Rapid analysis of acetaminophen in serum by gas chromatography-mass spectrometry with extractive derivatization using a diatomaceous earth tube

A new analytical method for acetaminophen (ACAP) in serum was developed by modifying an existing method used for amphetamines, which used extractive derivatization followed by gas chromatographymass spectrometry. After a serum sample was adjusted to pH 12.8, it was applied onto a diatomaceous earth tube; the analyte was simultaneously extracted and heptafluorobutyrylated during elution with a solvent containing a derivatizing reagent. Three internal standard (IS) candidates were tested: N-acetyl-d ₃-paminophenol, N-acetyl-m-aminophenol, and N-acetyl-4-amino-m-cresol. All ISs gave good linear relationships (r ² > 0.999) for ACAP in the concentration range from 1 to 200μg/ml. The detection limit for ACAP using each IS was estimated to be 0.05–0.1μg/ml. Intraday precision was satisfactory with a coefficient of variation of less than 8.3%. The use of this method with any of the three ISs is recommended in forensic and clinical toxicology because of its rapidity and good reproducibility.     

Acute humoral rejection and C4d immunostaining in ABO blood type-incompatible liver transplantation.

Complement C4d deposition in graft capillaries has been reported to be associated with antibody-mediated rejection in kidney and other solid organ transplantation. The correlation of C4d deposits and humoral rejection in liver transplants, however, is not well understood. We investigated the C4d immunostaining pattern in 34 patients whose liver biopsy was taken within the first 3 postoperative weeks for suspected acute rejection after ABO blood type-incompatible liver transplantation. The staining pattern was classified as positive (portal stromal staining), indeterminate (endothelial staining only), and negative (no staining). Positive C4d immunostaining was seen in 17 (50%) patients and was significantly associated with high (x64 or more) postoperative antidonor A/B antibody (immunoglobulin M (IgM)) titers (88 vs. 35%, P = 0.002) and poorer overall survival rate (41 vs. 88%, P = 0.007). Ten of 11 (91%) cases with histological acute humoral rejection (periportal edema and necrosis (PEN) or portal hemorrhagic edema) were positive for C4d, all of which showed high postoperative antibody titers. The other histologies associated with C4d positivity was purulent cholangitis (n = 4), coagulative hepatocyte necrosis (n = 1), acute cellular rejection (n = 1), and hepatocanalicular cholestasis (n = 1). Full clinical recovery was observed in only 6 of 17 (35%) C4d-positive patients, and tended to be associated with a lower rejection activity index (RAI). In conclusion, our study indicates that C4d deposits in the portal stroma can be a hallmark of acute humoral rejection in ABO-incompatible liver transplantation, and allograft damage can be reversible in a minority of cases.     

Two simple methods for enantiomeric analyses of urinary amphetamines by GC/MS using deuterium-labeled l-amphetamines as internal standards

Two simple methods for enantiomeric analyses of amphetamines in urine by gas chromatography-mass spectrometry (GC-MS) using l-amphetamine-d ₃ and l-methamphetamine-d ₆ as internal standards are presented. One method (method A) employs extractive derivatization on a diatomaceous column with (S)-(-)-N-(trifluoroacetyl)prolyl chloride (TPC) followed by separation with a conventional capillary column. The second method (method B) uses headspace solid-phase microextraction (HD-SPME) after derivatization with heptafluoro-n-butyryl chloride (HFB), followed by separation with an enantiomeric capillary GC column. By the two methods, all enantiomers were well separated in each chromatogram, and good linearity was obtained in practical concentration ranges (0.1–1.6μg/ml for method A and 0.05–1μg/ml for method B) for every compound by selected-ion monitoring. The precision studies indicated satisfactory coefficients of variation (<5%) for every enantiomer at 0.1μg/ml by both methods. Both methods were also evaluated by applying them to an actual poisoning case. Both methods are recommended for use in forensic analysis, because of their simplicity, high precision, and sufficient sensitivity.     

Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 (UGT2B21) and 2B22 in COS-7 cells enhances UGT2B21-catalyzed chloramphenicol glucuronidation.

Our previous study suggested that hetero-oligomer formation of guinea pig liver UDP-glucuronosyltransferases (UGTs) 2B21 and 2B22 enhances UGT2B21-catalyzed morphine-6-glucuronidation. In this work, further evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 was provided by studies of the glucuronidation of chloramphenicol with dual expression in COS-7 cells. UGT2B21 expressed in COS cells was capable of glucuronidating the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, borneol, testosterone, androsterone, and estriol, whereas it had some effect on chloramphenicol. On the contrary, UGT2B22 does not exhibit any significant activity toward these typical substrates tested in this study. When UGT2B21 and UGT2B22 were expressed simultaneously, the chloramphenicol glucuronidation was enhanced to 4.5-fold, whereas the activities toward other substrates were little affected except that for the 6-hydroxyl group of morphine. The protein expression level of UGT2B21 was comparable when UGT2B21 was expressed with or without UGT2B22. These results suggest that simultaneous expression of UGT2B21 and UGT2B22 enhances UGT2B21-catalyzed chloramphenicol glucuronidation. Hetero-oligomer formation of UGT2B21 and UGT2B22 may act by fine-tuning the catalytic glucuronidation of chloramphenicol.     

Growth inhibition of human colon cancer cell line HCT116 by bis[2-(acylamino)phenyl] disulfide and its action mechanism

Our laboratory has been investigating the use of compounds which disrupt beta-catenin/T cell factor (TCF) binding to treat human colon cancer. There are several cysteine residues on the surface of beta-catenin where it binds to TCF. Some bis[2-(acylamino)phenyl] disulfides might have the ability to form a disulfide bond with the cysteine residues of beta-catenin, leading to inhibition of the growth of human colon cells. Bis[2-(acylamino)phenyl] disulfides were screened to inhibit the growth of cancer cells. Among them, bis[2-(2,2-dimethylpropanoylamino)phenyl] disulfide (1) had promising inhibitory effects (HCT116, IC50: 9.7 microM; DLD-1, IC50: 6.9 microM) on cell proliferation, and did not show any cytotoxicity among normal human fibroblast CCD-1059SK cells even at 200 microM. This derivative reduced the beta-catenin/TCF4 association in the HCT116 cells to ca. 50% at 150 microM. Furthermore, it activated markedly the phosphorylation of c-Jun N-terminal kinase (JNK) connected to stress-activated apoptosis at a lower concentration (30 microM). In view of cell cycle analyses, Hoechst staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick end-labeling (TUNEL) assays along with the above results, it is likely that 1 inhibited the growth of HCT116 cells through pathways including the JNK-mediated apoptosis.     

Enzymatic degradation of keratin films and keratin fibers prepared from human hair

Protease degradation of matrix (Ma) protein and microfibril (Mf) protein, which are the major components of human hair, were investigated. Ma or Mf protein was partially extracted from human hair and four types of keratins were prepared: Ma film, Mf film, Ma-rich fiber, and Mf-rich fiber. Of the two keratin films treated with protease, Mf film showed much higher degradation than Ma film. Of the two keratin fibers treated with protease, Mf-rich fiber showed much higher degradation than Ma-rich fiber. Protease potently degraded Mf protein. Furthermore, there was a high correlation between the extent of degradation and the water absorption of keratins.     

Monitoring of cholinesterase-inhibiting activity in water from the Tone canal, Japan, as a biomarker of ecotoxicity

The cholinesterase (ChE)-inhibiting activity of water and the concentrations of representative inhibitors were monitored in the Tone canal, Japan, during April to December 2006. The ChE-inhibiting activity, measured by using horse serum as enzyme source, increased from late April to early June, and from September to October. Although the trends in the ChE-inhibiting activity of the samples were consistent with concentration changes of organophosphorus pesticides, ChE-inhibiting activity was not observed in samples replicated on the basis of the chemical concentrations detected. The water samples were treated with chlorine to enhance the ChE-inhibiting activity by conversion of thiophosphate pesticides to phosphate pesticides. The ChE-inhibiting activity increased in almost all the chlorine-treated samples, although organophosphorus pesticides were either not detected or detected in traces in the samples by gas chromatographic–mass spectrometric analysis. These results suggested that assay of ChE-inhibiting activity is important for evaluating the ecotoxicity of environmental water, because toxicological investigations based solely on inhibitor concentrations may underestimate the contamination. Furthermore, the combined method of oxidation by chlorination and the ChE assay is very effective for screening and monitoring of organophosphorus pesticides in environmental water.