Evolution of LC-MS/MS in clinical laboratories.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has attracted significant attention in clinical practice owing to its numerous advantages. However, the widespread adoption of this technique is hindered by certain limitations, such as inappropriate analyte selection, low levels of automation, and a lack of specific reference intervals and quality control programs. This review comprehensively summarizes the current challenges associated with LC-MS/MS and proposes potential resolutions. The principle of utility should guide the selection of biomarkers, prioritizing their practical value over sheer quantity. To achieve full-process automation, methodological innovation is crucial for developing high-throughput equipment. Establishing reference intervals for mass spectrometry-based assays across multiple centers and diverse populations is essential for accurate result interpretation. Additionally, the development of commercial quality control materials assumes pivotal importance in ensuring assay reliability and reproducibility. Harmonization and standardization efforts should focus on the development of reference methods and materials for the clinical use of LC-MS/MS. In the future, commercial assay kits and laboratory-developed tests (LDTs) are expected to coexist in clinical laboratories, each offering distinct advantages. The collaborative efforts of diverse professionals is vital for addressing the challenges associated with the clinical application of LC-MS/MS. The anticipated advancements include simplification, increased automation, intelligence, and the standardization of LC-MS/MS, ultimately facilitating its seamless integration into clinical routines for both technicians and clinicians.

Nightshift work can induce oxidative DNA damage: a pilot study.

BACKGROUND: Regular sleep is very important for human health; however, the short-term and long-term effects of nightshift with sleep deprivation and disturbance on human metabolism, such as oxidative stress, have not been effectively evaluated based on a realistic cohort. We conducted the first long-term follow-up cohort study to evaluate the effect of nightshift work on DNA damage. METHODS: We recruited 16 healthy volunteers (aged 33 ± 5 years) working night shifts at the Department of Laboratory Medicine at a local hospital. Their matched serum and urine samples were collected at four time points: before, during (twice), and after the nightshift period. The levels of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), two important nucleic-acid damage markers, were accurately determined based on a robust self-established LC‒MS/MS method. The Mann-Whitney U or Kruskal-Wallis test was used for comparisons, and Pearson's or Spearman's correlation analysis was used to calculate the correlation coefficients. RESULTS: The levels of serum 8-oxodG, estimated glomerular filtration rate-corrected serum 8-oxodG, and the serum-to-urine 8-oxodG ratio significantly increased during the nightshift period. These levels were significantly higher than pre-nightshift work level even after 1 month of discontinuation, but no such significant change was found for 8-oxoG. Moreover, 8-oxoG and 8-oxodG levels were significantly positively associated with many routine biomarkers, such as total bilirubin and urea levels, and significantly negatively associated with serum lipids, such as total cholesterol levels. CONCLUSION: The results of our cohort study suggested that working night shifts may increase oxidative DNA damage even after a month of discontinuing nightshift work. Further studies with large-scale cohorts, different nightshift modes, and longer follow-up times are needed to clarify the short- and long-term effects of night shifts on DNA damage and find effective solutions to combat the negative effects.

Research in the genetics of pheochromocytoma and paraganglioma: a bibliometric analysis from 2002 to 2022.

Over the past two decades, there has been a significant growth in articles focusing on the genetics of pheochromocytoma and paraganglioma (PPGL). We used bibliometric methods to investigate the historical changes and trend in PPGL research. There was a total of 1263 articles published in English from 2002 to 2022 included in our study. The number of annual publications and citations in this field has been increasing in the past 20 years. Furthermore, most of the publications originated from the European countries and the United States. The co-occurrence analysis showed close cooperation between different countries, institutions, or authors. The dual-map discipline analysis revealed that majority articles focused on four disciplines: #2 (Medicine, Medical, Clinical), #4 (Molecular, Biology, Immunology), #5 (Health, Nursing, Medicine), and #8 (Molecular, Biology, Genetics). The hotspot analysis revealed the keywords that have been landmark for PPGL genetics research in different time periods, and there was continued interest in gene mutations, especially on SDHX family genes. In conclusion, this study displays the current status of research and future trends in the genetics of PPGL. In future, more in-depth research should concentrate on crucial mutation genes and their specific mechanisms to assist in molecular target therapy. It is hoped that this study may help to provide directions for future research on genes and PPGL.

A robust LC-MS/MS method to measure 8-oxoGuo, 8-oxodG, and NMN in human serum and urine.

OBJECTIVE: To establish and validate a robust LC-MS/MS method for simultaneously measuring 8-oxoGuo, 8-oxodG, and NMN in serum and urine to evaluate the oxidative stress status. METHODS: A Waters TQ-XS triple quadrupole mass spectrometer system coupled with an Acquity UPLC Primer HSS T3 column was chosen. The clinical performance was verified according to the CLSI C62-A and EP-15 guidelines. Furthermore, matched serum and urine samples from 22 apparently healthy check-ups, 20 patients with atherosclerosis, and 18 individuals with dementia were evaluated. RESULTS: The recovery for serum 8-oxoGuo, urine 8-oxoGuo, serum 8-oxodG, urine 8-oxodG, serum NMN, and urine NMN was 88.8-112.4%, 102.4-114.1%, 88.5-107.7%, 94.9-102.6%, 98.4-108.9%, and 88.5-108.6%, respectively. Based on the inter-assay results, total coefficient of variation, matrix effect, and carryover, the LC-MS/MS method was deemed robust. The limit of quantification was 0.017, 0.018, and 0.150 nmol/L for 8-oxoGuo, 8-oxodG, and NMN, respectively, which are suitable for accurate measurements in human serum and urine samples. Higher 8-oxoGuo and 8-oxodG levels and lower NMN levels, indicative of significantly higher oxidative stress status, were found in patients with dementia compared to healthy subjects. CONCLUSION: We established and validated a robust LC-MS/MS method to simultaneously measure 8-oxoGuo, 8-oxodG, and NMN in serum and urine.

Total serum vitamin B12 (cobalamin) LC-MS/MS assay as an arbiter of clinically discordant immunoassay results.

OBJECTIVES: Measurement of the serum levels of vitamin B12 (VB12) is key for evaluating VB12 deficiency-dependent anemia. Immunoassay, the major method for determining VB12, tends to give false-normal results because of the presence of anti-intrinsic factor (IF-Ab) or other factors such as heterophilic antibodies et al. This study aimed to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that is helpful for distinguish false normal VB12 results measured by the immunoassay. METHODS: Different forms of VB12 were derivatized into CN-B12, which was collected through solid-phase extraction and analyzed via LC-MS/MS. 236 serum samples were measured both by LC-MS/MS and immunoassay, results were compared, and the IF-Ab effect was evaluated. RESULTS: The LC-MS/MS assay afforded a linear slope from 20 to 4,000 pmol/L for CN-B12. OH-VB12, methyl-VB12, and CoA-VB12 showed recovery within 89.3-109.5%. The intra-assay CV of VB12 was 2.6-4.1%, whereas the total CV was 9.3-9.8%. Passing-Bablok regression between LC-MS/MS and immunoassay results showed that the slope was 1.085 and the intercept was -15.691. The Bland-Altman plot showed that the mean difference and difference% were -34.6 pmol/L and 0.3%, respectively. Inter-rater agreement analysis showed that the linear weighted kappa value was 0.885, implying good agreement between the two methods. However, two samples were falsely elevated and one sample was falsely normal in the immunoassay compared with LC-MS/MS. The LC-MS/MS method helped in the distinction of false-normal VB12 results shown by the immunoassay. CONCLUSIONS: The VB12 LC-MS/MS method can be used as an arbiter of clinically discordant immunoassay results.

Analysis of two intestinal bacterial metabolites (trimethylamine N-oxide and phenylacetylglutamine) in human serum samples of patients with T2DM and AMI using a liquid chromatography tandem mass spectrometry method.

BACKGROUND: Trimethylamine N-oxide (TMAO) and phenylacetylglutamine (PAGln) are associated with acute myocardial infarction (AMI) and type 2 diabetes mellitus (T2DM). This study developed and validated a simple method firstly for simultaneously quantifying serum TMAO and PAGln using liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Serum samples from patients with T2DM, AMI, and healthy subjects were analyzed using a new LC-MS/MS method to evaluate TMAO and PAGln levels. Statistical analyses were performed to evaluate TMAO and PAGln distributions among different groups. RESULTS: Retention and separation of the two metabolites were achieved within 5 min. For both analytes, the assay was linear in a 0.02-10 µg/mL range, with >0.99 average linear correlation coefficients, and quantification limit values of approximately 0.010 µg/mL. The average recoveries of TMAO and PAGln were 96.3 % and 96.4 %, respectively. The intra-run and total coefficient variations were 3.5-4.8 % and 3.9-5.7 % respectively for TMAO; and 4.0-5.1 % and 4.6-6.3 % respectively for PAGln. TMAO and PAGln showed a moderate correlation (P < 0.001) and their levels in patients with T2DM were significantly higher than those in healthy individuals (P < 0.001). TMAO levels were higher in patients with T2DM than in patients with AMI (P < 0.01). Patients with AMI had higher PAGln levels than healthy individuals (P < 0.05). After adjusting for sex and age, the top tertile of PAGln was positively correlated with T2DM and AMI while that of TMAO was positively correlated with T2DM. CONCLUSIONS: Overall, a robust isotope dilution LC-MS/MS method was established, which may be beneficial for assessing the association between two metabolites with AMI and T2DM.

Oncogene or tumor suppressor gene: An integrated pan-cancer analysis of NBPF1.

Neuroblastoma breakpoint family, member 1 (NBPF1), appears to be a double-edged sword with regard to its role in carcinogenesis. On the one hand, the tumor-suppressing functions of NBPF1 have been definitively observed in neuroblastoma, prostate cancer, cutaneous squamous cell carcinoma, and cervical cancer. On the other hand, there is evidence that NBPF1 regulates the colony formation, invasion, and maintenance of liver cancer cells and hence functions as an oncogene. The roles of NBPF1 are strictly dependent on the biological context and type of organization. However, a systematic pan-cancer analysis has thus far not been undertaken, and the significance of NBPF1 in the occurrence and progression of many malignancies is uncertain. In this paper, bioinformatics techniques were employed to analyze NBPF1 expression across different cancers and investigate the relationship between NBPF1 and clinical features, prognosis, genetic alteration, and tumor immune microenvironment, respectively. Our results show that NBPF1 is variably expressed in distinct tumor tissues and is also closely linked to clinical outcomes. In particular, compared to other tumor types, there was a strong negative correlation between NBPF1 expression and various components of the tumor microenvironment in adrenocortical carcinoma (ACC). We thus developed an NBPF1-derived immune risk model based on NBPF1-related immune genes; ACC patients with a high-risk score tended to have a poorer prognosis, accompanied by immune hyporesponsiveness. NBPF1 can be used as a prognostic biomarker for multiple cancers. Moreover, anti-NBPF1 immunotherapy may be suitable for treating ACC patients.

Design, methods and baseline characteristics of the Beijing Hospital Atherosclerosis Study: a prospective dynamic cohort study.

Background: The atherosclerotic coronary artery disease (CAD) risk assessment based on conventional risk factors have only moderate performance, and residual risks still exist. Thus, we reported here a cohort study that aims to identify and validate the new biosignatures (especially the metabolomics, lifestyle biomarkers and biological age), and elucidate their predictive effect on CAD and subsequent cardiovascular events. Methods: This prospective, single-center, cohort study commenced in March 2017 and seeks to examine patients undergoing coronary angiography (CAG) at the Beijing Hospital. Patients' baseline demographic and medical history data are captured by questionnaires. Blood samples are taken before CAG for clinical laboratory tests and metabolomics analyses. Traditional CAD risk factors are analyzed by routine assays. CAD-related metabolites from different metabolic pathways and lifestyle biomarkers are measured by liquid chromatography-tandem mass spectrometry methods. Biological ages are calculated based on the laboratory and metabolomics data. The enrolled patients attend annual follow-up examinations for 10 years. The primary end points are the composite end points of major adverse cardiovascular events, including death from any cause, non-fatal myocardial infarction, and non-fatal stroke. Quality management and control are carried out through the entire research process, including standardized baseline and follow-up investigation, intra- and inter-run quality controls for laboratory measurements, etc. Results: Baseline data of the enrolled 2,970 patients from 2017 to 2020 were collected and are presented in this article. Among them, there were more males (62.5%) than females, and the patients tended to be old and overweight. The percentages of diagnosed hypertension and diabetes were 67.3% and 35.2%, respectively. A total of 8.5% had a family history of premature CAD. Their lipid profiles were within the normal range, probably due to the use of statins. Conclusions: This study has successfully initiated an investigation into the roles of new biosignatures in predicting CAD among Chinese Han patients undergoing CAG. To the best of our knowledge, this cohort is the first study systematically focusing on the association of lifestyle biomarkers and biological age with CAD risk. Findings from this study will provide biomarkers to discriminate the presence of CAD and to predict subsequent cardiovascular events.

High-throughput analysis of total homocysteine and methylmalonic acid with the efficiency to separate succinic acid in serum and urine via liquid chromatography tandem mass spectrometry.

Vitamin B12 (VB12) deficiency may lead to hyperhomocysteinemia and methylmalonic acidemia development which are risk factors of cardiovascular disease and nervous system impairment, respectively. However, few analytical methods are available to simultaneously quantify total homocysteine (tHcy) and methylmalonic acid (MMA) due to complex analytical requirements, such as sensitivity at nanomolar concentration, separation performance for succinic acid (SA), an endogenous isomer of MMA, and retention properties for polar compounds. Therefore, we developed and validated a simple and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of tHcy and MMA with the efficient separation of SA in human serum and urine. The clinical performance of the assay was validated according to CLSI C62-A guidelines. The recovery for serum tHcy was 95.2-105.8%, urine tHcy was 98.1-111.5%, serum MMA was 94.6-99.4%, and urine MMA was 101.6-105.6%. In addition, the LC-MS/MS method was found to be reliable based on the value of inter-assay imprecision and total imprecision coefficient variation (CV), matrix effect, and carryover. Standards and samples were stable in -20 °C for at least 2 months. The limits of quantifications (LOQs) were 0.074 nmol/mL for tHcy and 0.040 nmol/mL for MMA, which are suitable for detecting tHcy and MMA concentrations in human serum and urine. The concentration of tHcy and MMA in samples collected from 148 subjects were measured using this method. The results suggested that the concentrations of serum tHcy and MMA considerably differed between VB12 sufficient and deficient groups. Serum tHcy and serum MMA concentrations were inversely correlated with VB12 status. Our method represents a rapid technique for estimating tHcy and MMA concentrations in serum and urine samples without the need for derivatization and may be used to assess VB12 status in clinical applications.

Genome-Wide Identification and Expression Analyses of AnSnRK2 Gene Family under Osmotic Stress in Ammopiptanthus nanus.

Sucrose non-fermenting-1 (SNF1)-related protein kinase 2's (SnRK2s) are plant-specific serine/threonine protein kinases and play crucial roles in the abscisic acid signaling pathway and abiotic stress response. Ammopiptanthus nanus is a relict xerophyte shrub and extremely tolerant of abiotic stresses. Therefore, we performed genome-wide identification of the AnSnRK2 genes and analyzed their expression profiles under osmotic stresses including drought and salinity. A total of 11 AnSnRK2 genes (AnSnRK2.1-AnSnRK2.11) were identified in the A. nanus genome and were divided into three groups according to the phylogenetic tree. The AnSnRK2.6 has seven introns and others have eight introns. All of the AnSnRK2 proteins are highly conserved at the N-terminus and contain similar motif composition. The result of cis-acting element analysis showed that there were abundant hormone- and stress-related cis-elements in the promoter regions of AnSnRK2s. Moreover, the results of quantitative real-time PCR exhibited that the expression of most AnSnRK2s was induced by NaCl and PEG-6000 treatments, but the expression of AnSnRK2.3 and AnSnRK2.6 was inhibited, suggesting that the AnSnRK2s might play key roles in stress tolerance. The study provides insights into understanding the function of AnSnRK2s.

Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens.

Background: Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR. Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR. Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR. Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

Expression of a Chromoplast-Specific Lycopene ß-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat.

Carotenoids are the principal pigments in the loquat. Although the metabolic pathway of plant carotenoids has been extensively investigated, few studies have been explored the regulatory mechanisms of loquat carotenoids because knowledge of the loquat genome is incomplete. The chromoplast-specific lycopene ß-cyclase gene (CYC-B) could catalyze cyclization of lycopene to ß-carotene. In this study, the differential accumulation patterns of loquat with different colors were analyzed and virus-induced gene silencing (VIGS) was utilized in order to verify CYC-B gene function. Using a cloning strategy of homologous genes, a CYC-B gene orthologue was successfully identified from the loquat. At a later stage of maturation, CYC-B gene expression and carotenoids concentrations in the 'Dawuxing' variety were higher than in 'Chuannong 1-5-9', possibly leading to the difference in pulp coloration of loquat. Interference of CYC-B gene expression in the loquat demonstrated clear visual changes. The green color in negative control fruits became yellow, while TRV2-CYC-B silenced fruits remained green. CYC-B gene expression and total carotenoid content in the pulp decreased by 32.5% and 44.1%, respectively. Furthermore, multiple key genes in the carotenoid metabolic pathway synergistically responded to downregulation of CYC-B gene expression. In summary, we provide direct evidences that CYC-B gene is involved in carotenoid accumulation and coloration in the loquat.

Association of serum glycine levels with metabolic syndrome in an elderly Chinese population.

BACKGROUND: Several studies have identified a negative association between serum glycine (Gly) levels and metabolic syndrome (MetS). However, this association has not been fully established in the elderly. METHODS: A total of 472 Chinese individuals (272 males and 200 females, 70.1 ± 6.6 years old) participated in a population-based, cross-sectional survey in Beijing Hospital. The MetS and its components were defined based on the 2006 International Diabetes Federation (IDF) standard for Asians. Serum Gly concentration was determined using isotope dilution liquid chromatography tandem mass spectrometry. RESULT: The proportion of patients with MetS decreased gradually with increasing Gly levels (p for trend < 0.001), and serum Gly concentrations declined gradually with increasing numbers of MetS components (p = 0.03 for trend). After adjusting for age and gender, lower Gly levels were significantly associated with MetS and central obesity, with OR (95% CI) of 0.40 (0.25-0.65) and 0.46 (0.28-0.74). The stratified analysis conducted according to age showed that the OR between serum Gly levels and MetS was greater in those older than 65 (OR = 0.66; 95% CI, 0.51-0.86) than in those younger than 65 (OR = 0.89; 95% CI, 0.54-1.46). In the stratified analysis, using other age cut-off points, the results consistently showed that the association between serum Gly levels and MetS was more remarkable in the older groups. CONCLUSIONS: Gly levels are associated with cardiometabolic characteristics and MetS in the elderly, and the association is more pronounced in very old people than in younger old people.

A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography.

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FERHDL) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.

Understanding intestinal glucose transporter expression in obese compared to non-obese subjects.

INTRODUCTION: The impact of Roux-en-Y gastric bypass (RYGB) on weight loss and co-morbid disease resolution is well established. However, the mechanisms underlying the procedure remain incompletely understood. Intestinal remodeling involving glucose transporters (GLUTs) may play a crucial role. Rat studies have demonstrated morphological adaptation of GLUTs within adipose and intestinal cells in association with the reprogramming of glucose metabolism. There is a limited understanding of the variations in expression amongst GLUT family receptors in the human intestine. The aim of this study was to evaluate and describe jejunal GLUT expression patterns in the obese versus non-obese. METHODS: Tissue samples were collected from 19 adults (age ≥18) patients with morbid obesity undergoing elective RYGB. Specimens were obtained from excess jejunum removed during the stapled jejuno-jejunal anastomosis. All subjects met National Institutes of Health criteria for bariatric surgery (body mass index or BMI ≥40 or ≥35 with obesity-related comorbidities). Exclusion criteria included age less than 18, age greater than 65, patients undergoing a revision procedure, and the presence of a seizure disorder (possible association with GLUT-1 deficiency syndrome). Five samples were obtained from non-obese subjects (average BMI 26.7) without diabetes who were consenting organ donors after brain death. Samples of jejunum from non-obese individuals were obtained at the time of organ procurement. Institutional Review Board and Gift of Hope approval was obtained. Specimens underwent quantitative real-time PCR and Western blotting. Western blot densitometry was performed using Image J software. Student T test was performed using SPSS statistics software. RESULTS: GLUT-1 and GLUT-7 expression were not detected in the jejunum of either group. No difference in expression pattern was observed for GLUT-2, GLUT-4, and GLUT-9 between the groups. Western blot band density of GLUT-5 to loading control (GADPH) mean ratio was 0.21 (SD = 0.20) in obese specimens compared to 0.56 (SD = 0.17) in non-obese. Densitometry revealed GLUT-5 levels in the jejunum of the obese were significantly lower than non-obese specimens (P < 0.05). CONCLUSION: The absence of GLUT-1 expression in both the obese and non-obese groups is consistent with the established view of GLUT-1 being abundantly present in fetal intestine but diminished to negligible levels by adulthood. Decreased GLUT-5 expression in samples from subjects with obesity compared to non-obese samples may represent a down-regulation of gene expression amongst the obese. The differential expression of GLUT-5 suggests a possible role in obesity. Studies of GLUT family expression will aid in understanding the impact of intestinal remodeling on obesity.

The Role of miR-212 and iNOS in Alcohol-Induced Intestinal Barrier Dysfunction and Steatohepatitis.

BACKGROUND: Alcoholic liver disease is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of intestinal tight junction proteins, such as Zonula Occludens-1 (ZO-1), plays an important role in alcohol-induced gut leakiness. However, the mechanism of alcohol-induced disruption of tight junction proteins is not well established. The goal of this study was to elucidate this mechanism by studying the role of microRNA 212 (miR-212) and inducible nitric oxide synthase (iNOS) in alcohol-induced gut leakiness. METHODS: The permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance and flux of fluorescein sulfonic acid. miR-212 was measured by real-time polymerase chain reaction. The wild-type, iNOS knockout, and miR-212 knockdown mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. The LNA-anti-miR-212 was used to inhibit miR-212 expression in mice. The alcohol-induced intestinal permeability, miR-212 expression, and liver injuries in mice were measured. RESULTS: Our in vitro monolayer and in vivo mice studies showed that: (i) alcohol-induced overexpression of the intestinal miR-212 and intestinal hyperpermeability is prevented using miR-212 knockdown techniques; and (ii) iNOS is up-regulated in the intestine by alcohol and that iNOS signaling is required for alcohol-induced miR-212 overexpression, ZO-1 disruption, gut leakiness, and steatohepatitis. CONCLUSIONS: These studies thus support a novel miR-212 mechanism for alcohol-induced gut leakiness and a potential target that could be exploited for therapeutic intervention to prevent leaky gut and liver injury in alcoholics.

Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation.

The circadian clock orchestrates temporal patterns of physiology and behavior relative to the environmental light:dark cycle by generating and organizing transcriptional and biochemical rhythms in cells and tissues throughout the body. Circadian clock genes have been shown to regulate the physiology and function of the gastrointestinal tract. Disruption of the intestinal epithelial barrier enables the translocation of proinflammatory bacterial products, such as endotoxin, across the intestinal wall and into systemic circulation; a process that has been linked to pathologic inflammatory states associated with metabolic, hepatic, cardiovascular and neurodegenerative diseases - many of which are commonly reported in shift workers. Here we report, for the first time, that circadian disorganization, using independent genetic and environmental strategies, increases permeability of the intestinal epithelial barrier (i.e., gut leakiness) in mice. Utilizing chronic alcohol consumption as a well-established model of induced intestinal hyperpermeability, we also found that both genetic and environmental circadian disruption promote alcohol-induced gut leakiness, endotoxemia and steatohepatitis, possibly through a mechanism involving the tight junction protein occludin. Circadian organization thus appears critical for the maintenance of intestinal barrier integrity, especially in the context of injurious agents, such as alcohol. Circadian disruption may therefore represent a previously unrecognized risk factor underlying the susceptibility to or development of alcoholic liver disease, as well as other conditions associated with intestinal hyperpermeability and an endotoxin-triggered inflammatory state.

Role for intestinal CYP2E1 in alcohol-induced circadian gene-mediated intestinal hyperpermeability.

We have shown that alcohol increases Caco-2 intestinal epithelial cell monolayer permeability in vitro by inducing the expression of redox-sensitive circadian clock proteins CLOCK and PER2 and that these proteins are necessary for alcohol-induced hyperpermeability. We hypothesized that alcohol metabolism by intestinal Cytochrome P450 isoform 2E1 (CYP2E1) could alter circadian gene expression (Clock and Per2), resulting in alcohol-induced hyperpermeability. In vitro Caco-2 intestinal epithelial cells were exposed to alcohol, and CYP2E1 protein, activity, and mRNA were measured. CYP2E1 expression was knocked down via siRNA and alcohol-induced hyperpermeability, and CLOCK and PER2 protein expression were measured. Caco-2 cells were also treated with alcohol or H2O2 with or without N-acetylcysteine (NAC) anti-oxidant, and CLOCK and PER2 proteins were measured at 4 or 2 h. In vivo Cyp2e1 protein and mRNA were also measured in colon tissue from alcohol-fed mice. Alcohol increased CYP2E1 protein by 93% and enzyme activity by 69% in intestinal cells in vitro. Alcohol feeding also increased mouse colonic Cyp2e1 protein by 73%. mRNA levels of Cyp2e1 were not changed by alcohol in vitro or in mouse intestine. siRNA knockdown of CYP2E1 in Caco-2 cells prevented alcohol-induced hyperpermeability and induction of CLOCK and PER2 proteins. Alcohol-induced and H2O2-induced increases in intestinal cell CLOCK and PER2 were significantly inhibited by treatment with NAC. We concluded that our data support a novel role for intestinal CYP2E1 in alcohol-induced intestinal hyperpermeability via a mechanism involving CYP2E1-dependent induction of oxidative stress and upregulation of circadian clock proteins CLOCK and PER2.

New molecular insights into inflammatory bowel disease-induced diarrhea.

Diarrhea is one of the common symptoms that significantly affects quality of life in patients with inflammatory bowel disease (IBD). The clinical manifestation of diarrhea is mainly dependant on the type of IBD and the location, extent and severity of intestinal inflammation. Understanding the pathophysiologic mechanisms of diarrhea in patients with IBD will be beneficial to developing effective treatments for IBD-associated diarrhea. In recent years, modern molecular techniques have been used intensively to dissect the role of the intestinal microbiota, epithelial barrier and the host immune system in the mechanisms of IBD-induced diarrhea. These studies have significantly advanced our knowledge of the mechanisms of IBD-induced diarrhea. In this article, we focus on the new and critical molecular insights into the contributions of the intestinal microbiota, epithelial tight junctions, proinflammatory cytokines and microRNA as potential mechanisms underlying to IBD-induced diarrhea.