alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Intestinal absorption and metabolism of retinoyl beta-glucuronide in humans, and of 15-[14C]-retinoyl beta-glucuronide in rats of different vitamin A status

In order to prove the hypothesis that humans and animals with adequate vitamin A status do not absorb and metabolize orally administered all-trans retinoyl β-glucuronide, unlabeled retinoyl glucuronide (0.1 mmol) was orally dosed to fasting well-nourished young men. Neither retinoyl glucuronide nor retinoic acid, a possible metabolite, appeared in the blood within 12 h after ingestion. Next, radiolabeled all-trans 15-[¹⁴C]-retinoyl β-glucuronide was chemically synthesized by a new procedure, and fed orally to rats of different vitamin A status. Analysis of blood and other tissues 5 or 24 h after the dose, showed the presence of radioactivity ( 0.5%) in the blood of vitamin A deficient rats, but not in sufficient rats. Livers of all rats contained small, but detectable amounts (0.3 to 1.1% of the dose) of radioactivity. The accumulation of radioactivity in the liver was highest in deficient rats. Analysis of the retinoids showed that the radioactivity in serum and liver was due to retinoic acid formed from retinoyl glucuronide. Within 24 h after the dose, 31 to 40% of the administered radioactivity was excreted in the feces, and 2 to 4.7% of the dose was excreted in the urine. Results of the present studies show that oral administration of retinoyl β-glucuronide did not give rise to detectable changes in blood retinoyl glucuronide and/or retinoic acid concentrations in humans or rats with adequate vitamin A status.     

Distribution of [14C]-trans-resveratrol,a cancer chemopreventive polyphenol,in mouse tissues after oral administration

Trans-resveratrol, a phenolic compound present in wine, has been reported to be a potential cancer chemopreventive agent. However, although it has numerous biological activities in vitro, there are few data about its bioavailability and tissue distribution in vivo. The objectives of this study were to investigate the absorption and tissue distribution of 14C-trans-resveratrol following oral administration to mice. Male Balb/c mice were given a single oral dose of 14C-trans-resveratrol and were sacrificed at 1.5, 3 or 6 h postdose. The distribution of radioactivity in tissues was evaluated using whole-body autoradiography, quantitative organ-level determination and microautoradiography. In addition, identification of radioactive compounds in kidney and liver was done with high-performance liquid chromatography. Autoradiographic survey of mice sections as well as radioactivity quantification in various organs revealed a preferential fixation of 14C-trans-resveratrol in the organs and biological liquids of absorption and elimination (stomach, liver, kidney, intestine, bile, urine). Moreover, we show that 14C-trans-resveratrol derived radioactivity is able to penetrate the tissues of liver and kidney, a finding supported by microautoradiography. The presence of intact 14C-trans-resveratrol together with glucurono- and/or sulfoconjugates in these tissues was also shown. This study demonstrates that trans-resveratrol is bioavailable following oral administration and remains mostly in intact form. The results also suggest a wide range of target organs for cancer chemoprevention by wine polyphenols in humans.     

Absorption and distribution of [1-14C]dolichol intubated into rats

[1-14C]Dolichol was mixed in vitro with sunflower seed oil and intubated into rats. Radioactivity began to appear in the blood at 3 h and peaked after about 6 h. The absorbed radioactivity was rapidly cleared from the blood. At 7.5 h postintubation two thirds of the radioactivity in the serum was associated with chylomicrons and about one quarter with the high density lipoproteins. At 12 h the proportion of the radioactivity in the chylomicrons had fallen to one third and that in the high density lipoproteins had increased to one half of the total. Less than 0.02% of the dose was found in the tissues after 12 h. Liver and blood each contained about one third of the total, with smaller amounts in the lungs and spleen. The heart, testes, brain, and kidneys contained only traces of radioactivity. After 12 h most of the radioactivity in the tissues and feces was present as [1-14C]dolichol. The radioactive compounds in the urine (about 0.05% of the dose) were more polar than [1-14C]dolichol as determined by thin-layer chromatography.     

Metabolism and toxicokinetics of the mycotoxin ochratoxin A in F344 rats

Male (n=18) and female (n=18) F344 rats were administered a single dose of OTA (0.5 mg/kg b.w.) in corn oil by gavage. Animals (n=3) were sacrificed 24, 48, 72, 96, 672 and 1,344 hours after OTA administration and concentrations of OTA and OTA-metabolites in urine, feces, blood, liver and kidney were determined by HPLC with fluorescence detection and/or by LC-MS/MS. Recovery of unchanged OTA in urine amounted to 2.1% of dose in males and 5.2% in females within 96 h. In feces, only 5.5% resp. 1.5% of dose were recovered. The major metabolite detected was OTalpha, low concentrations of OTA-glucosides were also present in urine. Other postulated metabolites were not observed. The maximal blood levels of OTA were observed between 24 and 48h after administration and were app. 4.6 µmol/l in males and 6.0 µmol/l in females. Elimination of OTA from blood followed first-order kinetics with a half-life of app. 230h calculated from 48h to 1344h. In liver of both male and female rats OTA-concentrations were less than 12 pmol/g tissue, with a maximum at 24h after administration. In contrast, OTA accumulated in the kidneys, reaching a concentration of 480 pmol/g tissue in males 24h after OTA-administration. In general, tissue concentrations in males were higher than in females. OTalpha was not detected in liver and kidney tissue of rats administered OTA and OTalpha concentrations in blood were low (10–15 nmol/1). The high concentrations of OTA in kidneys of male rats may explain the organ- and gender-specific toxicity of OTA.     

Intracellular metabolic fate of radioactivity after injection of technetium-99m-labeled hydrazino nicotinamide derivatized proteins.

Hydrazino nicotinate (HYNIC) has been shown to produce technetium-99m (99mTc)-labeled proteins and peptides of high stability with high specific activities. However, persistent localization of radioactivity was observed in nontarget tissues such as the liver and kidney after administration of [99mTc]HYNIC-labeled proteins and peptides, which compromises the diagnostic accuracy of the radiopharmaceuticals. Since lysosomes are the principal sites of intracellular catabolism of proteins and peptides, 99mTc-HYNIC-labeled galactosyl-neoglycoalbumin (NGA) was prepared using tricine as a co-ligand to investigate the fate of the radiolabel after lysosomal proteolysis in hepatocytes. When injected into mice, over 90% of the injected radioactivity was accumulated in the liver after 10 min injection. At 24 h postinjection, ca. 40% of the injected radioactivity still remained in liver lysosomes. Size-exclusion HPLC analyses of liver homogenates at 24 h postinjection showed a broad radioactivity peak ranging from molecular masses of 0.5-50 kDa. RP-HPLC analyses of liver homogenates suggested the presence of multiple radiolabeled species. However, most of the radioactivity migrated to lower molecular weight fractions on size-exclusion HPLC after treatment of the liver homogenates with sodium triphenylphosphine-3-monosulfonate (TPPMS). The TPPMS-treated liver homogenates showed a major peak at a retention time similar to that of [[99mTc](HYNIC-lysine)(tricine)(TPPMS)] on RP-HPLC. Similar results were obtained with urine and fecal samples. These findings suggested that the chemical bonding between 99mTc and HYNIC remains stable in the lysosomes and following excretion from the body. The persistent localization of radioactivity in the liver could be attributed to the slow elimination rate of the final radiometabolite, [[99mTc](HYNIC-lysine)(tricine)2], from lysosomes, and subsequent dissociation of one of the tricine co-ligands in the low pH environment of the lysosomes in the absence of excess co-ligands, followed by binding proteins present in the organelles. The findings in this study also suggested that the development of appropriate co-ligands capable of preserving stable bonding with the Tc center is essential to reduce the residence time of radioactivity in nontarget tissues after administration of [99mTc]HYNIC-labeled proteins and peptides.     

Metabolie Fate of Glycyl-14C-prolylhydroxyproline in Young Rats

To examine the origin of urinary hydroxyproline peptides, the metabolism of the radioactive tripeptide, glycyl-¹⁴C-prolylhydroxyproline, was investigated in normal young rats in vivo. The radioactive tripeptide was synthesized from glycine, l-(U-¹⁴C)proline and hydroxy-l-proline in our laboratory. The distributions of the radioactivity in body protein, lipid and soluble fractions were 23.7, 1.8 and 0.12% of the injected dose, respectively, 56 hr after the intraperitoneal injection of the ¹⁴C-tripeptide. The excretions of the radioactivity into expired carbon dioxide and urine were 29.6 and 34.2% of the injected dose, respectively, and large proportions of both the ¹⁴C excretions occurred during the first 12hr.

The results suggest that not a small amount of the glycylprolylhydroxyproline peptide injected is hydrolyzed in tissues of animals and the free proline derived is used for protein synthesis and/or further degraded to expired carbon dioxide.>     

Plasma kinetics and urine profile of ethyl glucosides after oral administration in the rat

In this in vivo study, the time course of plasma concentration and the urinary excretion of ethyl alpha-D-glucoside (alpha-EG) and ethyl beta-D-glucoside (beta-EG) were investigated in rats after a single oral dose of 4 mmol/kg body weight. Maximal plasma concentrations of both alpha-EG and beta-EG (EGs) reached approximately 3 mM at 1 h after oral administration and then decreased rapidly. Approximately 80% of EGs administered were excreted into the urine during the first 6 h. Within 24 h, cumulative urinary alpha-EG and beta-EG excretions were estimated to be 87.2+/-7.9% and 85.4+/-5.0%, respectively. Traces of both EGs were detected in plasma and urine 24 h after oral ingestion. The results of this study indicate that almost all of both EGs was rapidly absorbed into the blood stream and easily excreted into the urine after oral administration, and that a small amount of them remained in the rat body 24 h after administration.     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.     

Transport and distribution of homocarnosine after intracerebroventricular and intravenous injection in the rat

Rats were injected intracerebroventricularly (i.c.v.) or i.v. with [¹⁴C]homocarnosine (250 nmol). Distribution of the dipeptide in brain structures, transport from the brain to the blood, distribution in peripheral organs, and excretion in the urine were studied by measuring radioactivity in tissue, plasma, and urine samples by liquid scintillation counting 15–120 min after injection. After i.c.v. injection, [¹⁴C]homocarnosine was taken up into all parts of the brain investigated (highest uptake in structures close to the site of injection), it was transported to the blood, and radioactive substances were found in low concentration in muscle, spleen, and liver, in high concentration in the kidneys, and very high concentration in the urine. Investigations using high pressure liquid chromatography (HPLC) showed that no degradation took place in the brain, all radioactivity was found in the homocarnosine fraction. In the plasma 86% of the radioactivity was found in the GABA fraction presumed to be formed by cleavage of the peptide, while in the kidneys 35% and in the urine 40% was found in the GABA fraction. After i.v. injection of [¹⁴C]homocarnosine, no radioactivity was measured in hippocampus, striatum, cerebellum and cerebral cortex 15 min after injection, however, 60 min after injection a very low activity was detected in these structures (estimated intravascular radioactivity subtracted). A low activity was also measured in the spinal cord both 15 and 60 min after injection. When homocarnosine and GABA were separated on HPLC, all radioactivity in brain tissue was found in the GABA fraction, indicating either that [¹⁴C]homocarnosine did not cross the blood-brain barrier in amounts that could be measured with the method used, or that peptide entering the brain was rapidly transported back to the blood. [¹⁴C]Homocarnosine was not taken up either into crude synaptosomal preparations from hippocampus, striatum, cerebellum, cortex and spinal cord, or into slices prepared from the hippocampus and striatum. Transport from the brain to the kidneys and excretion in the urine seems to be a major route for disposal of this peptide in the rat.     

New metabolites of riboflavin appeared in rat urine

By using paper and silica gel thin layer chromatography with various solvent systems, flavin compounds appeared in rat urine after administration of radioactive riboflavin were analyzed in detail. Two new metabolites having radioactivity were separated and their structures were determined to be 7-carboxy lumichrome and 8-carboxy lumichrome. The sum of radioactivities of these two compounds was about 46% of total radioactivity excreted in the urine during 24 h.     

Pharmacokinetics of methimazole in normal subjects and hyperthyroid patients

Serum and urinary concentrations of methimazole (MMI) were measured by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD) in 10 normal subjects and 43 hyperthyroid patients after intravenous and oral administration of the drug. The pharmacokinetic parameters of MMI were estimated in 5 normal subjects and 15 hyperthyroid patients according to a two-compartment model after intravenous injection of a 10 mg dose. The mean half-life of the distribution phase (T1/2 alpha) was 2.7 +/- 1.0 h (mean +/- SD) and 3.1 +/- 1.4 h and that of the slower-phase (T1/2 beta) was 20.7 +/- 9.6 h and 18.5 +/- 12.9 h in normal subjects and hyperthyroid patients, respectively. There were no significant differences between pharmacokinetic parameters of normal subjects and those of hyperthyroid patients. No correlations between free T4 index (FT4I) and pharmacokinetic parameters were observed. Maximum serum MMI concentrations (Cmax) (213 +/- 84 and 299 +/- 92 ng/ml) were attained 1.8 +/- 1.4 h and 2.3 +/- 0.8 h after a single dose of 10 mg in 5 normal subjects and in 15 hyperthyroid patients, respectively. In hyperthyroid patients the time taken to reach the peak concentration (Tmax) after a single dose of 10 mg was similar to that after a single 15 mg and 30 mg dose. The pharmacokinetic parameters, except Cmax and the area under the curve (AUC), were not affected by the administered dose and those, except Cmax, were not affected by the thyroid function. All urine was collected at intervals of 3 h for the first 12 h and then at 24 h and 48 h after intravenous and oral administration of MMI. In all subjects, MMI rapidly appeared in the urine and the rate of excretion was highest in the first 3 h. The cumulative urinary excretion of MMI was 5.5-8.5% of administered doses in normal subjects and hyperthyroid patients. These findings in the present study are compatible with the assumption that the extent of absorption of MMI is high, if not complete, and hyperthyroidism does not affect the kinetics of MMI, and that interindividual variation is observed in the time taken to reach the peak concentration after oral administration.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

Validation of a high-performance liquid chromatography assay for urinary nedocromil sodium following oral and inhaled administration

The validation of a solid-phase extraction and an ion pair high-performance liquid chromatographic assay for the determination of nedocromil sodium (NCS) in urine samples following oral and inhaled administration to healthy volunteers is described. NCS and its internal standard sodium cromoglycate (SCG) were extracted from urine samples using solid-phase extraction and then quantified using high-performance liquid chromatography (HPLC). A 25-cm C₈ Spherisorb 5 μm stationary phase with a mobile phase containing a long alkyl chain ion-pair reagent (methanol–0.045 M phosphate buffer–0.05 M dodecyl triethyl ammonium phosphate; 550:447.6:2.4, v/v) was used. The mean (S.D.) intra-day accuracy and precision of the HPLC assay was 99.9 (1.6) and 7.05 (4.9)%, respectively. These values for the inter-day data were 102.4 (4.07) and 10.5 (2.7)%, respectively, over the concentration range investigated. The method described permits the detection of NCS in human urine at concentrations as low as 0.04 μg ml⁻¹ where the signal-to-noise ratio is greater than 3:1. In 10 healthy volunteers a significantly greater amount of NCS was excreted in the urine following inhalation than after oral dosing (p<0.001). The mean (S.D.) amount of NCS renally excreted at 0.5, 1.0 and 24 h following inhalation of four 2-mg doses of NCS from a metered dose inhaler (MDI) was 0.513 (0.24), 1.163 (0.49) and 4.00 (1.73)% of the nominal dose. Similar values after oral administration of 8 mg of NCS were 0.026 (0.03), 0.079 (0.06) and 0.930 (0.74)%, respectively.     

Identification and determination of the enantiomers of moprolol and their metabolites in human urine by high-performance liquid chromatography and gas chromatography—mass spectrometry

A simple and sensitive high-performance liquid chromatographic (HPLC) method using chiral derivatization was developed to screen and determine the enantiomers of moprolol and their metabolites in human urine. The recovery of (+)- and (−)-moprolol from urine was 70.8–81.1% at different concentrations. The coefficients of variation (C.V.) were less than 3.2 and 6.5% for intra- and inter-assays, respectively. Moprolol could be detected in urine up to 24 h after oral administration of a 50-mg dose of moprolol. Unconjugated and conjugated enantiomers of moprolol and their metabolites were analyzed by gas chromatography (GC). A gas chromatographic—mass spectrometric (GC—MS) confirmatory method was established to identify the metabolites of moprolol. The double derivatization procedure for moprolol and their metabolites with S-(−)-menthyl chloroformate [(−)-MCF] and N-methyl(trimethylsilyl)trifluoroacetamide (MSTFA) gave very good GC—MS properties of the derivatized compounds and provided reliable structural information for their confirmation analysis. This is the first published report on the use of a GC—MS method for the detection of the enantiomers of moprolol and their metabolites in human urine.     

Pharmacokinetics of oestrone-3-O-sulphamate

The sulphatase pathway is thought to be the major route of oestrogen synthesis in breast tumours in postmenopausal women. There is currently considerable interest in developing a potent steroid sulphatase inhibitor to block oestrogen synthesis by this route. One of the most potent inhibitors discovered so far is oestrone-3-O-sulphamate (EMATE) which is active in vivo. In this study we report the preparation of a formulation for the administration of EMATE by the oral route. A method, using high-performance liquid chromatography (HPLC), was also established to measure concentrations of EMATE in rat plasma after its oral or i.v. administration. Using the oral formulation and HPLC assay, EMATE was readily detected in rat plasma after oral administration. Plasma EMATE concentrations were related to the dose of drug administered orally over the 10–40 mg/kg range. To examine the pharmacokinetics of EMATE, the compound (40 mg/kg, single dose) was administered either orally (in the formulation) or i.v. (in propylene glycol) with plasma samples being collected for up to 6 h. After oral administration, EMATE was rapidly absorbed, with the peak plasma concentration being detected at 30 min, after which plasma concentrations rapidly decreased. After i.v. administration a plasma EMATE concentration was detected at 1 h similar to that after oral administration. The clearance of EMATE from plasma followed a bi-phasic curve, showing an initial half-life of 30 min, followed by a slower half-life of 4 h 30 min. Little evidence was obtained for any metabolism of EMATE to oestrone. Rat liver sulphatase activity was almost completely inhibited (>99%) within 30 min of oral or i.v. administration of EMATE.     

Dose-linear pharmacokinetics, tissue distribution, and excretion of a recombinant fusion protein 125I-GST-TatdMt possessing potent anti-obesity activity

This study first examined the pharmacokinetic disposition of GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in rats after i.v. injection. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. The radioiodinated 125I-GST-TatdMt was administered to rats at doses of 9 microg (1640 nCi), 18 mug (3388 nCi), and 35 microg (6420 nCi). Upon administration, the total radioactivity in serum declined bi-exponentially, with the average terminal elimination half-life ranging from 13.7 to 15.7 h. There was a linear relationship between dose and AUC(INF) (r2=1.000) and between dose and Co (r2=0.999). The fraction of administered radioactivity excreted in feces was low (mean range 1.5-2.8%), while the majority of the radioactivity was excreted in urine (mean range 54.9-61.4%). The radioactivity found in the liver, lungs, spleen, and kidneys were higher than in serum, but the tissue-to-serum ratios were relatively low (<1.64). The radioactivity in testes, adipose tissue, heart, and brain was lower than in serum (tissue-to-serum ratios 0.046-0.27). The findings of this study indicate dose-linear pharmacokinetics of 125I-GST-TatdMt in rats over the i.v. dose range studied.     

gamma-Tocopherol biokinetics and transformation in humans

BACKGROUND: The uptake and biotransformation of gamma-tocopherol (gamma-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of gamma-T biology in healthy volunteers and their response to gamma-T supplementation. METHODS: A single bolus of 100 mg of deuterium labeled gamma-T acetate (d(2)-gamma-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d(2) and d(0)-gamma-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-gamma-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of gamma-T supplementation on alpha-T concentrations in plasma and alpha-T and gamma-T metabolite formation were also assessed by HPLC or GC-MS analysis. RESULTS: At baseline, mean plasma alpha-T concentration was approximately 15 times higher than gamma-T (28.3 vs. 1.9 micromol/l). In contrast, plasma gamma-CEHC concentration (0.191 micromol/l) was 12 fold greater than alpha-CEHC (0.016 micromol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 micromol, respectively) suggesting that the clearance of alpha-CEHC from plasma was more than 40 times that of gamma-CEHC. After d(2)-gamma-TAC administration, the d(2) forms of gamma-T and gamma-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d(0) species were hardly affected. Mean total concentrations of gamma-T and gamma-CEHC in plasma and urine peaked, respectively, between 0-9, 6-12 and 9-24 h post-supplementation with increases over baseline levels of 6-14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d(2)-gamma-T equivalents was approximately 7 mg. Baseline levels of gamma-T correlated positively with the post-supplementation rise of (d(0) + d(2)) - gamma - T and gamma-CEHC levels in plasma, but correlated negatively with urinary levels of (d(0) + d(2))-gamma-CEHC. Supplementation with 100 mg gamma-TAC had minimal influence on plasma concentrations of alpha-T and alpha-T-related metabolite formation and excretion. CONCLUSIONS: Ingestion of 100mg of gamma-TAC transiently increases plasma concentrations of gamma-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of gamma-T nor the concentration and metabolism of alpha-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios alpha-T to gamma-T and gamma-CEHC to alpha-CEHC.     

Investigations into the chirality of the metabolic sulfoxidation of cimetidine

The individual enantiomers of cimetidine sulfoxide were resolved by preparative chromatography using a Chiralcel OC stationary phase and were characterized by the determination of optical rotation and circular dichroism spectra. Cimetidine sulfoxide was isolated from the urine of two healthy male volunteers following oral administration of cimetidine (400 mg). Urine was collected every 2 h for 12 h postdosing, after which time HPLC analysis indicated negligible recovery of the drug as the sulfoxide. Some 7% of the dose was recovered as cimetidine sulfoxide over this period. The enantiomeric composition of cimetidine sulfoxide was determined by sequential achiral—chiral chromatography using the OC phase. Over the collection period the enantiomeric ratio was found to be constant in all samples at (+/?) of 71 ± 2.5:29 ± 2.5. The enantiomeric composition of cimetidine sulfoxide was also determined in rat urine (24 h) following the administration of cimetidine (30 mg/kg po) to male Wistar rats (n = 7). The enantiomeric ratio in this case was found to be (+/?) 57 ± 2.3:43 ± 2.3. These preliminary data indicate that sulfoxidation of cimetidine is stereoselective with respect to the (+)-enantiomer and that species variation in enantiomeric composition occurs. © 1994 Wiley-Liss, Inc.     

Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

To evaluate vitamin E metabolism, a method was developed to quantitate liver alpha- and gamma-tocopherol metabolites, alpha-carboxyethyl hydroxychroman [alpha-CEHC; 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman] and gamma-CEHC [2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman], respectively. Vitamin E supraenriched livers were obtained from rats that were injected with vitamin E daily for 18 days. Liver samples (approximately 50 mg) were homogenized, homogenate CEHC-conjugates were hydrolyzed, CEHCs were extracted with ethyl ether, and then CEHCs were quantitated using liquid chromatography-mass spectrometry (LC-MS). Precision, based on intersample variability, ranged from 1% to 3%. Recovery of alpha- and gamma-CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of alpha- and gamma-CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver alpha-tocopherol, the MS peak for liver alpha-CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 +/- 0.01 to 15 +/- 2 nmol/g). Liver alpha-CEHC concentrations were correlated with serum alpha-CEHC, liver alpha-tocopherol, and serum alpha-tocopherol (P < 0.001 for each comparison). alpha-CEHC represented 0.5-1% of the liver alpha-tocopherol concentration. Thus, LC-MS can be successfully used to quantitate alpha- and gamma-CEHC in liver samples. These data suggest that in times of excess liver alpha-tocopherol, increased metabolism of alpha-tocopherol to alpha-CEHC occurs.