Non-invasive urinary metabolomic profiles discriminate biliary atresia from infantile hepatitis syndrome

Introduction

Neonatal cholestatic disorders are a group of hepatobiliary diseases occurring in the first 3 months of life. The most common causes of neonatal cholestasis are infantile hepatitis syndrome (IHS) and biliary atresia (BA). The clinical manifestations of the two diseases are too similar to distinguish them. However, early detection is very important in improving the clinical outcome of BA. Currently, a liver biopsy is the only proven and effective method used to differentially diagnose these two similar diseases in the clinic. However, this method is invasive. Therefore, sensitive and non-invasive biomarkers are needed to effectively differentiate between BA and IHS. We hypothesized that urinary metabolomics can produce unique metabolite profiles for BA and IHS.

Objectives

The aim of this study was to characterize urinary metabolomic profiles in infants with BA and IHS, and to identify differences among infants with BA, IHS, and normal controls (NC).

Methods

Urine samples along with patient characteristics were obtained from 25 BA, 38 IHS, and 38 NC infants. A non-targeted gas chromatography–mass spectrometry (GC–MS) metabolomics method was used in conjunction with orthogonal partial least squares discriminant analysis (OPLS-DA) to explore the metabolomic profiles of BA, IHS, and NC infants.

Results

In total, 41 differentially expressed metabolites between BA vs. NC, IHS vs. NC, and BA vs. IHS were identified. N-acetyl-d-mannosamine and alpha-aminoadipic acid were found to be highly accurate at distinguishing between BA and IHS.

Conclusions

BA and IHS infants have specific urinary metabolomic profiles. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be used to discriminate BA from IHS.

     

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.     

金双歧治疗新生儿高胆红素血症的临床疗效研究

目的 探讨微生态制剂金双歧(双歧杆菌乳酸杆菌三联活菌片)对新生儿高胆红素血症的临床疗效,以找出能及早有效地纠正新生儿高胆红素血症,预防新生儿胆红素脑病发生的新方法.方法 通过随机抽样、收集青白江区人民医院2007年3月至2011年3月72例新生儿高胆红素血症患儿的临床资料并进行回顾性分析,将72例新生儿高胆红素血症患儿随机分为治疗组40例和对照组32例.对照组给予常规治疗,治疗组在常规治疗基础上,加用金双歧1片(0.5亿)/次,每天3次,温开水溶碎后喂服或经胃管注入,共7～10d.检测治疗前后两组血清胆红素水平、计算日平均经皮测胆红素下降值、平均住院时间和黄疸消退时间.结果 金双歧辅助治疗新生儿高胆红素血症,治疗后治疗组与对照组血清总胆红素水平分别为(58.21 +4.36) μmol/L和(64.16±5.29) μmol/L(P ＜0.01);治疗组和对照组日平均经皮测胆红素下降值、平均住院时间和黄疸退尽平均时间分别为( 10.35±2.46)、(7.68±1.35)μmol/L;( 6/54±2/59)、(9/05 +3/12)d; (6/38±1/38)、( 8/75±2/68)d;差异均有统计学意义(P＜0.01).结论 应用金双歧治疗新生儿高胆红素血症可迅速降低血胆红素水平,缩短治疗时间,预防胆红素脑病的发生疗效确切、安全,可作为治疗新生儿高胆红素血症的方法之一,值得临床应用推广.     

Rapid determination of serological cytokine biomarkers for hepatitis B virus-related hepatocellular carcinoma using antibody microarrays

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with an increasing incidence. The exploration of biomarkers for HCC is one of the main aims for improving the efficacy of diagnosis and treatment. The microarray technology provides a high-throughput platform for parallel exploration of biomarkers for clinics. In this study, we used antibody microarrays to screen the novel cytokine biomarkers of hepatitis B virus (HBV)-related HCC. Cytokine-secreting patterns in sera were determined from 109 cases including 43 HBV-related HCC patients, 33 chronic hepatitis B patients, and 33 normal controls by RayBio Biotin label-based human antibody array. The correlation analysis was performed with conventional clinical diagnostic biomarkers, including serum alanine aminotransferase, alpha-fetoprotein (AFP) and hepatitis B surface antigen. Our results showed that in HBV-related HCC group, which had the highest percentage of AFP positive (>20 ng/ml) ratio, six cytokines were found differentially expressed in HCC patients (P < 0.05), compared with either normal controls or chronic hepatitis B group. Two macrophage-related cytokines, macrophage-derived chemokine (MDC) and macrophage-stimulating protein α (MSPα), displayed significant difference in the HCC group. Furthermore, an HCC diagnostic model for prediction was constructed, by which the combination of MDC and MSPα together with AFP had improved the diagnostic sensitivity from 60% (AFP alone) to 73.2% with similar specificity. Our results suggested that MDC and MSPα screened by antibody microarrays might serve as novel cytokines biomarkers for potential auxiliary diagnosis of HBV-related HCC.     

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4’-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.     

Early predictive value of cord blood bilirubin and dynamic monitoring of transcutaneous bilirubin for hyperbilirubinemia of newborns

ObjectiveTo study the early predictive value of cord blood bilirubin and dynamic monitoring of transcutaneous bilirubin for hyperbilirubinemia of newborns.Methods389 newborns delivered from June 2014 to December 2015 were enrolled as the research subjects; detailed records were made about the general data of newborns and mothers, and after cord blood bilirubin being graded, the incidence of hyperbilirubinemia was counted, and the prediction efficiency of cord blood bilirubin was analyzed by receiver operator characteristic (ROC) curve. At the same time, the transcutaneous bilirubin was detected continuously when the neonate was born and 24 h, 48 h and 72 h after birth, and the relativity between transcutaneous bilirubin at 72 h and serum bilirubin was analyzed.ResultsNo significant difference was found in the hyperbilirubinemia group and the non-hyperbilirubinemia group concerning general data of the newborns and their mothers. With the concentration of cord blood bilirubin increased, the incidence of hyperbilirubinemia also increased; separate prediction of hyperbilirubinemia by cord blood bilirubin showed a sensitivity and specificity of 71.4% and 65.6% respectively, and they need further dynamic monitoring. The daily mean of transcutaneous bilirubin in hyperbilirubinemia group was significantly higher than that in non-hyperbilirubinemia group at 24 h, 48 h and 72 h, and the measurement value of transcutaneous bilirubin at 72 h had a high correlation with serum bilirubin. When transcutaneous bilirubin value is higher than 18, the incidence of hyperbilirubinemia should be considered.ConclusionsThe increase of cord blood bilirubin effectively predict the occurrence of neonatal hyperbilirubinemia. There is a good correlation between levels of transcutaneous bilirubin and serum bilirubin. Moreover, combined detection of transcutaneous bilirubin and cord blood bilirubin can significantly improve the prediction accuracy of hyperbilirubinemia.     

Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.     

Identification of HSP90 as Potential Biomarker of Biliary Atresia Using Two-Dimensional Electrophoresis and Mass Spectrometry

Biliary atresia (BA) is a devastating cholestatic liver disease targeting infants. Current diagnosis depends on surgical exploration of the biliary tree. The aim of the present study was to identify potential biomarkers for the diagnosis of biliary atresia (BA). Two-dimensional electrophoresis was utilized for the identification of proteins that were differentially expressed in liver biopsies of 20 BA patients and 12 infants with non-BA neonatal cholestasis (NC) as controls. Using mass spectrometry, we identified 15 proteins with expressions significantly altered. Out of the 15 proteins identified, heat shock protein (HSP) 90 was the most significantly altered and was down-regulated in BA samples compared to NC samples using immunoblotting analysis. Our findings suggest that HSP90 might be a potential biomarker for the diagnosis of BA and may be used for monitoring further development and therapy for BA. This study demonstrated that a comprehensive strategy of proteomic identification combined with further validation should be adopted in biomarker discovery.     

Plasma Superoxide Dismutase Measurement in Children with Viral Hepatitis

Seventy-seven blood samples from normal controls aged 0–8 years and 93 blood samples from children of similar ages with various viral hepatitis were investigated by measuring plasma superoxide dismutase (EC 1.15.1.1) using chemiluminescence immunoassay (CLIA). Total and Cu,Zn-SOD activities of normal controls of group 2(1–8 years old) were significantly higher than that of normal controls of group 1 (0–1 year old) (P < 0.01. P < 0.01), while there were no differences of Mn-SOD activities between the two groups. Total. Cu,Zn-and Mn-SOD activities significantly increased in the acute phase (0–4 weeks after onset) and dropped to the normal levels in the restoration phase (4th week later) for 29 children with cytomegalovirus hepatitis (CMVH), in comparison with group 1. Only Mn-SOD activities were significantly increased in the acute phase (with increased ALT levels) and restoration phase (with normal ALT levels) for 18 children with hepatitis A (HA). Total and Cu,Zn-SOD activities significantly decreased and Mn-SOD activities significantly increased in both the active (with increased ALT levels) and the inactive phases (with normal ALT levels) for 36 children with chronic persistent hepatitis (CPH). Only Cu,Zn-SOD activities fell significantly in both active and inactive phases for 10 children with chronic active hepatitis (CAH).     

Biomarker Discovery in Human Prostate Cancer: an Update in Metabolomics Studies

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in Western countries. Current screening techniques are based on the measurement of serum prostate specific antigen (PSA) levels and digital rectal examination. A decisive diagnosis of PCa is based on prostate biopsies; however, this approach can lead to false-positive and false-negative results. Therefore, it is important to discover new biomarkers for the diagnosis of PCa, preferably noninvasive ones. Metabolomics is an approach that allows the analysis of the entire metabolic profile of a biological system. As neoplastic cells have a unique metabolic phenotype related to cancer development and progression, the identification of dysfunctional metabolic pathways using metabolomics can be used to discover cancer biomarkers and therapeutic targets. In this study, we review several metabolomics studies performed in prostatic fluid, blood plasma/serum, urine, tissues and immortalized cultured cell lines with the objective of discovering alterations in the metabolic phenotype of PCa and thus discovering new biomarkers for the diagnosis of PCa. Encouraging results using metabolomics have been reported for PCa, with sarcosine being one of the most promising biomarkers identified to date. However, the use of sarcosine as a PCa biomarker in the clinic remains a controversial issue within the scientific community. Beyond sarcosine, other metabolites are considered to be biomarkers for PCa, but they still need clinical validation. Despite the lack of metabolomics biomarkers reaching clinical practice, metabolomics proved to be a powerful tool in the discovery of new biomarkers for PCa detection.     

Identification of complement C3a as a candidate biomarker in human chronic hepatitis C and HCV-related hepatocellular carcinoma using a proteomics approach

Although the significant risk factors for hepatocellular carcinoma (HCC) are well known from epidemiological studies, diagnosis of this disease at an early stage is difficult, and HCC remains one of the leading causes of cancer death worldwide. Thus, to identify any useful HCC-related biomarkers is still a need. We performed SELDI-TOF MS to identify differentially expressed proteins in HCC serum using weak cation exchange protein chips. Protein characterization was performed by 2-DE separation and nano flow LC-MS/MS. A total of 55 sera were collected from HCC patients and compared with those from 48 patients with chronic hepatitis and 9 healthy individuals. A candidate marker of about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus (HCV)-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. The combination of SELDI-TOF MS and 2-DE provides a solution to identify disease-associated serum biomarkers.     

Discovery of early urinary biomarkers in preclinical study of gentamicin-induced kidney injury and recovery in rats

LC/MS- and NMR-based global metabolomics analyses were utilized to study the changes in rat urine in response to gentamicin treatment. Sprague?CDawley rats were dosed with gentamicin sulfate at 0, 75, 150 or 300?mg/kg/day for one, two or three consecutive days. Four animals from each group were sacrificed to harvest kidney tissue and to collect urine on days 1, 2, 3, 7, 10, 15, 18, 22, 29, 36 and 44 for a total of 11 different time points. Both uni- and multivariate statistical analyses were employed to identify the significantly changed metabolites in urine at the different dose levels and time points. Increases and decreases in amino acids including tyrosine, valine and hydroxyproline reflected histopathology changes of kidney injury development and/or kidney injury recovery. Glucosuria was noted much earlier than changes in the classic kidney function biomarkers, blood urea nitrogen and serum creatinine. Dopamine-related compounds, homovanillic acid sulfate (HVA-SO₄) and homoveratric acid sulfate (HVrA-SO₄) were significantly increased at early time points and could be early indicators of a renal adaptive response to gentamicin-induced renal injury. Furthermore, the drug efficacy of gentamicin was evaluated through the detection of changes in gut microflora-related compounds (e.g. indole-containing metabolites). Metabolomics was successful in identifying valine, hydroxyproline, HVA-SO₄ and HVrA-SO₄ that might serve as potential early injury biomarkers or adaptive markers of gentamicin-induced renal injury, and in assessing gentamicin efficacy through changes in compounds reported to be related to gut microflora. However, caution should be taken in direct translation of the biomarkers reported in clinical settings because a much higher dose of gentamicin than the normal therapeutic dose (~1?C2?mg/kg) was used to cause kidney damaged.     

Genetic variability of interleukin4 gene in Taiwanese children with biliary atresia

Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology that leads to biliary cirrhosis and is the most common cause of liver transplantation in children. A still undetermined hepatobiliary viral infection may elicit an uncontrollable autoimmune response against the biliary epithelial cells in genetically predisposed children and culminates in atresia of the biliary trees. Interleukin 4 (IL4) is crucial for the differentiation of naive T helper cells into the T helper 2 effector cells that promote humoral immunity. This study aims to investigate whether polymorphisms of the IL4 gene are associated with susceptibility to BA. Genomic DNA was extracted from whole blood samples of 53 Taiwanese children with BA and 904 ethnically-matched healthy controls. The IL4 -590 C/T, -33 C/T, and 8375 A/G polymorphisms were genotyped using the Pre-Developed TaqMan Allelic Discrimination Assay in a real-time polymerase chain reaction system. No significant difference between children with BA and healthy controls were found when comparing genotype, allele, carrier, and haplotype frequencies of these IL4 gene variants. These results suggest that the tested polymorphisms of IL4 gene are unlikely to contribute significantly to BA susceptibility in Taiwanese children.     

Significant hepatic expression of IL-2 and IL-8 in biliary atresia compared with other neonatal cholestatic disorders

ObjectivesAlthough the exact etiology of biliary atresia (BA) is still elusive, inflammation plays a key role. Release of proinflammatory cytokines from activated immune cells perpetuates the injury and causes biliary destruction. We aimed to study interleukin (IL)-2 and IL-8 expression in liver tissue of BA patients compared with other neonatal cholestatic disorders.MethodsThe study included 59 infants with neonatal cholestasis in two groups; BA group (n = 31) and non-BA group (n = 28) with cholestatic disorders other than BA as controls. Demographic, clinical, laboratory, and histopathological parameters were collected. IL-2 and IL-8 immunostaining was performed. Immunostaining in portal cellular infiltrate was scored as positive or negative and expressed as the mean cell count in three portal tracts.ResultsThe mean value of IL-2 and IL-8 positive inflammatory cells was significantly higher in BA than in non-BA group (P-values of 0.004 and 0.002 respectively). IL-2 correlated significantly with IL-8 immunostaining in both BA and non-BA group (P < 0.0001 for both). Furthermore, both cytokines in both groups correlated significantly with inflammatory activity in liver biopsy while there was no significant correlation with the other studied parameters. Yet, there was a trend of increased expression of IL-2 and IL-8 with increasing stage of fibrosis in BA group. This trend was not observed in non-BA group.ConclusionThe significantly higher expression of IL-2 and IL-8 in patients with BA compared to non-BA suggests a potential role for these cytokines in the pathogenesis in therapy of this devastating neonatal hepatic disorder.     

微生态制剂对新生儿高胆红素血症的预防作用

目的 :探讨微生态制剂预防新生儿高胆红素血症的效果。方法 :74例正常足月新生儿随机分为干预组 39例和对照组 35例 ,出生后 12 h内干预组在常规预防的同时 ,加服微生态制剂金双歧。结果 :新生儿高胆红素血症干预组的发生率为 33.33% ,对照组的发生率为 5 7.14 % ,干预组的发生率较对照组低 (P<0 .0 5 )。结论 :微生态制剂金双歧预防新生儿高胆红素血症有一定效果。     

Clinical relevance of glycerophospholipid,sphingomyelin and glutathione metabolism in the pathogenesis of pharyngolaryngeal cancer in smokers: the Korean Cancer Prevention Study-II

Introduction

Although smoking is a major risk factor for pharyngolaryngeal cancer, most smokers do not develop pharyngolaryngeal cancer.

Objectives

In the prospective Korean Cancer Prevention Study-II (KCPS-II), we investigated the application of metabolomics to differentiate smokers with incident pharyngolaryngeal cancer (pharyngolaryngeal cancer group) from smokers who remained free from cancer (controls) during a mean follow-up period of 7 years and aimed to discover valuable early biomarkers of pharyngolaryngeal cancer.

Methods

We used baseline serum samples from 30 smoking men with incident pharyngolaryngeal cancer and 59 age-matched cancer-free smoking men. Metabolic alterations associated with the incidence of pharyngolaryngeal cancer were investigated by performing metabolomics on baseline serum samples using ultra-performance liquid chromatography-linear-trap quadrupole-Orbitrap mass spectrometry.

Results

Compared to the control group, the pharyngolaryngeal cancer group showed significantly higher oxidized LDL levels. Seventeen metabolites were differentially abundant between the two groups. At baseline, compared to controls, smokers with incident pharyngolaryngeal cancer during follow-up showed significantly higher levels of pyroglutamic acid (glutathione metabolism) but lower levels of lysophosphatidylcholines (lysoPCs) C14:0, C15:0, C16:0, C17:0, C18:0, and C20:5; glycerophosphocholine; PC C36:5; lysoPEs C16:0, C20:1, and C22:0 (glycerophospholipid metabolism); SM (d18:0/16:1); and SM (d18:1/18:1) (sphingomyelin metabolism). Furthermore, smokers with incident pharyngolaryngeal cancer showed significantly higher levels of oleamide and lower levels of tryptophan and linoleyl carnitine at baseline than cancer-free smokers.

Conclusion

This prospective study showed the clinical relevance of dysregulated metabolism of glutathione, glycerophospholipids and sphingolipids to the pathogenesis of pharyngolaryngeal cancer among smokers. These data suggest that the dysregulation of these metabolic processes may be a key mechanism underlying pharyngolaryngeal cancer progression and development.

     

Combined effects of the UGT1A1 and OATP2 gene polymorphisms as major risk factor for unconjugated hyperbilirubinemia in Indian neonates

Genetic association studies have linked a number of single nucleotide polymorphisms (SNPs) with unconjugated hyperbilirubinemia. The present study was undertaken to validate the association of SNPs with development of hyperbilirubinemia in Indian neonates. Genotyping of five SNPs in two candidate genes was performed in 126 infants with hyperbilirubinemia and 181 controls by PCR-RFLP, Gene Scan analysis and direct DNA sequencing. Genetic polymorphisms of the UGT1A1 promoter, specifically the − 3279 T ? G phenobarbital responsive enhancer module (rs4124874) and (TA)7 dinucleotide repeat (rs8175347) as well as the coding region variants (rs2306283 and rs4149056) of the OATP2 gene were significantly higher among the cases than the controls. The presence of the mutant haplotypes either in homozygous, heterozygous or compound heterozygous state had a significant effect on neonatal hyperbilirubinemia as well as on the requirement of phototherapy than those with the wild haplotype. Further, a significantly higher number of hyperbilirubinemic cases had ≥ 3 variants than the controls (73.80% vs 40.36%, p < 0.0001) and the mean total serum bilirubin levels and requirement of phototherapy also increased according to the number of variants co-expressed. This study demonstrates that UGT1A1 and OATP2 polymorphisms were associated with altered bilirubin metabolism and could be genetic risk factors for neonatal hyperbilirubinemia.     

Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease

Metabolomics is a powerful new technology that allows for the assessment of global metabolic profiles in easily accessible biofluids and biomarker discovery in order to distinguish between diseased and nondiseased status information. Deciphering the molecular networks that distinguish diseases may lead to the identification of critical biomarkers for disease aggressiveness. However, current diagnostic methods cannot predict typical Jaundice syndrome (JS) in patients with liver disease and little is known about the global metabolomic alterations that characterize JS progression. Emerging metabolomics provides a powerful platform for discovering novel biomarkers and biochemical pathways to improve diagnostic, prognostication, and therapy. Therefore, the aim of this study is to find the potential biomarkers from JS disease by using a nontarget metabolomics method, and test their usefulness in human JS diagnosis. Multivariate data analysis methods were utilized to identify the potential biomarkers. Interestingly, 44 marker metabolites contributing to the complete separation of JS from matched healthy controls were identified. Metabolic pathways (Impact-value≥0.10) including alanine, aspartate, and glutamate metabolism and synthesis and degradation of ketone bodies were found to be disturbed in JS patients. This study demonstrates the possibilities of metabolomics as a diagnostic tool in diseases and provides new insight into pathophysiologic mechanisms.     

Serum MicroRNAs as Potential Biomarkers for Early Diagnosis of Hepatitis C Virus-Related Hepatocellular Carcinoma in Egyptian Patients

Circulating microRNAs are deregulated in liver fibrosis and hepatocellular carcinoma (HCC) and are candidate biomarkers. This study investigated the potential of serum microRNAs; miR-19a, miR-296, miR-130a, miR-195, miR-192, miR-34a, and miR-146a as early diagnostic biomarkers for hepatitis C virus (HCV)-related HCC. As how these microRNAs change during liver fibrosis progression is not clear, we explored their serum levels during fibrosis progression in HCV-associated chronic liver disease (CLD) and if they could serve as non-invasive biomarkers for fibrosis progression to HCC. 112 Egyptian HCV-HCC patients, 125 non-malignant HCV-CLD patients, and 42 healthy controls were included. CLD patients were subdivided according to Metavir fibrosis-scoring. Serum microRNAs were measured by qRT-PCR custom array. Serum microRNAs were deregulated in HCC versus controls, and except miR-130a, they were differentially expressed between HCC and CLD or late fibrosis (F3-F4) subgroup. Serum microRNAs were not significantly different between individual fibrosis-stages or between F1-F2 (early/moderate fibrosis) and F3-F4. Only miR-19a was significantly downregulated from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC. Individual microRNAs discriminated HCC from controls, and except miR-130a, they distinguished HCC from CLD or F3-F4 patients by receiver-operating-characteristic analysis. Multivariate logistic analysis revealed a panel of four microRNAs (miR-19a, miR-195, miR-192, and miR-146a) with high diagnostic accuracy for HCC (AUC = 0.946). The microRNA panel also discriminated HCC from controls (AUC = 0.949), CLD (AUC = 0.945), and F3-F4 (AUC = 0.955). Studied microRNAs were positively correlated in HCC group. miR-19a and miR-34a were correlated with portal vein thrombosis and HCC staging scores, respectively. In conclusion, studied microRNAs, but not miR-130a, could serve as potential early biomarkers for HCC in high-risk groups, with miR-19a as a biomarker for liver fibrosis progression to cirrhosis to HCC. We identified a panel of four serum microRNAs with high accuracy in HCC diagnosis. Additional studies are required to confirm this panel and test its prognostic significance.     

Quantitative proteomic signature of liver cancer cells: tissue transglutaminase 2 could be a novel protein candidate of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common diseases worldwide, with extremely poor prognosis due to failure in diagnosing it early. Alpha-fetoprotein (AFP) is the only available biomarker for HCC diagnosis; however, its use in the early detection of HCC is limited, especially because about one-third of patients afflicted with HCC have normal levels of serum AFP. Thus, identifying additional biomarkers that may be used in combination with AFP to improve early detection of HCC is greatly needed. A quantitative proteomic analysis approach using stable isotope labeling with amino acids in cell culture (SILAC) combined with LTQ-FT-MS/MS identification was used to explore differentially expressed protein profiles between normal (HL-7702) and cancer (HepG2 and SK-HEP-1) cells. A total of 116 proteins were recognized as potential markers that could distinguish between HCC and normal liver cells. Certain proteins, such as AFP, intercellular adhesion molecule-1 (ICAM-1), IQ motif containing GTPase activating protein 2 (IQGAP2), claudin-1 (CLDN1) and tissue transglutaminase 2 (TGM2), were validated both in multiple cell lines and in 61 specimens of clinical HCC cases. TGM2 was overexpressed in some of the AFP-deficient HCC cells (SK-HEP-1 and Bel-7402) and in about half of the tumor tissues with low levels of serum AFP (17/32, AFP-negative HCC). Trace amounts of TGM2 were found to be expressed in the samples with high serum AFP (26/29, AFP-positive HCC). Moreover, TGM2 expression in liver tissues showed an inverse correlation with the level of serum AFP in HCC patients. Notably, TGM2 existed in the supernatant of the AFP-deficient SK-HEP-1, SMMC-7721 and HLE cells, and it was found to be induced in AFP-producing cells (HepG2) by specific siRNA silence assay. Serum TGM2 levels of 109 HCC patients and 42 healthy controls were further measured by an established ELISA assay; the levels were significantly higher in HCC patients, and they correlated with the histological grade and tumor size. These data suggest that TGM2 may serve as a novel histological/serologic candidate involved in HCC, especially for the individuals with normal serum AFP. These novel findings may provide important clues to identify new biomarkers of HCC and indirectly improve early detection of the disease.