Novel Matrix Normalization Technique for Accurate LC-ESI-MS/MS Detection and Quantification of Drugs and Their Metabolites in Toxicology Research and Practice.

Accurate identification and quantification of drugs and their metabolites (analytes) in biological matrices is an analytical foundation of clinical and forensic toxicology. For decades, liquid chromatography interfaced by electrospray ionization with tandem mass spectrometry (LC-ESI-MS/MS) has been a widely used technology for analysis in the field of toxicology, as well as in many other fields of bioscience. It is also known that ion response in LC-ESI-MS/MS analysis is influenced by coeluting biological compounds and that preanalytical sample clean-up is often insufficient in removing these interferences.

Application and Clinical Value of Definitive Drug Monitoring in Pain Management and Addiction Medicine.

Objective: To assess routine application and clinical value of definitive urine drug monitoring (UDM) for drug detection, inconsistent drug use, and prescription adherence, along with a comparison to immunoassay screening (IAS).

Methods: Direct-to-definitive UDM performance was analyzed retrospectively in 5000 patient specimens. Drug findings, medication inconsistencies, and detection sensitivity were assessed, and definitive UDM versus IAS monitoring was studied.

Matrix Normalization Techniques for Definitive Urine Drug Testing.

Analytical performance of stable isotope-labeled internal standardization (SIL-IS) and threshold accurate calibration (TAC) methods of matrix normalization are compared for quantitation of 51 drugs and metabolites (analytes) in urine with analysis by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS-MS). Two SIL-IS methods of analysis were performed, one method using analyte-specific internal standardization (ASIL-IS) and another method using a shared stable isotope from another analyte for internal standardization (SSIL-IS). Variance in inter-specimen matrix effect, without the use of a matrix normalization method, was studied by UPLC-MS-MS analysis of 338 urine donor samples and showed >200% variation in ion response for some analytes.

Butyrate and other short-chain fatty acids increase the rate of lipolysis in 3T3-L1 adipocytes.

We determined the effect of butyrate and other short-chain fatty acids (SCFA) on rates of lipolysis in 3T3-L1 adipocytes. Prolonged treatment with butyrate (5 mM) increased the rate of lipolysis approximately 2-3-fold. Aminobutyric acid and acetate had little or no effect on lipolysis, however propionate stimulated lipolysis, suggesting that butyrate and propionate act through their shared activity as histone deacetylase (HDAC) inhibitors.

Curcumin is a direct inhibitor of glucose transport in adipocytes.

Curcumin has been reported to inhibit insulin signaling and translocation of GLUT4 to the cell surface in 3T3-L1 adipocytes. We have investigated the effect of curcumin on insulin signaling in primary rat adipocytes. Curcumin (20 μM) inhibited both basal and insulin-stimulated glucose transport (2-deoxyglucose uptake), but had no effect on insulin inhibition of lipolysis.

Transferrin and iron induce insulin resistance of glucose transport in adipocytes.

Normal serum can increase the rate of lipolysis in isolated adipocytes. Recently, we reported that the lipolytic effect of serum could be partly explained by effects of iron and transferrin. To further investigate these effects on fat cell metabolism, we have investigated effects of serum, iron, and transferrin on glucose transport in isolated rat adipocytes.

Turnover and characterization of UDP-N-acetylglucosaminyl transferase in a stably transfected HeLa cell line.

To estimate the turnover of UDP-N-acetylglucosaminyl transferase (OGT), we exposed stably transfected HeLa cells to tetracycline for 16h to induce OGT gene expression and increase cytosolic enzyme levels. Removal of tetracycline led to a progressive decrease in OGT activity (after a 6h lag period), yielding an estimated OGT half-life of 13h. A similar half-life (12h) was obtained by measuring the loss of biosynthetically labeled OGT ([35S]methionine pulse-chase experiments).

Transferrin and iron contribute to the lipolytic effect of serum in isolated adipocytes.

Previous reports have demonstrated that normal serum can increase the rate of adipocyte lipolysis in vitro. However, the nature of the lipolytic activity has remained obscure. We have investigated the lipolytic activity of human serum using isolated rat adipocytes.

Stimulation of lipolysis by tumor necrosis factor-alpha in 3T3-L1 adipocytes is glucose dependent: implications for long-term regulation of lipolysis.

Tumor necrosis factor-alpha (TNF-alpha) and hyperglycemia both impair insulin sensitivity in vivo. This may be secondary to stimulation of adipose tissue lipolysis and consequent increased circulating free fatty acids (FFAs). Here we report that neither TNF-alpha nor glucose alone has a pronounced effect on lipolysis in 3T3-L1 adipocytes.

Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.

We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h.

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose, glutamine, and glucosamine on glycerophosphate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase mRNA levels.

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH).

Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.

UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification).