OBJECTIVE: To explore the diagnostic value of the serum metabolites identified by high-performance liquid chromatography-mass spectrometry (HPLC/MS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A total of 126 patients admitted to Tianjin Third Central Hospital were enrolled, including 27 patients with HBV-related hepatitis with negative viral DNA (DNA-N), 24 with HBV-related hepatitis with positive viral DNA, 24 with HBV-related liver cirrhosis, 27 with HBV-related HCC undergoing surgeries or radiofrequency ablation, and 24 with HBV-related HCC receiving interventional therapy, with 25 healthy volunteers as the normal control group. Serum samples were collected from all the subjects for HPLC/MS analysis, and the data were pretreated to establish an orthogonal partial least- squares discriminant analysis (OPLS-DA) model. The differential serum metabolites were preliminarily screened by comparisons between the HBV groups and the control group, and the characteristic metabolites were identified according to the results of non-parametric test. The potential clinical values of these characteristic metabolites were evaluated using receiver operator characteristic curve (ROC) analysis. RESULTS: A total of 25 characteristic metabolites were identified in the HBV- infected patients, including 9 lysophosphatidylcholines, 2 fatty acids, 17α-estradiol, sphinganine, 5-methylcytidine, vitamin K2, lysophosphatidic acid, glycocholic acid and 8 metabolites with few reports. The patients with HBV- related HCC showed 22 differential serum metabolites compared with the control group, 4 differential metabolites compared with patients with HBV-related liver cirrhosis; 10 differential metabolites were identified in patients with HBV-related HCC receiving interventional therapy compared with those receiving surgical resection or radiofrequency ablation. From the normal control group to HBV-related HCC treated by interventional therapy, many metabolites underwent variations following a similar pattern. CONCLUSIONS We identified 25 characteristic metabolites in patients with HBV-related HCC, and these metabolites may have potential clinical values in the diagnosis of HBV-related HCC. The continuous change of some of these metabolites may indicate the possibility of tumorigenesis, and some may also have indications for the choice of surgical approach.
Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; Case-Control Studies ; Chromatography, High Pressure Liquid ; DNA, Viral ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Liver Cirrhosis ; virology ; Liver Neoplasms ; blood ; diagnosis ; virology ; Mass Spectrometry ; Metabolome ; Metabolomics ; ROC Curve

OBJECTIVETo screen and identify serum biomarkers for childhood hepatoblastoma (HB).

METHODSThe serum samples from 30 children with hepatoblastoma (HB), 20 children with systemic inflammatory response syndrome, and 20 normal children were treated with magnetic bead-based weak cation exchange chromatography. The platform of surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) was used to eliminate the interference of inflammatory factors and to screen out the differentially expressed proteins in serum between tumor group and normal group. After the purification and separation of target proteins were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry was used to determine their amino acid sequences. The SwissProt database was searched for matched proteins. Finally, real-time PCR and ELISA were used to verify and measure the expression of target proteins.

RESULTSAfter SELDI-TOF-MS was used for screening and elimination of the interference of inflammatory factors, a differentially expression protein with a mass-to-charge ratio of 9 348 Da was found in serum between HB group and normal group, and the HB group had significantly lower expression of this protein than the normal group (p<0.05). This protein was identified as apolipoprotein A-1 (Apo A-I). Real-time PCR and ELISA verified the low mRNA and protein expression of Apo A-I in serum in the HB group and high expression in serum in the normal group.

CONCLUSIONSApo A-I can be used as a non-inflammatory protein marker for HB and has a certain value in the early diagnosis of HB.

Apolipoprotein A-I ; blood ; genetics ; Biomarkers ; blood ; Child, Preschool ; Early Detection of Cancer ; Female ; Hepatoblastoma ; blood ; diagnosis ; Humans ; Infant ; Liver Neoplasms ; blood ; diagnosis ; Male ; Real-Time Polymerase Chain Reaction ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Recent studies have shown that circulating microRNAs are a potential biomarker in various types of malignancies. The aim of this study was to investigate the feasibility of using serum exosomal microRNAs as novel serological biomarkers for hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). We measured the serum exosomal microRNAs and serum circulating microRNAs in patients with CHB (n=20), liver cirrhosis (LC) (n=20) and HCC (n=20). Serum exosomal microRNA was extracted from 500 mul of serum using an Exosome RNA Isolation kit. The expression levels of microRNAs were quantified by real-time PCR. The expression levels of selected microRNAs were normalized to Caenorhabditis elegans microRNA (Cel-miR-39). The serum levels of exosomal miR-18a, miR-221, miR-222 and miR-224 were significantly higher in patients with HCC than those with CHB or LC (P<0.05). Further, the serum levels of exosomal miR-101, miR-106b, miR-122 and miR-195 were lower in patients with HCC than in patients with CHB (P=0.014, P<0.001, P<0.001 and P<0.001, respectively). There was no significant difference in the levels of miR-21 and miR-93 among the three groups. Additionally, the serum levels of circulating microRNAs showed a smaller difference between HCC and either CHB or LC. This study suggests that serum exosomal microRNAs may be used as novel serological biomarkers for HCC.
Adult ; Aged ; Biomarkers, Tumor/blood/genetics ; Carcinoma, Hepatocellular/blood/diagnosis/*genetics ; Exosomes/genetics ; Female ; Gene Expression Profiling ; Humans ; Liver/pathology ; Liver Neoplasms/blood/diagnosis/*genetics ; Male ; MicroRNAs/blood/*genetics ; Middle Aged

BACKGROUND/AIMS: We tried to investigate the expression characteristics of KAI1, a suppressor of wide-spectrum tumor metastasis, and vascular endothelial growth factor (VEGF), the most common angiogenesis factor, and then to analyze their diagnostic value for hepatocellular carcinoma (HCC). METHODS: The protein and mRNA expression levels of KAI1 or VEGF in HCC tissues and in self-controlled para-carcinoma tissues were analyzed by Western blot and real-time polymerase chain reaction, respectively. Serum levels of KAI1 and VEGF in the patients with HCC, benign liver disease or in healthy controls were quantitatively detected by enzyme-linked immunosorbent assay. RESULTS: The expression level of KAI1 was downregulated, while the expression level of VEGF was upregulated in the tissues or serum of the patients with HCC. The expression level of serum KAI1 in HCC patients was correlated with TNM staging, intrahepatic metastasis, lymph node or peritoneal metastasis, and portal vein thrombus. In addition to the factors that were correlated with KAI1 expression, VEGF expression was also closely related to the alpha-fetoprotein level of the patients. The area under the receiver operating characteristic curve for the diagnosis of HCC was 0.907 for KAI1 and 0.779 for VEGF. The sensitivity of serum KAI1 levels in the diagnosis of HCC was 86.96%; the accuracy was 83.06%, while the sensitivity, the accuracy and the negative predictive value were improved to 91.86%, 84.68%, and 78.79% according to the combined detection of KAI1 and VEGF, respectively. CONCLUSIONS: A combined detection of KAI1 and VEGF may greatly improve the efficiency of diagnosis and form a reliable panel of diagnostic markers for HCC.
Adult ; Aged ; Aged, 80 and over ; Antigens, CD82/blood/genetics/*metabolism ; Carcinoma, Hepatocellular/blood/*diagnosis/genetics ; Case-Control Studies ; Female ; Gene Expression Regulation ; Humans ; Liver Diseases/genetics ; Liver Neoplasms/blood/*diagnosis/genetics ; Male ; Middle Aged ; Vascular Endothelial Growth Factor A/blood/genetics/*metabolism ; alpha-Fetoproteins/analysis

Since the discovery of HBsAg in the early 1960s, presence of HBsAg in serum has only served to diagnose hepatitis B. Recent development in the quantitative measurement of serum HBsAg has enabled us to improve our understanding on the management of chronic hepatitis B. The surface antigen (sAg) level is at its highest in immune tolerance phase and decreases to the lowest level in immune control/inactive phase when HBeAg is cleared from the serum. Combination of serum sAg titer less than 1,000 IU/mL and serum HBV DNA less than 2,000 IU/mL can identify true inactive carrier from e antigen (eAg) negative hepatitis with diagnostic accuracy of 95%. During the natural course of chronic hepatitis B, changes or absolute level of sAg less than certain level can predict spontaneous sero-clearance of HBsAg. Although the decline of sAg is very slow in interferon (IFN)/pegylated interferon (PEG-IFN) or oral nucleos(-t)ide treated patients, interferon based therapy results in a greater decrease of sAg level and sAg loss. Lack of any decline in sAg titer during PEG-IFN therapy could identify the group of patients who do not response to IFN/PEG-IFN therapy. With the aid of serum HBV DNA, quantitative measurement of serum HBsAg level can be used to optimize the management of chronic hepatitis B in our daily practice.
Antiviral Agents/therapeutic use ; DNA, Viral/blood ; Hepatitis B Surface Antigens/*blood ; Hepatitis B e Antigens/blood ; Hepatitis B, Chronic/*diagnosis/drug therapy/genetics ; Humans ; Interferons/therapeutic use ; Liver Neoplasms/diagnosis ; Prognosis

Since the discovery of HBsAg in the early 1960s, presence of HBsAg in serum has only served to diagnose hepatitis B. Recent development in the quantitative measurement of serum HBsAg has enabled us to improve our understanding on the management of chronic hepatitis B. The surface antigen (sAg) level is at its highest in immune tolerance phase and decreases to the lowest level in immune control/inactive phase when HBeAg is cleared from the serum. Combination of serum sAg titer less than 1,000 IU/mL and serum HBV DNA less than 2,000 IU/mL can identify true inactive carrier from e antigen (eAg) negative hepatitis with diagnostic accuracy of 95%. During the natural course of chronic hepatitis B, changes or absolute level of sAg less than certain level can predict spontaneous sero-clearance of HBsAg. Although the decline of sAg is very slow in interferon (IFN)/pegylated interferon (PEG-IFN) or oral nucleos(-t)ide treated patients, interferon based therapy results in a greater decrease of sAg level and sAg loss. Lack of any decline in sAg titer during PEG-IFN therapy could identify the group of patients who do not response to IFN/PEG-IFN therapy. With the aid of serum HBV DNA, quantitative measurement of serum HBsAg level can be used to optimize the management of chronic hepatitis B in our daily practice.
Antiviral Agents/therapeutic use ; DNA, Viral/blood ; Hepatitis B Surface Antigens/*blood ; Hepatitis B e Antigens/blood ; Hepatitis B, Chronic/*diagnosis/drug therapy/genetics ; Humans ; Interferons/therapeutic use ; Liver Neoplasms/diagnosis ; Prognosis

OBJECTIVETo explore changes of serum protein fingerprinting in primary liver cancer (PLC) patients of different Chinese medical syndromes before and after interventional treatment detected by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS).

METHODSTotally 154 PLC patients were assigned to 5 groups, i.e., Gan depression syndrome (GDS, 37 cases), Pi deficiency syndrome (PDS, 45 cases), dampness heat syndrome (DHS, 18 cases), blood stasis syndrome (BSS, 28 cases), yin deficiency syndrome (YDS, 26 cases). The mass spectra of serum proteins was analyzed by using SELDI-TOF-MS. Then the correlation between Chinese medical syndrome types and the mass spectra of serum proteins was explored before and after interventional treatment.

RESULTSCompared with the healthy control group, the expression of serum proteins peak was down-regulated in GDS with M/Z being 6 589 and 4 182 Da, in DHS with M/Z being 5 710 Da, in YDS with M/ Z being 6 992 Da, while it was up-regulated in PDS with M/Z being 5 816 Da and in BSS with M/Z being 4 297 Da, showing statistical difference (P < 0.01). Compared with before intervention, the expression of serum proteins peak was down-regulated in GDS with M/Z being 6 589 and 4 182 Da, in PDS with M/Z being 5 816 Da, in DHS with M/Z being 5 710 Da in BSS with M/Z being 4 297 Da, while it was up-regulated in YDS with M/Z being 6 992 Da, showing statistical difference (P < 0.05, P < 0.01).

CONCLUSIONThere was statistical difference in changes of serum protein fingerprinting in PLC patients of different Chinese medical syndromes before and after interventional treatment.

Adult ; Aged ; Aged, 80 and over ; Blood Proteins ; genetics ; Female ; Humans ; Liver Neoplasms ; blood ; diagnosis ; Male ; Medicine, Chinese Traditional ; methods ; Middle Aged ; Peptide Mapping ; Yang Deficiency ; diagnosis ; Yin Deficiency ; diagnosis ; Young Adult

Parvovirus B19 infection is known to cause chronic anemia in immunocompromised hosts, including organ transplant recipients. We report the first case of liver transplant recipient with parvovirus B19-induced pure red cell aplasia in Korea. A 57-yr-old female patient with hepatocellular carcinoma due to hepatitis C virus received a liver transplantation. Two months later, anemia developed and she received periodic red blood cell transfusions. However, chronic anemia persisted and bone marrow examination was performed 8 months after transplantation. Bone marrow aspiration smears showed markedly reduced erythroid precursors with atypical giant pronormoblasts and nuclear remnants with viral inclusions, and characteristic lantern cells were observed in biopsy sections. In addition, parvovirus B19 DNA PCR was positive. She was diagnosed as parvovirus B19-induced pure red cell aplasia and her anemia was improved following intravenous immunoglobulin therapy.
Blood Transfusion ; Bone Marrow/pathology ; Carcinoma, Hepatocellular/etiology/therapy ; DNA, Viral/analysis ; Female ; Hepatitis C/complications/diagnosis ; Humans ; Immunocompromised Host ; Immunoglobulins/therapeutic use ; Liver Neoplasms/etiology/therapy ; Liver Transplantation ; Middle Aged ; Parvoviridae Infections/complications/*diagnosis ; *Parvovirus B19, Human/genetics ; Red-Cell Aplasia, Pure/*diagnosis/therapy/virology

OBJECTIVETo study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein.

METHODSGolph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein.

RESULTSThere were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40.

CONCLUSIONThe polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.

Animals ; Antibodies, Monoclonal ; analysis ; immunology ; Base Sequence ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Membrane Proteins ; biosynthesis ; blood ; genetics ; immunology ; metabolism ; Molecular Sequence Data ; Polymerase Chain Reaction ; Rabbits

BACKGROUND/AIMS: Alcohol and the hepatitis B virus (HBV) exert synergistic effects in hepatocelluar carcinogenesis. We aimed to elucidate the clinical significance of the antibody to hepatitis B core antigen (anti-HBc) and occult HBV infection on the development of hepatocellular carcinoma (HCC) in patients with alcoholic liver cirrhosis (LC). METHODS: Patients with alcoholic LC alone (n=193) or combined with HCC (n=36), who did not have HBsAg or antibody to hepatitis C virus were enrolled. Clinical data and laboratory data including anti-HBc were investigated at enrollment. The polymerase chain reaction was applied to HBV DNA using sera of patients with HCC or LC after age and sex matching. RESULTS: Patients with HCC were older (60+/-11 years vs. 53+/-10 years, mean+/-SD, P<0.001), more likely to be male (100% vs. 89%, P=0.03), and had a higher positive rate of anti-HBc (91.2% vs. 77.3%, P=0.067), and a higher alcohol intake (739+/-448 kg vs. 603+/-409 kg, P=0.076) than those with LC. Age was the only significant risk factor for HCC revealed by multiple logistic regression analysis (odds ratio, 1.056; P=0.003). The positive rate of anti-HBc and alcohol intake did not differ in age- and sex-matched subjects between the LC (n=32) and HCC (n=31) groups. However, the detection rate of serum HBV DNA was higher in the HCC group (48.4%) than in the LC group (0%, P<0.001). CONCLUSIONS: Anti-HBc positivity is not a risk factor for HCC. However, occult HBV infection may be a risk factor for HCC in patients with alcoholic LC.
Adult ; Aged ; Antibodies, Viral/blood ; Carcinoma, Hepatocellular/diagnosis/epidemiology/*etiology ; DNA, Viral/analysis ; Female ; Hepatitis B/*complications/diagnosis ; Hepatitis B Core Antigens/*immunology ; Hepatitis B Surface Antigens/immunology ; Hepatitis B virus/genetics/immunology/isolation & purification ; Hepatitis C/complications/diagnosis ; Humans ; Liver Cirrhosis, Alcoholic/*complications/diagnosis/epidemiology ; Liver Neoplasms/diagnosis/epidemiology/*etiology ; Male ; Middle Aged ; Risk Factors