A standard addition method to quantify histamine by reductive amination and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Histamine is an organic nitrogenous compound that acts as a neurotransmitter in the uterus, spinal cord, and brain and is involved in local immune responses. In this study, we developed a fast and simple derivatization method based on reductive amination that can be used to quantify histamine by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Histamine isotope analogs were synthesized via reductive amination. Histamine was modified with H2-formaldehyde to form N-dimethylated histamine to act as a standard or with D2-formaldehyde to form N-dimethylated histamine-d4 to act as an internal standard. Using this method, we achieved a limit of detection of 3.6 ng/mL, a limit of quantification of 7.9 ng/mL, and a linear calibration curve with a coefficient of determination (R2) of 0.9987. Furthermore, the intra-day relative standard deviations ranged from 0.9% to 3.7% and the inter-day relative standard deviations ranged from 2.0% to 17.6%. After derivatization, N-dimethylated histamine showed 382.5% signal enhancement compared to unmodified histamine in mass spectrometry detection. To demonstrate the applicability of this method for biological samples, we utilized standard addition method to quantify histamine in fetal bovine serum and achieved a recovery of 86.7%.

Quantification of the ß2-Adrenergic Feed Additives Ractopamine and Salbutamol by Reductive Amination-Assisted Modification.

HIGHLIGHTS: A reductive amination-assisted method was used to synthesize standards and internal standards of ractopamine and salbutamol. Standard and internal standard analogs were fabricated by isotopic formaldehydes and sodium cyanoborohydride. A quantitative method of modified ractopamine and salbutamol was successfully validated. The reductive amination-assisted method enhances the signal for MS detection.

The use of chemical probes to detect the proteomics of renal tubular injury induced by maleic acid.

Maleic acid (MA), an industrial raw material, was found to be illegally added to edible starch-based food products in Taiwan in 2013, a practice unheard of in most of the world. MA has been associated with renal dysfunction in many experimental animal studies. In this study, we developed chemical probes to investigate protein-protein interactions between MA and renal proteins. In the fabrication of the MA probes, we used silicon dioxide (SiO2) modified with a silanized linker (3-aminopropyl triethoxyslane, APTES) to generate MA with APTES-SiO2 particles. The probes were then incubated with the cell lysates of normal human kidney cell lines (HK-2) and subjected to MS/MS for identifying several MA-related proteins, including nucleophosmin, neutral alpha-glucosidase AB, translocon-associated protein subunit alpha, elongation factor 1-gamma, 60S acidic ribosomal protein P0-like, and heat shock protein (HSP 90-alpha and beta). Based on our findings, we believed that the probe can potentially be used to identify and detect the target proteins and help characterize a network of MA protein-protein interactions.

Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS.

Oseltamivir phosphate (OP) is the first line therapy for influenza, and its primary metabolite oseltamivir carboxylate (OC) is the active agent via inhibition of neuraminidase of influenza virus. Dosages of OP and OC might affect human causing nausea and vomiting and it is therefore necessary to evaluate their toxicity and safety. The separation system: liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique to monitor OP and OC. However, quantification of OP and OC needs isotopic analogs as internal standards to monitor the stability of the sample pretreatment procedures and instruments. In this study, we demonstrated a modified method (i.e., reductive amination) to synthesize OP and OC deuterated and hydrogenated analogs as internal standards (ISs) and for illustration of calibration curves, respectively. This modification allowed to overcome ISs selection and to enhance the signal intensities via high yield reductive amination in MS detection. We utilized the multiple reaction monitoring (MRM) mode to target m/z values of precursor and product ions. N-dimethylated OP and N-dimethylated OC showed linearity ranging from 1 to 1000 ng/mL with coefficient of determination (R2) values of 0.9995 and 0.9999, respectively. Additionally, the relative standard deviations (RSD) of intra-day ranged from 0.3% to 5.2%, and the RSD of inter-day ranged from 2.0% to 18.8%, respectively. This quantitative method utilized spiked OP and OC at low (20 ng/mL), intermediate (100 ng/mL), and high (500 ng/mL) concentrations in human serum samples. The average recoveries for OP and OC were 84.6%-107.7% and 94.9%-98.5%, respectively.

MicroRNA-196b inhibits late apoptosis of pancreatic cancer cells by targeting CADM1.

Pancreatic cancer (PC), as the leading cause of cancer death worldwide, is one of the deadliest tumors with a very low 5-year survival rate. Therefore, it is urgent to seek new biomarkers of PC for more accurate and reliable treatments. To identify the differentially expressed miRNAs (DEM) in PC tissues, we performed the systematic microarray and qRT-PCR analyses. We found miR-196b was the top dysregulated DEM in PC tissues as compared with the corresponding adjacent tissues, and positively correlated with poor differentiation, tumor size, lymphatic invasion and TNM stage. Furthermore, the late apoptosis rate was significantly reduced, while the cell proliferation was increased in PANC-1 and ASPC-1 cell-lines after treatment with miR-196b mimics. The qRT-PCR and Western blot analysis demonstrated that the level of CADM1 in PANC-1 cells response to the alteration of miR-196b. Moreover, blockade of CADM1 could decrease the late apoptosis in PANC-1 cells as up-regulated by inhibition of miR-196b. Finally, luciferase report assay confirmed that CADM1 was the direct target gene of miR-196b. Overexpression of miR-196b in PC tissues can increase the late apoptosis of pancreatic cancer cells by targeting CADM1. These findings suggested miR-196b is a potential target for diagnosis and therapeutics of human pancreatic cancer.

Reductive amination derivatization for the quantification of garlic components by isotope dilution analysis.

In this work, we synthesized internal standards for four garlic organosulfur compounds (OSCs) by reductive amination with 13C, D2-formaldehyde, and developed an isotope dilution analysis method to quantitate these organosulfur components in garlic samples. Internal standards were synthesized for internal absolute quantification of S-allylcysteine (SAC), S-allylcysteine sulfoxide (alliin), S-methylcysteine (SMC), and S-ethylcysteine (SEC). We used a multiple reaction monitoring (MRM) to detect 13C, D2-formaldehyde-modified OSCs by ultrahigh-performance liquid phase chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and obtained MS spectra showing different ratios of 13C, D2-formaldehyde-modified and H2-formaldehyde-modified compounds. The resulting labeled and unlabeled OSCs were exhibited correlation coefficient (R2) ranged from 0.9989 to 0.9994, respectively. The average recoveries for four OSCs at three concentration levels ranged from 89% to 105%. By 13C, D2-formaldehyde and sodium cyanoborohydride, the reductive amination-based method can be utilized to generate novel internal standard for isotope dilution and to extend the quantitative application.

Reductive amination-assisted quantitation of tamoxifen and its metabolites by liquid phase chromatography tandem mass spectrometry.

Tamoxifen, a hormonal therapy drug against estrogen receptor-positive breast cancer, can be metabolized by cytochrome P450 enzymes such as CYP3A4 and CYP3A5, and converted to N-desmethyltamoxifen, which is subsequently, metabolized by CYP2D6 and inverted to form 4-hydroxy-N-desmethyltamoxifen (endoxifen). Conventional mass spectrometry (MS) analyses of tamoxifen and its metabolites require isotopic internal standards (ISs). In this study, endoxifen and N-desmethyltamoxifen amine groups were modified by reductive amination with formaldehyde-D2 to produce new metabolite molecules. Both endoxifen and N-desmethyltamoxifen generated their corresponding D2-methyl modified analogs. This method is expected to simplify MS detection and overcome the difficulty in selecting adequate ISs when tamoxifen metabolites are analyzed by absolute quantification. It identified tamoxifen, D2-methyl modified endoxifen, and D2-methyl modified N-desmethyltamoxifen with a linearity ranging from 2 to 5000 ng/mL with correlation coefficient (R(2)) values of 0.9868, 0.9849, and 0.9880, respectively. Furthermore, this reductive amination-based method may enhance the signal intensities of D2-methyl modified N-desmethyltamoxifen and endoxifen, thus facilitating the MS detection.

A novel reductive amination method with isotopic formaldehydes for the preparation of internal standard and standards for determining organosulfur compounds in garlic.

Garlic (Allium sativum) is a long-cultivated plant that is widely utilized in cooking and has been employed as a medicine for over 4000 years. In this study, we fabricated standards and internal standards (ISs) for absolute quantification via reductive amination with isotopic formaldehydes. Garlic has four abundant organosulfur compounds (OSCs): S-allylcysteine, S-allylcysteinine sulfoxide, S-methylcysteine, and S-ethylcysteine are abundant in garlic. OSCs with primary amine groups were reacted with isotopic formaldehydes to synthesize ISs and standards. Cooked and uncooked garlic samples were compared, and we utilized tandem mass spectrometry equipped with a selective reaction monitoring technique to absolutely quantify the four organosulfur compounds.

[The clinical, endoscopic and pathologic features of Crohn's disease in the differentiation from intestinal tuberculosis].

OBJECTIVE: To investigate the clinical, endoscopic and pathologic features in the differential diagnosis between Crohn's disease(CD) and intestinal tuberculosis (ITB). METHODS: The complete clinical data of 107 patients with CD and 69 patients with ITB in our hospital from January 2011 to January 2012 were retrospectively analyzed. The diagnostic value of the clinical and endoscopic scoring system was evaluated. RESULTS: CD occurred mainly in male. The salient features of CD included long duration of disease high incidence of colectomy. Comparing with patients with ITB, patients with CD have more cases of diarrhea, hematochezia, abdominal mass, intestinal obstruction, intestinal hemorrhage, perianal lesions, and extraintestinal manifestations (all P < 0.05).It's more frequent to have positive results of anti-Saccharomyces cerevisiae antibody (ASCA), perinuclear antineutrophil cytoplasmic antibody (pANCA) and fecal occult blood in CD patients, as well as low albumin, high C-reactive protein ( CRP), elevated platelet count and hematocrit (P < 0.05 or P < 0.01). The salient features of ITB included low fever, night sweats, active parenteral tuberculosis, increased erythrocyte sedimentation rate (ESR), chest X-ray abnormalities, the positive PPD (purified protein derivatives tuberculin) and T-SPOT (P < 0.05 or P < 0.01). Based on the imaging, CD often involved the small intestine, such as the intestinal stricture and abdominal abscess (P < 0.05), while mesenteric lymphadenopathy was more common in ITB (P < 0.05). The endoscopic examination showed that some patterns of disease involvement such as fissure-shape ulcer [41.12% (44/107) vs 5.80% (4/69)], cobblestone sign[15.89% (17/107) vs 4.35% (3/69)], lesions over four segment [24.30% (26/107) vs 7.25% (5/69)], rectum involvement [17.76% (19/107) vs 5.80% (4/69)], ileocecal valve stenosis [21.50% (23/107) vs 8.70% (6/69)] and mucosal bridge[5.61% (6/107) vs 0(0/69)] were more frequent in CD patients than those in ITB patients(P < 0.01 or P < 0.05). However circular ulcers[37.68% (26/69) vs 9.35% (10/107)], rat-bite-like ulcers[24.64% (17/69) vs 12.15% (13/107)], persistent open ileocecal valves [39.13% (27/69) vs 19.63% (21/107)], tuberous and polypoid lesions[36.23% (25/69) vs 20.56% (22/107), 37.68% (26/69) vs 22.43% (24/107)] were more common in ITB (P < 0.01 or P < 0.05). In terms of pathological findings, certain characteristic features such as transmural inflammation [5.61% (6/107) vs 0(0/69)], fissure-liked ulcers [14.02% (15/107) vs 4.35% (3/69)], non-caseous granulomas [5.61% (6/107) vs 0(0/69)], lymphoid hyperplasia [16.82% (18/107) vs 5.80% (4/69)] and crypt abscess [9.35% (10/107) vs 1.45% (1/69)] were more common in CD than those in ITB(P < 0.05). According to the clinical and endoscopic scoring system, the positive diagnostic rate of CD was 50.47% (54/107) and of ITB was 66.67% (46/69) (P < 0.05) . CONCLUSIONS: The differential diagnosis between CD and ITB should be considered carefully based on clinical, endoscopic, pathological characteristics. The clinical and endoscopic scoring system may contribute to distinguish CD and ITB.

Utility of in vitro interferon-γ release assay in differential diagnosis between intestinal tuberculosis and Crohn's disease.

OBJECTIVE: To evaluate the diagnostic utility of interferon-γ release assay (T-SPOT.TB) for the differential diagnosis between Crohn's disease (CD) and intestinal tuberculosis (ITB). METHODS: A total of 103 CD and 88 ITB patients, confirmed by histology and anti-tuberculosis treatment response from 2003 to 2011, were included. Their characteristics and clinical features were recorded. Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) of IS6110, in vitro T-SPOT.TB, tuberculin skin test (TST), immunoglobulin G (IgG) antibody to MTB (protein chip), serum anti-Saccharomyces cerevisiae antibodies (ASCA IgG, chronic inflammatory bowel disease profile) and acid-fast staining of biopsied colonic tissue specimens were performed. Statistical analysis was conducted to determine their concordance with the diagnosis and its sensitivity, specificity, positive (PPV) and negative predictive value (NPV). RESULTS: Abnormal pulmonary X-ray, ascites and lesions of both cecum and ascending colon were more associated with ITB, while intestinal surgery and lesions of both ileum and adjacent colon were more commonly seen in CD. Significant diagnostic concordance was found using T-SPOT.TB (κ = 0.786) by consistency test. The sensitivity, specificity, PPV and NPV of T-SPOT.TB were 86%, 93%, 88% and 91%, respectively, and the sensitivity and NPV were significantly higher than other examinations (P < 0.05). CONCLUSION: T-SPOT.TB is a valuable assay in differentiating ITB from CD, particularly in the diagnostic exclusion of ITB based on its high specificity and NPV.

Syndecan-1 and E-cadherin expression in differentiated type of early gastric cancer.

AIM: To elucidate the role and alterations of syndecan-1 and E-cadherin expression in different cellular phenotypes of differentiated-type gastric cancers (DGCs). METHODS: A total of 120 DGCs at an early stage, and their adjacent mucosa, were studied both by immunohistochemistry. Syndecan-1 and E-cadherin were assessed by immunohistochemical staining with anti-syndecan-1 and anti-E-cadherin antibodies, respectively. Based on immunohistochemistry, DGCs and their surrounding mucosa were divided into four types: gastric type (G-type), ordinary type (O-type), complete-intestinal type (CI-type), and null type (N-type). RESULTS: Syndecan-1 expression was significantly lower in G-type cancers (29.4%) than in O-type (79.6%) and CI-type cancers (90%) (P<0.05, respectively), but E-cadherin did not show this result. In addition, syndecan-1 expression was significantly reduced in DGCs comprised partly of poorly differentiated adenocarcinoma or signet-ring cell carcinoma, compared to DGCs demonstrating papillary and/or tubular adenocarcinoma (P<0.05). G-type intestinal metaplasia (IM) surrounding the tumors was observed in 23.8% of G-type, 4.9% of O-type, and 6.7% of CI-type cancers (P<0.05; G-type vs O-type). Reduction of syndecan-1 expression was significant in G-type IM (25%) compared to non-G-type IM (75%; P<0.05). CONCLUSION: Loss of syndecan-1 plays a role in the growth of G-type cancers of DGCs at an early stage, and the reduction of syndecan-1 expression in IM surrounding the tumors may influence the growth of G-type cancer.

Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor.

AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression. METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-ras G to A point mutation in codon 12. RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-ras G to A point mutation in codon 12 had methylated MGMT (P = 0.011). The frequencies of K-ras G to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively. CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

[Analysis on mutation of adrenoleukodystrophy gene in exon 1 and exon 5].

OBJECTIVE: To elucidate the molecular mechanism of X-linked adrenoleukodystrophy(ALD) in Chinese. METHODS: Polymerase chain reaction in exon 1, exon 5 and their flanking sequences and direct DNA sequencing of ALD gene were performed in four patients, their mothers and twenty normal individuals as controls. RESULTS: A splice mutation was identified in the interface of exon 5 and intron 5 (1875 G-->A). This splice mutation in 5' end of intron 5 might lead to abnormal splice in exon 5 and exon 6 and bring about unstable and abnormal ALD protein; the lignoceryl CoA ligase could not transport very long chain fatty acids (VLCFA) into peroxisome and could not function normally; consequently, defective beta-oxidation of VLCFA in peroxisome could result in an accumulation of VLCFAS in the central nervous system, adrenal gland and blood. CONCLUSION: The splice mutation in 5' end of intron 5 leading to abnormal splice in exon 5 and exon 6 appears to be one of the causes of X-linked recessive adrenoleukodystrophy.

The relation between HLA-DQA1 genes and genetic susceptibility to duodenal ulcer in Wuhan Hans.

AIM:To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS:Seventy patients with duodenal ulcer and fifty healthy controls were examined for HLA-DQA1 genotypes.HLA-DQA1 typing was carried out by digesting the locus specific polymerase chain reaction amplified products with alleles specific restriction enzymes (PCR-RFLP), i.e.,Apal I, Bsaj- I, Hph I, Fok I, Mbo II and Mnl I.RESULTS:The allele frequencies of DQA10301 and DQA1-0102 in patients with duodenal ulcer were significantly higher and lower respectively than those in healthy controls (0.40 vs 0.20, P = 0.003, P(corret) = 0.024) and (0.05 vs 0.14, P = 0.012, but P(corret) > 0.05), respectively.CONCLUSION: DQA1(*)0301 is a susceptible gene for duodenal ulcer in Wuhan Hans, and there are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients and healthy controls.