Regulation of murine contact sensitivity to urushiol components by serum factors.

Mice epicutaneously painted with components of poison ivy urushiol oil exhibit contact sensitivity (as detected by ear swelling reactions) that persist for about 25 days. Sera taken from mice at times when the contact sensitization response is waning suppressed the induction of sensitization to 3-n-pentadecylcatechol (PDC), a urushiol component, in recipients. The suppressive serum factor was present in greatest amount 25 days after sensitization, but was no longer detectable 40 days post sensitization. Suppression was antigen-specific, absorbed out with PDC-immune, but not normal lymph node cells, and transferable with a single 0.6 ml dose 7 days prior to sensitization of recipients. Suppression was transferable by the purified IgG fraction of desensitized mice. Results indicate that contact sensitivity to urushiol in mice is regulated by serum factors.

Quantification of recombinant human interleukin-1 alpha by a specific two cell immunobioassay.

The detection of picogram quantities of recombinant human IL-1 alpha in human and rat serum was accomplished by a sensitive and specific two cell immunobioassay. The specificity is provided by an IL-1 alpha specific mouse IgM monoclonal antibody which is non-neutralizing thus allowing for the addition of the EL-4 NOB-1 cell line directly to the IL-1 alpha monoclonal antibody complex. The above cell line is then converted to an IL-2 producer line in response to the captured IL-1 alpha. Supernatant from the EL-4 NOB-1 cells is then added to the IL-2 dependent CTLL-2 line and cell proliferation measured by thymidine incorporation. This assay has the advantage of specificity provided by the antibody capture step, sensitivity provided by the EL-4 NOB-1 line (1-50 pg/ml) and finally ease of maintenance of the responder cell line which requires no feeder cells or mitogens. Data are reported on the sensitivity, precision, reproducibility and specificity of the assay, the stability of rhIL-1 alpha in serum and the recovery of rhIL-1 alpha from serum. We also report on the use of this procedure to assay samples from rats given ascending doses of rhIL-1 alpha.

Regiospecific attack of nitrogen and sulfur nucleophiles on quinones derived from poison oak/ivy catechols (urushiols) and analogues as models for urushiol-protein conjugate formation.

Attempts to characterize potential biologically important covalent interactions between electrophilic quinones derived from catechols present in poison oak/ivy (urushiol) and biomacromolecules have led to the analysis of model reactions involving sulfur and amino nucleophiles with 3-heptadecylbenzoquinone. Characterization of the reaction products indicates that this quinone undergoes regiospecific attack by (S)-N-acetylcysteine at C-6 and by 1-aminopentane at C-5. The red solid obtained with 1-aminopentane proved to be 3-heptadecyl-5-(pentylamino)-1,2-benzoquinone. Analogous aminobenzoquinones were obtained with the quinones derived from the 4- and 6-methyl analogues of 3-pentadecylcatechol. All three adducts absorbed visible light at different wavelengths. When the starting catechols were incubated with human serum albumin almost identical chromophores were formed. These results establish that cathechols responsible for the production of the poison oak/ivy contact dermatitis in humans undergo a sequence of reactions in the presence of human serum albumin that lead to covalent attachment of the catechols to the protein via carbon-nitrogen bonds. Estimations of the extent of this binding indicate that, at least with human serum albumin, the reaction is quantitative.

Valproylcarnitine: a novel drug metabolite identified by fast atom bombardment and thermospray liquid chromatography-mass spectrometry.

Urine samples from three children at different stages of chronic valproate therapy were partially purified using a cation exchange column. A signal consistent with either valproylcarnitine or octanoylcarnitine was observed in one of these extracts by direct fast atom bombardment-mass spectrometry analysis. These isomeric acylcarnitines were synthesized, separated and characterized by thermospray high performance liquid chromatography-mass spectrometry. This new technique was then employed to positively identify intact valproyl-carnitine in the patients' urine samples. The implications of this finding with regard to a mechanism to account for carnitine deficiency in patients receiving valproate are discussed.

Disposition and metabolism of Ro 24-4736 in the rat.

Ro 24-4736, a new platelet activating factor antagonist, is currently under preclinical and clinical development. The tissue distribution of the 14C-label in male rats following a single intravenous dose of 1.0 mg/kg of 14C-Ro 24-4736 indicated appreciable uptake by the liver, kidney, heart and gastrointestinal tract. Peak plasma and tissue concentrations were seen at 5 minutes after dosing except for the small intestine (4 hrs) and abdominal fat, stomach and large intestine (4 hrs). Thereafter, the 14C-label rapidly declined in all tissues. At 48 hours, only 3.5% of the dose was present in the tissues, and 6.1% in the lumen of the gastrointestinal tracts. The excretion of 14C was essentially completed; 94% of the administered 14C was excreted in the feces and 4.0% in the urine. Overall recoveries of the administered 14C label ranged from 96 to 116%. The purified major 14C-labelled component in the fecal extracts yielded essentially the same NMR spectrum as authentic Ro 24-4736 which accounted for 11% of the dose. In vitro incubations of Ro 24-4736 with rat liver 9S supernatant in an NADPH generating system produced two metabolites. NMR spectra indicated that one metabolite was hydroxylated at carbon-1 while the other one contained a hydroxyl at carbon-10 of the parent molecule. Interestingly, the sites of hydroxylation were at carbons C1, and C10 bearing the protons guarding the bay area of the phenanthrenoid ring, rather than carbons of the phenyl-methyl-thienotriazolodiazepine moiety.

Identification of high-affinity anti-IL-1 alpha autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1 alpha.

A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 alpha (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 alpha added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 alpha added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 alpha in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 alpha binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 alpha-specific IgG. The binding of 125I-labeled IL-1 alpha to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 x 10(-11) M) but not by unlabeled IL-1 beta. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 alpha-specific IgGs was 4.7 x 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 alpha in human serum by various assay systems and also could be a regulator of circulating IL-1 alpha.

Separation and quantification of choline and acetylcholine by thermospray liquid chromatography/mass spectrometry.

The development of sensitive analytical techniques for use in the identification and qualification of molecules of biological origin remains a constant challenge. Often the compounds of interest are present in a complex mixture and require extensive sample preparation prior to analysis. Thermospray liquid chromatography/mass spectrometry (LC/MS) has been shown to be a method which allows for both separation of target molecules from complex mixtures and sensitive specific detection using a mass spectrometer. In this work thermospray LC/MS is used for the direct detection of intact, underivatized choline and acetylcholine from mouse brain homogenate. The results of our analysis corroborate previous analyses using a variety of indirect measurement techniques and thereby show that this simple direct analysis has wide potential applicability.

Plasma kinetics of vitamin A in humans after a single oral dose of [8,9,19-13C]retinyl palmitate.

The kinetics of vitamin A and its major metabolites were investigated in humans. Eleven healthy male subjects ingested 105 mumol (100,000 IU) of [8,9,19-13C]retinyl palmitate in an oily solution. Twenty-seven blood samples were collected during the 1-week study. Plasma samples were analyzed for retinyl esters and for [12C]- and [8,9,19-13C]retinol. Retinol isotopes were quantified using a newly developed GC-MS method. Total retinyl esters peaked at about 4.45 mumol/L from 3.5 to 12 h after dosing. As a result of the perturbation of the tracee system, the plasma concentration of [12C]retinol increased and then decreased as the concentration of [8,9,19-13C]retinol increased, indicating rapid distribution kinetics. A broad single peak (1.16 +/- 0.32 mumol/L) was observed for [8,9,19-13C]retinol at about 10 to 24 h postdose; this likely reflects hepatic secretion of [8,9,19-13C]retinol associated with retinol-binding protein. Then, declining levels of the tracer and increasing levels of the tracee were observed. At its peak, the ingested [8,9,19-13C]retinol reached about 51% of the observed total plasma retinol concentration. This percentage dropped to 13.4% on day 7 indicating slow final elimination from plasma. Our data support the concept that the liver follows the principle "last in/first out' in maintaining vitamin A homeostasis.

The formation of glutathione conjugate derived from propranolol.

Mixed function oxidases in liver microsomes from 3-methylcholanthrene-pretreated guinea pigs converted S-propranolol to 4-hydroxypropranolol, 5-hydroxypropranolol, and desisopropyl-propranolol. The addition of glutathione and cytosol from rat liver to the system resulted in the formation of a water-soluble metabolite. Evidence that the metabolite was a glutathione adduct was obtained by showing that treatment with gamma-glutamyltranspeptidase changed its HPLC retention time. The formation of the glutathione adduct required glutathione S-transferases in cytosol and was accompanied by a decrease in the formation of 5-hydroxypropranolol, but not of 4-hydroxypropranolol or desisopropylpropranolol. The formation of the propranolol-glutathione adduct was confirmed by two independent approaches. When [14C]glutathione and [3H]S-propranolol were used, the relationship between 14C and 3H in the metabolite indicated that it contained equal amounts of glutathione and propranolol. When a pseudoracemic mixture of S-propranolol and [2,4-2H2]R-propranolol was used, thermospray LC/MS analysis of the glutathione adduct revealed two peaks having different retention times. The first peak, representing a [2,4-2H2]R-propranolol-GSH adduct, gave fragment ions at m/z 294, 278, 262, while the second, which represented the S-propranolol-GSH adduct, gave fragment ions having 2 mass units less than those obtained with [2,4-2H2]R-propranolol. There was no evidence of any loss of deuterium during the formation of these fragment ions. The material in both peaks gave ions of m/z 308, 147, and 130, which is consistent with the presence of a glutathione group.

Metabolic kinetics and quantitative analysis by isotope dilution thermospray LC/MS.

The use of thermospray LC/MS for quantitative analysis using isotope dilution is reviewed. Assays for acetylcholine, glucose, sorbitol, cortisol, testosterone and 1,25-(OH)2-vitamin D in biological fluids are discussed.

Thermospray liquid chromatography/mass spectrometry for the analysis of L-carnitine and its short-chain acyl derivatives.

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.

Quantification of acitretin in human plasma by microbore liquid chromatography-negative chemical ionization mass spectrometry.

A highly sensitive liquid chromatographic-mass spectrometric procedure has been developed to quantitate plasma concentrations of acitretin, a dermatologic agent used to treat severe psoriasis. The assay utilizes the combination of normal-phase microbore high-performance liquid chromatography, negative chemical ionization mass spectrometry, selective ion monitoring and stable isotope dilution. The method has been used to measure acitretin and its metabolite, 13-cis-acitretin, over a range of 1-20 ng/ml in human plasma. The inter-assay precision was 5.3% for acitretin and 3.9% for 13-cis-acitretin, while the intra-assay precisions for acitretin and 13-cis-acitretin were 10.8 and 12.7%, respectively. Reproducibility of the assay for acitretin and 13-cis-acitretin, which was determined by the relative standard deviation of multiple analyses of the same quality assurance sample, was 5.9 and 8.1%, respectively.

Pharmacokinetics and pharmacodynamics of recombinant human interleukin-12 in male rhesus monkeys.

The pharmacokinetics and pharmacodynamics of recombinant human interleukin-12 (rHuIL-12) were investigated in male rhesus monkeys. The monkeys received a 40-min i.v. infusion of 42.5 micrograms/kg of recombinant human interleukin-12 on day 1 followed by a s.c. injection of the same dose on day 5. Serum samples were collected at appropriate time points and assayed for interleukin (IL-12) by an IL-12 capture bioassay, interferon (IFN-gamma) by an IFN-gamma enzyme-linked immunosorbent assay, and neopterin by a neopterin radioimmunoassay. After i.v. infusion, the systemic clearance rate of this protein was very slow (average, 3 ml/hr/kg). The volume of distribution at steady state ranged from 59 to 90 ml/kg. After the s.c. dose, the mean Cmax was 61 ng/ml and the mean Tmax was 18 hr. The absolute bioavailability was moderate (20-30%) after s.c. injection. By compartmental analysis, by using a two-compartment model the T 1/2 lambda 1 ranged from 0.2 to 5 hr and the T 1/2 lambda 2 ranged from 13 to 19 hr. When determining the percentage of the area of the serum concentration-time curve, per phase, > 85% of the protein was found in the lambda 2 phase. We selected IFN-gamma as one of the pharmacodynamic markers to study because it is produced by T-lymphocytes and natural killer cells in response to IL-12. In addition, once IFN-gamma is produced, it primes macrophages for tumor killing that in turn secrete neopterin. We show that within 24 to 48 hr after the i.v. dose, IFN-gamma concentrations are elevated in these monkeys (average, 300 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of chemical reactivity of urushiol-type haptens on sensitization and the induction of tolerance.

Contact sensitization to components of the urushiol oils of poison oak and poison ivy appears to require covalent bond formation between the o-quinones derived from urushiol catechols and nucleophilic groups on proteins. Previous studies using a murine delayed hypersensitivity model demonstrated that 5-methyl-3-pentadecylcatechol (5-Me-PDC) is an epicutaneous tolerogen to the parent compound and a weak sensitizer to itself. To investigate further the structural requirements for sensitization vs suppression, 5,6-dimethyl-3-pentadecylcatechol (5,6-di-Me-PDC) and 4,5,6-trimethylpentadecylcatechol (4,5,6-tri-Me-PDC) were synthesized. The former compound is blocked at both preferred sites for covalent bond formation and the latter is completely blocked towards conjugate addition reactions. These compounds were tested for sensitizing and suppressive ability. Epicutaneous application of both analogs suppressed subsequent induction of sensitization to 3-pentadecylcatechol (PDC) and 3-heptadecylcatechol (HDC). Lymph node cells from animals treated with 5,6-di-Me-PDC could transfer suppression. The dimethyl analog, 5,6-di-Me-PDC, but not the trimethyl analog also exhibited weak sensitizing capacity. The urushiol analogs 5-pentadecylresorcinol (PDR) and 3-heptadecylveratrole (HDV) which cannot form o-quinones were found to be ineffective sensitizers as well. HDV in addition produced no blastogenesis in draining lymph nodes whereas lymph node cell proliferation induced by 4,5,6-tri-Me-PDC followed the same kinetics as previously observed for HDC. PDR elicited weak proliferation with a different time course. These and previous studies indicate that blocking the C5-position on the catechol ring favors the induction of suppression, although some sensitizing capacity may be retained. Covalent bond formation may not be necessary for the induction of active suppressor cell populations.

An unusual metabolite of testosterone. 17 beta-Hydroxy-4,6-androstadiene-3-one.

A testosterone metabolite, 17 beta-hydroxy-4,6-androstadiene-3-one, possessing an absorbance maximum at 284 nm, was formed during incubation of testosterone with liver microsomes from dexamethasone-treated rats. The metabolite was identified by HPLC, UV spectroscopy, and thermospray liquid chromatography/mass spectrometry. The formation of this metabolite by rat liver microsomes required NADPH and oxygen and was inhibited markedly by SKF 525-A, 2,4-dichloro-6-phenylphenoxyethylamine, or CO/O2 (8:2, v/v), but not by cyanide, an inhibitor for stearyl-CoA desaturase. Pretreatment of rats with phenobarbital, pregnenolone 16 alpha-carbonitrile, and dexamethasone enhanced the formation of this metabolite in parallel with the increase in formation of 6 beta-hydroxytestosterone (r2 = 0.99). Although 16-methylprogesterone, a known 6 beta-hydroxylase inhibitor, competitively inhibited the formation of the metabolite and 6 beta-hydroxytestosterone by liver microsomes from dexamethasone-treated rats, the metabolite was not formed from either 6 beta-hydroxytestosterone or 7-hydroxytestosterone during incubation with liver microsomes. These findings are consistent with the view that cytochrome P-450 isozymes that catalyze 6 beta-hydroxylation of steroids in rat liver microsomes also catalyze the dehydrogenation of testosterone to form a double bond between the C-6 and C-7 positions.

Comparative tissue distribution of 125I- and U-14C-labeled recombinant human interleukin-2 in the rat.

The distribution of recombinant human interleukin-2 (rhIL-2) after intravenous administration of 125I- or U-14C-labeled rhIL-2 is reported in the major organs of the rat. Five minutes after the administration of U-14C-labeled rhIL-2, the radioactivity had been rapidly cleared from the plasma, while 38% of the dose was found in the kidneys, confirming that the major site of clearance for rhIL-2 is the kidney. After 1 h, a large fraction of the radioactivity had disappeared from the kidney and was found in the carcass. When the same experiments were carried out with 125I-labeled rhIL-2, comparable distribution results were obtained: Preferential accumulation of 125I radioactivity (37.4%) was found in the kidney at 5 min after iv administration of 125I-labeled rhIL-2. One hour after dosing 125I label was predominantly present in the carcass (46%) and skin (15%). Similar percentages of the dose of 125I or 14C radioactivity were present in other organs or tissues. The present study indicates a similar distribution of the radiolabel in selected tissue and organs regardless of whether 14C or 125I was employed.

Furosemide 1-O-acyl glucuronide. In vitro biosynthesis and pH-dependent isomerization to beta-glucuronidase-resistant forms.

A biosynthetic acyl-type glucuronic acid conjugate of furosemide was isolated from in vitro incubation of pregnenolone-16 alpha-carbonitrile-induced rat liver microsomes containing UDP-glucuronyltransferase activity, furosemide, and UDP-glucuronic acid. Furosemide 1-O-acyl glucuronide (FG) was specifically hydrolyzed by beta-glucuronidase (BG) and was also labile to alkaline hydrolysis. FG concentration decreased at an apparent first order rate when incubated at 37 degrees C in buffer solution of pH values greater than 6.0 with only moderate hydrolysis of the conjugate at pH values less than 8.5. Formation of rearrangement forms of FG that were resistant to BG but labile to alkaline hydrolysis accounted for most of the disappearance of FG at this pH range. Radiochemical labeling of the conjugate with either 14C-furosemide or 14C-UDP-glucuronic acid was detected in the BG-resistant isomerization products of FG as they were separated by HPLC. The structure of FG and its isomerization products was further verified by negative ion thermospray liquid chromatography/mass spectrometry. The abundant (M - 1)-ion at mass 505, the aglycone fragment at m/z 329, and the characteristic sugar fragment ion of mass 175 were found in the spectra of FG and three additional isomers. An ion at m/z 221 was noted only in the case of the parent conjugate and thus may prove to be a characteristic ion for 1-O-acyl-linked glucuronides under negative ion thermospray. In vivo as well as in vitro rearrangement of FG to BG-resistant forms might affect the results of furosemide disposition studies which use BG hydrolysis to determine FG formation.

Quantification of recombinant interleukin-2 in human serum by a specific immunobioassay.

A sensitive and specific assay for recombinant interleukin-2 (rIL-2) in human serum is described. The assay is based on a sequential sandwich immunobioassay that uses a microtiter plate coated with anti-rIL-2 monoclonal antibody (specific for recombinant human IL-2) to capture rIL-2 from serum, and an IL-2 dependent T-cell line that proliferates in a dose-dependent fashion. The lower limit of quantitation of the assay is 2 units/mL (1 unit = approximately 50 pg) using 0.1 mL of serum and the calibration curves ranged from 2 to 50 units/mL. Data are reported on the sensitivity, precision, reproducibility, and specificity of the assay; the stability of rIL-2 in serum; and the recovery of rIL-2 from serum. We also report on the use of the procedure to assay clinical samples from patients with AIDS undergoing treatment with rIL-2.

Thermospray liquid chromatography/mass spectrometry studies on mechanisms of nucleic acid alkylation by some deuterated carcinogens.

Alkylation of liver nucleic acids of rats treated with nitrosobis(2-oxopropyl)amine (BOP), nitrosomethyl(2-oxopropyl)amine (NMOP), and (methylazoxy)methane (AOM) was studied by using deuterium-labeled compounds. Six hours after treatment of each rat with 5-10 mg of compound, nucleic acids were isolated from the liver of two rats and hydrolyzed in dilute acid, and the fraction containing 7-methylguanine (7-MeG) was separated by ion-exchange high-pressure liquid chromatography. After purification by repeated chromatography, the sample was analyzed by liquid chromatography in ammonium acetate coupled with thermospray positive ion mass spectrometry. AOM containing a deuterated methyl group proximal to the oxygen-bearing nitrogen gave 7-MeG containing three deuterium atoms (169 amu), whereas AOM having the methyl group distal to oxygen deuterated produced 7-MeG without deuterium (166 amu). This indicates that methylation of nucleic acids is through oxidation of AOM to (methylazoxy)methanol and the corresponding aldehyde. BOP-d4, in which both alpha-methylenes contained deuterium, gave rise to 7-MeG containing two atoms of deuterium (168 amu). NMOP-d5, with deuterium in the N-methyl and in the alpha-methylene, gave rise only to 7-MeG containing three deuterium atoms. The methyl-d3 group must originate from the N-methyl of NMOP, probably through formation of a methyldiazonium ion. This result lends support to a mechanism involving a Baeyer-Villiger oxidation of the ketone, rather than oxidation of the N-methyl and formation of an (oxopropyl)diazonium ion, which could give rise to a methylating agent following cyclization.