-catalyzed oxidation of ascorbate, a sensitive assay for the redox activity of these metal ions. We found that DHLA, but not LA, significantly inhibited ascorbate oxidation mediated by Fe(III)-citrate, suggesting that reduced thiols are required for iron binding. DHLA also strongly inhibited Cu(II)(histidine)(2)-mediated ascorbate oxidation in a concentration-dependent manner, with complete inhibition at a DHLA:Cu(II) molar ratio of 3:1. In contrast, no inhibition of copper-catalyzed ascorbate oxidation was observed with LA. To investigate whether LA and DHLA remove copper or iron from the active site of enzymes, Cu,Zn superoxide dismutase and the iron-containing enzyme aconitase were used. We found that neither LA nor DHLA, even at high, millimolar concentrations, altered the activity of these enzymes. Our results suggest that DHLA chelates and inactivates redox-active transition metal ions in small-molecular, biological complexes without affecting iron- or copper-dependent enzyme activities.     

Simultaneous determination of celecoxib,hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.     

Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation

The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC(50) of 15 microM with linoleic acid and 5 microM with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC(50) of 4 and 100 microM, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC(50) of, respectively, 10 and 5 microM. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2'-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC(50) of 850 microM. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenase-dependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15-lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects.     

Alpha-lipoic--dihydrolipoic acids--active bioantioxidant and bioregulatory system

alpha-Lipoic (LA) acid (thioctic acid) is an intramolecular disulfide that may be simply endogenically turned into dithiol. Dihydrolipoic acid (DHLA)/ LA and DHLA are bioantioxidants. They are synthesized in the body and taken with diet. Water- and lipide-soluble LA is highly-effective against the reactive oxygen species. LA (DHLA) protect the biomembranes, mitochondria from oxidative stresses of various kinds. LA, DHLA and lipoamide function as cofactors of polyenzyme mitochondrial complexes of 2-oxoacid dehydrogenases, of glycin decarboxylases and of some other enzymes. LA (DHLA) is ubiquinone reactivator and synergist by vitamin A, C, E. LA optimizes glucose metabolism, it is effective in insulin-resistant diabetes and its complications, in neutopathies and neurodegenerative diseases.     

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.     

Antithrombotic potential of dihomo-gamma-linolenic acid in man.

The effects of orally ingested dihomo-gamma-linolenic acid (DHLA), the natural biosynthetic precursor of prostaglandin E1 (PGE1), were assessed in human volunteers. Single doses of DHLA (0.1--2g) increased the proportion of DHLA relative to arachidonic acid in plasma and platelets and also increased the ex-vivo capacity of platelets to produce PGE1 and PGE2. More pronounced effects were observed during sustained treatment (five days to four weeks) when DHLA also accumulated in red cell membranes. These biochemical changes were accompanied by potentially antithrombotic changes in haemostatic function. The most common effect, which was consistently detected after 0.1-g single doses of DHLA or its methyl ester, was a decrease in plasma heparin-neutralising activity. Inhibition of platelet aggregation induced by adenosine diphosphate was also detected, though this was generally less pronounced. Sustained treatment in one subject also produced definite inhibition of ristocetin-induced platelet aggregation. There was only one possible adverse effect--a transient cough in a subject with a history of asthma. DHLA therefore seems to have considerable potential as an agent for preventing and treating human thromboembolic disease.     

Inhibition of gelatinase B (matrix metalloproteinase-9) by dihydrolipoic acid

Alpha-lipoic acid (LA) is a disulphide-containing fatty acid that is absorbed from the diet and transported to tissues. Once it has been taken up by mammalian cells, LA is reduced to dihydrolipoic acid (DHLA), a vicinal dithiol, and rapidly effluxed into the extracellular milieu. We hypothesized that DHLA may be an effective inhibitor of human gelatinase B (GelB). Purified human GelB was incubated with 0 to 200 micromol/L DHLA, and residual enzyme activity was measured by HPLC using a fluorogenic substrate (matrix metalloproteinase substrate III). DHLA inhibited GelB in a dose-dependent fashion with an IC50 of 20 micromol/L. Oxidation of DHLA resulted in a loss of DHLA's capacity to inhibit GelB. The DHLA-mediated inhibition of GelB was independent of the zinc concentration in the reaction buffer. DHLA had no inhibitory effect on gelatinase A. Zymographs of activated neutrophil lysates demonstrated that higher concentrations of DHLA also prevent the activation of GelB proenzyme. Bronchoalveolar lavage fluid from mice fed a diet enriched with LA showed significantly increased GelB inhibitory capacity (p = 0.0002 vs. regular diet). We conclude that DHLA can modulate neutrophil-derived GelB activity through direct inhibition of enzyme activity and by preventing the activation of GelB proenzyme.     

Comparison of antioxidant effectiveness of lipoic acid and dihydrolipoic acid

The abilities of dihydrolipoic acid (DHLA) to scavenge peroxynitrite (ONOO^?), galvinoxyl radical, 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) cation radical (ABTS^+?), and 2,2′‐diphenyl‐1‐picrylhydrazyl radical (DPPH) were higher than those of lipoic acid (LA). The effectiveness of DHLA to protect methyl linoleate against 2,2′‐azobis(2‐amidinopropane hydrochloride) (AAPH)‐induced oxidation was about 2.2‐fold higher than that of LA, and DHLA can retard the autoxidation of linoleic acid (LH) in the β‐carotene‐bleaching test. DHLA can also trap ～0.6 radicals in AAPH‐induced oxidation of LH. Moreover, DHLA can scavenge ～2.0 radicals in AAPH‐induced oxidation of DNA and AAPH‐induced hemolysis of erythrocytes, whereas LA can scavenge ～1.5 radicals at the same experimental conditions. DHLA can protect erythrocytes against hemin‐induced hemolysis, but accelerate the degradation of DNA in the presence of Cu²⁺. Therefore, the antioxidant capacity of –SH in DHLA is higher than S‐S in LA. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:216–223, 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20378     

Rapid and sensitive determination of amprolium in chicken plasma by high-performance liquid chromatography with post-column reaction

A rapid, sensitive and reproducible reversed-phase HPLC assay was developed for the determination of amprolium (APL) in chicken plasma. Protein in plasma sample was precipitated with 0.33 M perchloric acid and supernatant solution was injected into the HPLC system. Following the chromatographic separation of APL and the beclotiamine (I.S.) on a C₁₈ column, the derivatives of APL and I.S. were formed by post-column reaction and detected by fluorescence detection (excitation at 400 nm, emission at 460 nm). The method showed excellent precision, accuracy and speed with a detection limit of 2 ng/ml. The intra- and inter-assay variance of this method were less than 11.2%. This method has been successfully applied to plasma determinations after oral administration of APL to chicken.     

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.     

Determination of dicentrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetics

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of dicentrine in rat plasma after 10 mg/kg intravenous administration. After addition of an internal standard (coumarin), plasma was deproteinized by acetonitrile for sample clean-up. The drugs were separated on a reversed-phase Nucleosil C₁₈ column (250 × 4 mm I.D., particle size 5 μm) and detected by photodiode-array detection at a wavelength of 308 nm. Acetonitrile-water (35:65, v/v, pH 2.5–2.8, adjusted with orthophosphoric acid) was used as the mobile phase. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve.     

Alpha-lipoic acid exhibits anti-amyloidogenicity for beta-amyloid fibrils in vitro

Inhibition of the formation of beta-amyloid fibrils (fAbeta), as well as the destabilization of preformed fAbeta in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of alpha-lipoic acid (LA) and the metabolic product of LA, dihydrolipoic acid (DHLA), on the formation, extension, and destabilization of fAbeta at pH 7.5 at 37 degrees C in vitro. LA and DHLA dose-dependently inhibited fAbeta formation from amyloid beta-protein, as well as their extension. Moreover, they destabilized preformed fAbetas. LA and DHLA could be key molecules for the development of therapeutics for AD.     

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.     

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison.

Methods

Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant.

Results

The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases.

Conclusions

DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0411-6) contains supplementary material, which is available to authorized users.     

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.     

Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid

Alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) may have a role as antioxidants against nitric oxide-derived oxidants. We previously reported that peroxynitrite reacts with LA and DHLA with second-order rate constants of 1400 and 500 M(-1) s(-1), respectively, but indicated that these direct reactions are not fast enough to protect against peroxynitrite-mediated damage in vivo. Moreover, the mechanism of the reaction of peroxynitrite with LA has been recently challenged (J. Biol. Chem.279:9693-9697; 2004). Pulse radiolysis studies indicate that LA and DHLA react with peroxynitrite-derived nitrogen dioxide (*NO2) (k2 = 1.3 x 10(6) and 2.9 x 10(7) M(-1) s(-1), respectively) and carbonate radicals (CO(3-)) (k2 = 1.6 x 10(9) and 1.7 x 10(8) M(-1) s(-1), respectively). Carbonate radical-mediated oxidation of LA led to the formation of the potent one-electron oxidant LA radical cation. LA inhibited peroxynitrite-mediated nitration of tyrosine and of a hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE), incorporated into liposomes but enhanced tyrosine dimerization. Moreover, while LA competitively inhibited the direct oxidation of glutathione by peroxynitrite, it was poorly effective against the radical-mediated thiol oxidation. The mechanisms of reaction defined herein allow to rationalize the biochemistry of peroxynitrite based on direct and free radical-mediated processes and contribute to the understanding of the antioxidant actions of LA and DHLA.     

Simultaneous determination of N-butyramide-tacrine and tacrine in mouse plasma and brain homogenate by high-performance liquid chromatography with a simple gradient solvent system

A novel reversed-phase HPLC method was developed for the simultaneous determination of tacrine (THA) and the newly synthesized prodrug (N-butyramide-THA, BTHA) in mouse plasma and brain homogenate. The assay involves deproteinisation and subsequent detection at 240 nm with a gradient solvent system. Retention times were 18.5 and 9.3 min for BTHA and THA, respectively. Average recoveries for the analytes were 80.7% (BTHA) and 76.6% (THA) from plasma, and 75.0% (BTHA) and 68.4% (THA) from brain homogenate. Linear responses were observed over a wide range (0.25-20 microg/ml for BTHA in plasma and in brain homogenate, 0.025-20 microg/ml for THA in both matrices). Both BTHA and THA degraded from the prodrug can be detected even 12 h after intravenous administration of BTHA, indicating that BTHA is a promising prodrug for brain targeting.     

Determination of Z-butylidenephthalide in plasma by high-performance liquid chromatography

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 μg/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.