Recent studies in our laboratories have confirmed that a major unidentified metabolite of nicotine in smokers' urine was susceptible to enzymatic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary ammonium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesized. Reaction of methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate with (S)-(-)-cotinine at 60 degrees C for 3 days affords the fully protected conjugate as the bromide salt. Deprotection was accomplished in 1 M NaOH overnight at 25 degrees C. The deprotected inner salt was isolated by Dowex-50W cation-exchange chromatography. Electrospray mass spectra of the inner salt revealed the presence of ions with m/z 353 (M + H)+, 375 (M + Na)+, and 391 (M + K)+ as well as ions resulting from loss of water and cleavage of the glycosidic bond. Proton and carbon nuclear magnetic resonance spectra established that the position of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and nuclear Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance liquid chromatographic method enabled the collection of a fraction containing (S)-(-)-cotinine N-glucuronide from a smoker's urine. The electrospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fraction was subjected to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMedDate not supplied

Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.

Urinary nicotine metabolic output was profiled for 11 smokers who smoked their regular cigarette brands ad libitum. Thermospray liquid chromatography/mass spectrometry was used to monitor nicotine and eight metabolites, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine that were determined indirectly using enzyme hydrolysis. These results were used to estimate an average, steady-state concentration in a 24-hr urine sample during ad libitum smoking and to assess interindividual variability in the excretion of these metabolites. The variability in absolute amount among the nine analytes ranged from 35 to 70% for these smokers. The glucuronide conjugates constituted an average of 29% of all urinary metabolites monitored in this study. trans-3'-Hydroxycotinine in the free form constitutes the largest single metabolite in smokers' urine, with an average of 35% of the total. The sums of nicotine metabolites determined here are very close to the Federal Trade Commission yields of nicotine for the total number of cigarettes smoked by these subjects during the urine collection interval. These results indicate that a large proportion of the nicotine absorbed while smoking can be accounted for as urinary metabolites of nicotine, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine.

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PubMedDate not supplied

Effect of mouthrinses on microhardness and wear of composite and compomer restoratives.

This study investigated the effect of commercially available mouthrinses on the microhardness and wear of composite (Esthet-X, Dentsply) and compomer (Dyract Posterior, Dentsply) restoratives. Fifty-four hardness and 36 wear specimens of each material were fabricated and stored in distilled water at 37 degrees C for two weeks. The specimens were then randomly divided into six equal groups and exposed to the following solutions for 24 hours at 37 degrees C: distilled water [WC] (control); Listerine Original [AP] (alcohol-containing essential oil/phenolic compound mouthrinse); Colgate Chloropharm [AC] (alcohol-containing chlorhexidine mouthrinse); Oral B Tooth & Gum Care [AF] (alcohol-containing fluoride mouthrinse); Oral B Tooth & Gum Care Alcohol Free [OF] (alcohol free fluoride mouthrinse) and Oral B Sensitive [PF] (phosphoric acid containing fluoride mouthrinse). After conditioning, the specimens were subjected to hardness testing using a digital microhardness tester (load = 500 gf; dwell time = 15 seconds) and wear testing with a reciprocal compression-sliding system (contact stress = 20 MPa). Wear depth was measured every 1,000 cycles up to 10,000 cycles using profilometry. Data was analyzed using ANOVA/Scheffe's test at significance level 0.05. Dyract was significantly softened by AP, while Esthet-X was significantly softened by AC and AP. The wear resistance of Dyract was significantly reduced after exposure to PF, while the wear resistance of Esthet-X was significantly reduced by AC. The effect of mouthrinses on hardness and wear was material dependent.

Analysis of Variance↗

PubMedDate not supplied

Immunoreactivity of nm23-H1 protein in renal cell carcinoma.

Formalin-fixed, paraffin-embedded tissue blocks from 25 surgical specimens of renal cell carcinoma (RCC) including 13 adjacent normal renal tissues were investigated. The specimens were examined by immunohistochemistry using the monoclonal antibody, nm23-H1. The positive immunostaining of nm23-H1 protein was confined primarily to the cytoplasm of both normal renal tubular epithelial cells and renal tumor cells. Immunostaining of nm23-H1 protein was reduced significantly in RCC as compared to the normal renal tissues (P = 0.003). The positive immunostaining of nm23-H1 protein was seen in 92% (12/13) of normal renal tissues and in 60% (15/25) of RCC. No relationship was found between immunostaining of nm23-H1 protein and the patient's clinicopathological factors including age, tumor size, tumor location, tumor grade and tumor stage. Furthermore, immunostaining of nm23-H1 protein was not correlated with patient survival. Although immunoreactivity of nm23-H1 protein in patients with RCC was not correlated with survival, nm23-H1 protein may play a role in human renal tubular tumorigenesis.

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