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.

A comparison of mass balance, pharmacokinetics and disposition of [14C(U)]- and [125I]recombinant human interleukin-2 in cynomolgus monkeys.

Recombinant human interleukin-2 (rHuIL-2) has been metabolically labeled with 14C amino acids in Escherichia coli and affinity purified on a rHuIL-2 receptor affinity column. The radiolabeled molecule had a specific radioactivity of 238 dpm/unit and the identical amino acid sequence and biological activity as unlabeled rHuIL-2. In this study, we used this labeled [14C(U)]rHuIL-2 and commercially available [125I]rHuIL-2 (identical in sequence to the [14C(U)]rHuIL-2) to compare the mass balance, pharmacokinetics, and disposition in cynomolgus monkeys. After a single intravenous bolus dose of 4 x 10(5) units/kg, serum samples were collected for 7 days and examined for biological activity, total radioactivity, and by molecular size exclusion chromatography. Urine and feces were analyzed for total radioactivity. When analyzed for biological activity, both [14C(U)]- and [125I]rHuIL-2 exhibited the following pharmacokinetic parameters: terminal elimination half-life of 1-2 hr, AUC0-infinity ranged from 2005 to 4659 units x hr/ml, clearance was 90-200 ml/hr/kg, and volume of distribution ranged from 103 to 163 ml/kg. Comparison of the pharmacokinetic profiles of the two radiolabels were very different from bioactivity, in that the elimination half-lives for radioactivity were approximately 8 days and 10 hr for [14C(U)]- and [125I]rHuIL-2, respectively. We conclude that the [14C(U)]rHuIL-2 was metabolized to constituent amino acids and recycled into newly synthesized proteins from our size exclusion chromatography studies.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Characterization of anti-IL-1 alpha autoantibodies in the sera from healthy humans.

Previously, we reported that pooled normal human serum contained anti-IL-1 alpha autoantibodies. Further characterization studies have been undertaken with sera from individual healthy humans. Molecular exclusion chromatography demonstrated that 9 of 38 test sera contained anti-IL-1 alpha autoantibodies that specifically bound 125I-IL-1 alpha. The autoantibodies formed immune complexes composed of either one IgG or two IgG molecules bound to one 125I-IL-1 alpha molecule. The data suggest that the autoantibodies recognize at least two separate antigenic sites on IL-1 alpha. The autoantibodies neutralized IL-1 alpha induced IL-2 secretion by mouse thymocytes (EL-4 NOB-1). Further, in an in vitro competitive receptor binding assay, the autoantibodies completely blocked 125I-IL-1 alpha binding to recombinant human IL-1 receptor expressed on CHO cells. Our previous studies have established a correlation between inhibition of 125I-IL-1 alpha binding to receptor bearing cells and neutralization of IL-1 bioactivity in vitro and in vivo. These data suggest that development of anti-IL-1 alpha autoantibodies may play a significant role in modulating the effects of high local or systemic concentrations of IL-1 alpha.

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.

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.

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.

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.

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.

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.

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.

Stable isotope dilution method using thermospray liquid chromatography/mass spectrometry for quantification of daily cortisol production in humans.

A thermospray high-performance liquid chromatography/mass spectrometry method for the separation and quantification of cortisol in human serum has been developed. The technique does not require derivatization, allows for both qualitative and quantitative determinations, provides increased specificity not available from conventional high-performance liquid chromatography, and has a detection limit of 5 pmol on-column. This isotope dilution mass spectrometry method, using d3-cortisol as an internal standard, allows precise determinations even at low isotopic ratios (2%-5 mole%). Evidence that this technique can be applied to the quantification of serum cortisol and to the determination of daily cortisol production in humans is presented.

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.

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.

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.

Thermospray HPLC/MS: a new mass spectrometric technique for the profiling of steroids.

The analysis of various steroid classes by thermospray HPLC-MS using solvent systems containing 0.1 M ammonium acetate has been described. For simple unconjugated 3-oxo-4-ene steroids the positive ion spectra are dominated by a parent ion M + H+ and with increasing numbers of hydroxyl group intense ions formed by sequential losses of water (M + H- n18)+ become important. Steroids with dihydroxyacetone side-chains readily lose these side-chains and the resulting (M + H-60)+ fragment is the base peak in their spectra. The (M + H-60)+ ion is not important for most steroids with glycerol-type side-chains. Although competition between thermal degradation and vaporization was observed at lower concentrations, the effect was minimized after optimizing conditions and the protonated molecular ion was easily detected when as little as 1-10 pmol of material were injected on-column. Steroid glucuronides when analyzed in the negative ion mode give simple spectra with base peak and parent ion (M-H)-. Lack of fragmentation permits facile and sensitive measurement of individual glucoronides by selected-ion-monitoring. Extensive fragmentation is seen in the positive ion mode with sequential losses of H2O from the molecular ions (M + NH4)+ and from the aglycone fragment ion. For simple unconjugated steroids the sensitivity of HPLC-MS in selected-ion-monitoring mode can be excellent. When the protonated molecular ion of testosterone was monitored the signal/noise ratio for 30 pg testosterone was about 10.

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.

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.