EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

The Essential Role of Circulating Thyroglobulin in Maintaining Dominance of Natural Regulatory T Cell Function to Prevent Autoimmune Thyroiditis.

Several key findings from the late 1960s to mid-1970s regarding thyroid hormone metabolism and circulating thyroglobulin composition converged with studies pertaining to the role of T lymphocytes in autoimmune thyroiditis. These studies cemented the foundation for subsequent investigations into the existence and antigenic specificity of thymus-derived natural regulatory T cells (nTregs). These nTregs prevented the development of autoimmune thyroiditis, despite the ever-present genetic predisposition, autoantigen (thyroglobulin), and thyroglobulin-reactive T cells. Guided by the hypothalamus-pituitary-thyroid axis as a fixed set-point regulator in thyroid hormone metabolism, we used a murine model and compared at key junctures the capacity of circulating thyroglobulin level (raised by thyroid-stimulating hormone or exogenous thyroglobulin administration) to strengthen self-tolerance and resist autoimmune thyroiditis. The findings clearly demonstrated an essential role for raised circulating thyroglobulin levels in maintaining the dominance of nTreg function and inhibiting thyroid autoimmunity. Subsequent identification of thyroglobulin-specific nTregs as CD4(+)CD25(+)Foxp3(+) in the early 2000s enabled the examination of probable mechanisms of nTreg function. We observed that whenever nTreg function was perturbed by immunotherapeutic measures, opportunistic autoimmune disorders invariably surfaced. This review highlights the step-wise progression of applying insights from endocrinologic and immunologic studies to advance our understanding of the clonal balance between natural regulatory and autoreactive T cells. Moreover, we focus on how tilting the balance in favor of maintaining peripheral tolerance could be achieved. Thus, murine autoimmune thyroiditis has served as a unique model capable of closely simulating natural physiologic conditions.

Clinical judgments of high-risk behavior during recovery.

This study utilized focus group research to explore high-risk behavior during recovery from drugs and alcohol. Participants in the focus group were professional substance abuse counselors. The findings identified specific high-risk behaviors and began an exploration of the processes that support them. Specific information is included on such issues as causes, time frames, developmental issues, and other factors associated with high-risk behavior. Attention was paid to existential issues in recovery, as well as childhood factors such as sexual abuse. High-risk behavior was seen as a means to avoid the existential dilemma of continuing in recovery or returning to drug use and as a possible means to leave this crisis unresolved without actual relapse to drug use, or dealing with the issues of advancing in recovery.

Determination of drug-plasma protein binding using human serum albumin chromatographic column and multiple linear regression model.

Reversible attachment to serum proteins plays a significant role in pharmacokinetics and pharmacodynamics, and a clear understanding of this process is fundamental in the development of the rational use of many therapeutics agents. Over the last few years, it has been demonstrated that immobilized human serum albumin (HSA) could be used to estimate plasma protein binding. A series of 40 structurally unrelated pharmaceutical compounds were chromatographed on an immobilized HSA column in order to construct a protein binding 'calibration curve' and multiple linear regression system. When studying the relationship between the chromatographic retention and the percentage of binding determined in vitro, a good correlation can be observed (r(2) = 0.799) using a wide variety of compounds with different binding affinities (from 0 to 99% binding). Using a quantitative structure-retention relationships (QSRR) approach to analysing chromatographic data, the correlation was improved compared to the traditional approach (r(2) = 0.824).

Metabolite profiling study of propranolol in rat using LC/MS/MS analysis.

Metabolite profiling is one of the most challenging fields in applied mass spectrometry. Mass spectrometry was used to characterize the metabolites of propranolol, a beta-adrenergic receptor antagonist containing numerous oxidation sites. Propranolol is extensively metabolized, with most metabolites appearing in urine. Urine samples were collected from young adult male Sprague-Dawley rats. Structural identification of various metabolites was performed by LC/MS/MS, using a PE SCIEX triple quadrupole instrument (PE SCIEX API 3000). Metabolites were itemized using several LC/MS/MS techniques, including Q3 full scan and precursor and constant neutral loss experiments. A looped experiment technique revealed the presence of mono- and di-hydroxylated metabolites as well as regio isomers of hydroxy- and dihydroxy-propranolol glucuronides and propranolol glucuronic acid. Propranolol glucuronide was not observed, while the presence of dealkylated metabolites was suggested but not confirmed.

In vivo pharmacokinetic screening in cassette dosing experiments; the use of on-line Pprospekt liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry technology in drug discovery.

Drug discovery is a fast growing field and the number of compounds generated daily by the pharmecutical industry is enormous. The necessity of developing new experimental strategies and analytical methods to rapidly screen the pharmacokinetics (PK) behavior of these compounds becomes a real challenge. A novel strategy to support in vivo PK screening in cassette doing experiments, using a fully automated system capable of analyzing between 320 to 960 samples a day by instrument in n-in-one experiment ( n = 64 in this work), has been developed. Using an on-line extraction technique, the average observed recovery was 64% using a single C18 procedure. A weighted (1/x) linear equation was used to perform standard calibration (0.5 to 500 ng/microL) and the average R value obtained was 0.994 (R2 = 0.997) for 63 analytes. The limit of detection, defined as a signal-to-noise ratio of 3 or greater, was found to be 25 pg for 41 of the 63 analytes (65%) and 250 pg for 57 of the 63 analytes (90%). The complete automation procedure using the Prospekt-LC-APCI/MS/MS system has substantially improved throughput in the area of drug discovery and bioanalysis.

Nontreatment and aggressive narcotic therapy among hospitalized pancreatic cancer patients.

OBJECTIVES: Strong feelings about patient autonomy as expressed in living wills, polls, and legislative referenda have been challenging the medical establishment to increase nontreatment, defined as foregoing a life-prolonging treatment, and even to provide treatments having life-shortening potential to selected patients. Because there are little data about the actual practice of these procedures, including aggressive narcotic therapy as defined herein, we studied the terminal management of 417 pancreatic cancer patients. DESIGN AND PARTICIPANTS: The medical records of 417 residents of King County, Washington, who died of pancreatic cancer in the time periods 1959-1962, 1969-1972, and 1985-1990, were reviewed to study the frequency of, and risk factors for, end-of-life nontreatment decisions and aggressive narcotic therapy decisions, defined here as the decision to administer treatment doses of narcotics or major sedatives to already comatose patients within 4 hours of death. RESULTS: Antibiotics were not provided to 71% of the 70 febrile patients (two readings >38.33-38.83 degrees C or one reading of 38.88 degrees C), intravenous fluid was not provided to 43% of 294 dehydrated patients (oral intake <500 mL/24 hours), transfusions were not provided to 39% of 57 severely anemic patients (hematocrit <20%), and laparotomy was not performed for 86% of 36 patients with abdominal emergencies (obstruction, bleeding, dehiscence). Also, 46% of the 118 patients who were comatose for at least 24 hours before death received aggressive narcotic therapy, as defined above. A total of 335 of the 417 patients had documentation of at least one of the above life-threatening conditions or were comatose for at least 24 hours before death, and 289 (86%) of these patients experienced nontreatment of one or more of these conditions or received aggressive narcotic therapy. Nontreatment decisions for febrile, dehydrated, or anemic patients tended to be more frequent if the patient was comatose (P=.004, .010, and .065, respectively), if there was a nontreatment statement in the medical record (P=.009, .035, and .001, respectively), or if the patient was described as terminal (P=.262, .029, and .002, respectively). Aggressive narcotic therapy in comatose patients was more common among patients who had regular visitors (P=.002), who had pre-coma pain (P=.006), who had nontreatment statements in their charts (P=.031), whose in-charge physician was an oncologist (P < .001), who were treated in a community nonprofit hospital compared with a Catholic hospital (P=.007), or who were treated in recent years (P=.011). CONCLUSION: Both nontreatment and aggressive narcotic therapy forms of medical management have been occurring commonly in terminal pancreatic cancer patients in King County, Washington, during the past 3 decades, the latter with greater frequency in recent years.

Mutagenic decomposition products of nitrosated 4-chloroindoles.

4-Chloro-6-methoxyindole, a constituent of fava beans, forms a potent direct-acting mutagen, 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime, when nitrosated. In order to better understand the properties of this mutagen, we have studied a readily-available analog, 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, prepared by nitrosation of 4-chloroindole. This analog is also mutagenic, and both mutagens decompose rapidly at neutral or higher pH to yield in each case a new, less potent mutagen which then reacts further to form a nonmutagenic final product. The two products arising from 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, on the basis of comparison of spectroscopic and chromatographic evidence with that from authentic standards, are 4-chloro-N-nitrosodioxindole and 4-chloroisatin; those arising from 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime appear to be the corresponding 6-methoxy analogs. The interplay of these pathways with respect to net biological activity, especially under gastric conditions, remains to be described.

Serum proteolytic activity during the growth of C6 astrocytoma.

Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Blood-vessel remodeling requires the reconstruction of the nonfibrous proteins and type IV collagen components of the basement membrane. This study has assessed the influence of the growth of C6 astrocytoma cells in the rat spheroid implantation model on serum general protease and type IV collagenase activity. The results demonstrate that general protease activity increased in serum, reaching maximum values on Day 6 and Day 13 following spheroid implantation, and that type IV collagenase activity increased in serum, obtaining maximum values on Day 8 and Day 15. The measurement of serum proteolytic activity may be of value in the detection of recurrent tumors.

Comparative in vitro metabolism of the cannabinoids.

The metabolism of delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-THC, delta-11-THC, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), cannabigerol (CBG) and the equatorial-isomer of hexahydrocannabinol (HHC) was studied in microsomal preparations obtained from rats, mice, guinea pigs, rabbits, hamsters, gerbils and a cat. Identification of metabolites was by GC/MS and quantification by gas chromatography. Major metabolites were monohydroxylated compounds but the pattern of hydroxylation varied considerably between the species, no doubt reflecting the variable nature of the cytochrome P-450 mixed-function oxidases. Although the primary carbon allylic to the endocyclic double bond of tricyclic cannabinoids was usually the major site of attack, the 4' (side-chain, omega-1 position) and the terpene ring were usually favoured by the cat and hamster respectively. The guinea pig generally produced more metabolites hydroxylated in the side-chain (all positions) than did the other species. The results from HHC were very similar to those from THC, namely hydroxylation at C-11 in most species, and the production of high concentrations of 8 alpha-hydroxy-HHC in the mouse and 8 beta-hydroxy-HHC in the hamster. As this molecule lacks the double bond of the THCs and, hence, the allylic nature of C-11 and C-8, the results suggest that it is the orientation of the molecule to the active site of the cytochrome P-450 mixed-function oxidase rather than the reactivity of the C-H bond that governs the position of hydroxylation.

Identification of cannabichromene metabolites by mass spectrometry: identification of eight new dihydroxy metabolites in the rabbit.

Metabolites of cannabichromene (CBC) produced by hepatic microsomal incubates from rabbits and mice were examined by gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) and (2H9)TMS derivatives. Most metabolites were hydroxylated compounds whose mass spectra gave very little information on metabolite structure as fragmentation was dominated by formation of the substituted chromenyl ion. This prevented charge localization and diagnostic fragmentation at the site of metabolic attack. This paper describes the identification of these metabolites by GC/MS techniques using both deuterium-exchange reactions and hydrogenation of the metabolites to tetrahydro derivatives; the latter method was used to suppress chromenyl ion formation and to enhance the relative abundance of diagnostic fragment ions. Twenty-one metabolites were identified. Metabolites were found hydroxylated in all positions of both aliphatic chains, with additional compounds formed by epoxidation and reduction of the aliphatic double bond in the methylpentenyl chain. Dihydroxy metabolites were hydoxylated in both the pentyl and methylpentenyl chains in positions common to those hydroxylated in the monohydroxy metabolites.

Electron impact-induced fragmentation of the trimethylsilyl derivatives of monohydroxy-hexahydrocannabinols.

Monohydroxylated derivatives of hexahydrocannabinols were synthesized by catalytic hydrogenation of hydroxytetrahydrocannabinols over a rhodium/alumina catalyst, reduction of tetrahydrocannabinol epoxides with lithium aluminium hydride, or by reaction of tetrahydrocannabinols with hydrogen peroxide. The electron impact-induced fragmentation of their trimethylsilyl ethers was investigated with the aid of deuterium labelling. Most of the compounds gave characteristically different mass spectra with abundant, diagnostically useful fragment ions. As hexahydrocannabinols containing hydroxy groups in all metabolically sensitive positions were readily prepared by the above methods, these provided reference samples for identification of new hydroxylated metabolites of isomeric tetrahydrocannabinols following hydrogenation. The method was validated by application to metabolites of delta-9(11)-tetrahydrocannabinol.

In vivo metabolism of the ethyl homologues of delta-8-tetrahydrocannabinol and delta-9-tetrahydrocannabinol in the mouse.

Ethyl-delta-8-tetrahydrocannabinol (ethyl-delta-8-THC) and ethyl-delta-9-THC were synthesized by condensation of 5-ethyl-1,3-dihydroxybenzene and 1S-cis-verbenol. The two cannabinoids were administered to male Charles River CD-1 mice and hepatic metabolites were extracted with ethyl acetate and isolated by chromatography on Sephadex LH-20. Metabolite identification was by gas chromatography/mass spectrometry as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS and dihydro/TMS derivatives. Metabolites from ethyl-delta-8-THC, of which six were identified, were similar with respect to the positions substituted on the terpene ring to those produced by higher homologues; the major metabolite, accounting for about 95% of the metabolic fraction, was ethyl-delta-8-THC-11-oic acid. Side-chain hydroxy metabolites were not detected. Metabolism of ethyl-delta-9-THC was also similar to that of the higher homologues with the exception that less metabolism occurred at C-8 and a higher percentage of the total metabolic fraction was accounted for by the 11-oic acid metabolite. Five metabolites were identified; minor metabolites were mainly dihydroxylated compounds and hydroxylated derivatives of ethyl-delta-9-THC-11-oic acid. A dihydro-metabolite, the C-9-axial-COOH isomer of ethyl-hexahydrocannabinol-11-oic acid, was produced by both compounds and a trace of ethyl-CBN-11-oic acid was produced by ethyl-delta-9-THC.

In vitro metabolism of cannabichromene in seven common laboratory animals.

Metabolism of cannabichromene (CBC) was studied in hepatic microsomal incubates from mouse, rat, rabbit, guinea pig, cat, hamster, and gerbil. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20, and identified by GC/MS as trimethylsilyl derivatives of both the metabolites themselves and their hydrogenated analogues. Thirteen metabolites were identified. The major metabolites were monohydroxy compounds with hydroxylation at all positions of the pentyl and methylpentenyl chains. An epoxide and its derived dihydrodiol were formed from the double bond in the methylpentenyl chains. Several unidentified decomposition products were found in the extracts from mouse, gerbil, and cat; these appeared to have been produced by the opening of the dihydropyran ring. Metabolism varied considerably between the species, although the trans-hydroxy metabolite 5'-hydroxy-CBC was the major metabolite in most cases. Metabolites hydroxylated in the pentyl chain were more abundant in mouse, rabbit, and cat; the hamster, gerbil, and cat produced the most epoxide-derived material.

Metabolites of the 1',2'-dimethylheptyl analogue of delta-8-tetrahydrocannabinol in the mouse and their identification by gas chromatography/mass spectrometry.

Metabolism of the 1,2-dimethylheptyl analogue of delta-8-tetrahydrocannabinol (delta-8-DMHP) was studied in vitro using mouse hepatic microsomes and in vivo in mouse liver. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined by low-resolution mass spectrometry as trimethylsilyl (TMS), (2H9)TMS and methyl ester/TMS derivatives. Reduction of metabolites with lithium aluminium deuteride also provided structural information. The electron-impact-induced mass spectrum of the TMS derivative of DMHP differed from that of its unbranched side-chain analogues in that prominent ions were produced by fragmentation of the side-chain at the expense of the retro-Diels-Alder fragmentation that was prominent in the spectra of the latter compounds. This, however, was found to reduce the relative abundance of ions diagnostic of side-chain hydroxy substitution in the spectra of the metabolites. In vitro, the only significant metabolite was 11-hydroxy-delta-8-DMHP. This is in contrast with metabolism of the corresponding delta-8-tetrahydrocannabinol (delta-8-THC, n-C5-side-chain) where a number of other monohydroxy metabolites are produced. Fifteen metabolites were found in vivo, of which nine were identified. Mass spectral information was not sufficient to determine the position of one of the hydroxy groups in the other six metabolites. The major site of hydroxylation was at C-11 and the resulting hydroxy metabolite was oxidized to delta-8-DMHP-11-oic acid. In this respect metabolism paralleled that of delta-8-THC. Dihydroxylation of the double bond also occurred, presumably via the epoxide.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of cannabigerol in several mammalian species.

Microsomal incubations were prepared from the livers of male mice, rats, cats, guinea-pigs, hamsters and gerbils and both male and female rabbits and were incubated with cannabigerol (CBG), a constituent of marihuana. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined as trimethylsilyl (TMS) and (2H9)TMS derivatives by gas chromatography/mass spectrometry. Structural elucidation was aided by hydrogenation of the metabolites to tetrahydro derivatives. Similar metabolites were produced by each of the species but the ratios of the individual compounds differed considerably. Twelve metabolites were identified. The major metabolites were monohydroxy compounds with the hydroxyl group at C-8', C-9', C-4' or at one of any position of the pentyl chain. Reduction of the delta-6' double bond was prominent in the cat to give 8'-hydroxy-6',7'-dihydro-CBG. The other major metabolic route was epoxidation of this double bond and hydrolysis to give 6',7'-dihydroxy-6',7'-dihydro-CBG. Although epoxidation of the other double bond was detected, the resulting metabolite was present in low concentration and hydrolysis was not observed. The mass spectral fragmentation of CBG and its metabolites was dominated by formation of the tropylium ion by cleavage of the C-1'--C-2' bond and by ions formed by cleavage of the C-3'--C-4' and C-4'--C-5' bonds. In addition, compounds containing hydroxylation at C-1"--C-4" (pentyl chain) gave rise to the same abundant diagnostic ions that have been observed for corresponding metabolites of other cannabinoids.

In vitro metabolism of cannabidiol in the rabbit: identification of seventeen new metabolites including thirteen dihydroxylated in the isopropenyl chain.

The metabolism of cannabidiol (CBD) was studied in liver microsomes from the female New Zealand white rabbit. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined as trimethylsilyl (TMS), methyl ester/TMS and (2H9)TMS derivatives by gas chromatography/mass spectrometry. Thirty-nine metabolites, mainly mono-, di- and tri-hydroxy compounds, were identified; 17 of these have not been reported before. New metabolites included 8,9-dihydroxy-8,9-dihydro-CBD (two isomers) and seven monohydroxy derivatives of each of these two compounds. The mass spectra of the TMS derivatives of metabolites not hydroxylated in the isopropenyl group were generally dominated by the ion produced by retro-Diels-Alder cleavage of the terpene ring. Other structurally informative ions included the tropylium ion and fragments diagnostic of hydroxylation at C-1", C-2", C-3", C-4" and C-7. The spectra of the TMS derivatives of metabolites hydroxylated in the isopropenyl group were generally dominated by the ion at m/z 143. This involved loss of CH2OTMS and a retro-Diels-Alder fragmentation analogous to that seen in the other metabolites, but with charge retention by the other (smaller) fragment. Other, related fragment ions also characterized these metabolites.

In vitro metabolism of cannabinol in rat, mouse, rabbit, guinea pig, hamster, gerbil and cat.

Metabolism of cannabinol (CBN) was studied in hepatic microsomal incubates from mouse, rat, rabbit, guinea pig, cat, hamster and gerbil. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and identified by GC/MS as TMS derivatives. Six monohydroxy metabolites were identified. These had hydroxy groups at C-11 and at all positions of the pentyl side-chain. Metabolism varied considerably between the species. 11-Hydroxylation was the most prominent route in the majority of species, but in the hamster and cat the major metabolic pathway was 4'-hydroxylation. Metabolites hydroxylated in the pentyl chain were generally more abundant in guinea pig, hamster and cat.