We describe a highly sensitive and specific method for the quantification of serum 7α-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is ...based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2–50 μl) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7α-picolinate), and then purified using a disposable C18 cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was ∼1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0–60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 ± 2.8 ng/ml, which coincided completely with the observed X̄0 ± SD = 23.3 ± 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.
Aim: Infection with hepatitis C virus (HCV) is the leading cause of liver cirrhosis that develops into hepatocellular carcinoma. Previous studies have shown in vitro that lipids within hepatocytes ...are crucially important for a series of HCV infection–proliferation–release processes. On the other hand, in the patients with HCV, the serum total cholesterol (Total‐C) and low‐density lipoprotein cholesterol (LDL‐C) levels have been reported to be lower. We conducted an epidemiological survey of a large cohort and investigated whether the lower serum lipid levels were caused by a direct or the secondary effects of HCV infection (i.e. hepatic damage or nutritional disorder).
Methods: Among 146 857 participants (male, 34%; female, 66%) undergoing public health examinations between 2002 and 2007 in Ibaraki Prefecture, Japan, the HCV positive rates determined by HCV antibody/antigen and/or RNA tests were 1.37% and 0.67% in males and females, respectively.
Results: In addition to Total‐C and LDL‐C, serum high‐density lipoprotein cholesterol and triglyceride concentrations were also significantly lower in the HCV positive subjects compared with the negative subjects, regardless of sex, age or nutritional state evaluated by body mass index. Multivariate analysis showed that HCV infection was the strongest among the factors to be significantly associated with the lower level of these lipids. Particularly, the hypolipidemia was also confirmed in the HCV positive subjects with normal aminotransferase levels (alanine aminotransferase ≤30 and aspartate aminotransferase ≤30).
Conclusion: This epidemiological survey in a large Japanese cohort suggests that the HCV infection itself might directly cause hypolipidemia, irrespective of host factors including age, hepatic damage and nutritional state.
We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This ...method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio = 3), ∼833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77–2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47 ± 0.07 nmol/mg protein, which coincided completely with the observed X̄0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.
Aim
Proton beam therapy is safe and more effective than conventional radiation therapy for the local control of nodular hepatocellular carcinoma (HCC). However, evaluating therapeutic response by ...imaging is not accurate during the early post‐irradiation period. Therefore, we examined whether the histopathological study of biopsy specimens obtained at 3 weeks after irradiation can be used to more accurately assess therapeutic response.
Methods
Fifteen HCC lesions from 13 patients were treated with proton beam irradiation. Tissue biopsy samples were obtained using abdominal ultrasound‐guided percutaneous fine‐needle aspiration from the center of the tumor before, 3 weeks after and 1 year post‐proton therapy. The specimens were examined after staining with hematoxylin–eosin (HE) and a MIB‐1 antibody.
Results
MIB‐1 labeling indices (LI) before treatment were 13.0 ± 8.5% (mean ± SD; range, 0.6–27.0), whereas those 3 weeks after proton therapy were significantly reduced to 3.2 ± 2.4% (range, 0.6–8.9) (P < 0.05). Although the tumor size was reduced, we did not observe a reduction in tumor blood flow by dynamic computed tomography or degenerative changes by HE. All lesions that displayed reduced MIB‐1 LI at 3 weeks post‐proton treatment were ultimately diagnosed as complete response at 1 year after treatment. In contrast, one case with increased MIB‐1 LI at 3 weeks had significant tumor size progression at 1 year post‐treatment.
Conclusion
The percutaneous fine‐needle aspiration biopsy of HCC is a safe and useful tool that can be used to evaluate the response to proton irradiation. In particular, MIB‐1 LI may provide additional information to assess the therapeutic response of HCC during the early post‐irradiated period.
Lymph node metastasis is an important prognostic factor for rectal carcinoma, but only a few attempts at defining the relationship between lymph node micrometastases and prognosis have been made. The ...purpose of this study was to examine the correlation between the presence of micrometastases and prognosis in patients with rectal carcinoma. Six hundred forty-four lymph nodes were dissected from 42 patients with Dukes' B rectal carcinoma and stained immunohistochemically using a monoclonal antibody, CAM5.2, that binds cytokeratin. Clinicopathological factors, rate of recurrence, and prognosis were compared among patients with and without micrometastases. Micrometastases were detected in 19 lymph nodes (19 of 644 = 2.9%) from 9 patients (9 of 42 = 21.4%). The presence of micrometastases was not related to clinicopathological factors. There were significant differences in recurrence rates (5 of 9 versus 5 of 33, P = 0.02), relapse-free survival rates (P = 0.04), and 10-year survival rates (P = 0.03) between patients with and without micrometastases. Immunohistochemistry successfully identified micrometastatic foci in lymph nodes missed with conventional staining methods. The existence of micrometastases influenced the prognosis in patients with Dukes' B rectal carcinoma.
Aim: Although the anti‐hepatitis C virus (HCV) antibody test has been recommended to the whole Japanese population, most countries have not implemented it. The present study aims to re‐evaluate the ...usefulness of markers examined in the general health examination for the initial screening of HCV carriers.
Methods: Of the overall population, 25 142 individuals (8876 males, 16 266 females) participated in health examinations with HCV tests in 2005, and the most commonly associated markers for HCV‐positive subjects were explored by multivariate analysis, based on blood biochemical, physical, sphygmomanometric and hematological parameters. Thereafter, the efficiencies of the markers were estimated from a total population of 85 013 individuals (29 502 males, 55 511 females) in 2003–2005.
Results: The most significantly associated markers for HCV positivity were aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Optimal limits of ALT and AST by receiver–operator characteristic (ROC) analysis were 24 and 27 IU (male, 33 and 28 IU; female, 22 and 26 IU), respectively. However, one‐quarter of HCV carriers were not found to be positive using the optimal limits of aminotransferases.
Conclusion: The present study confirmed the limitation of serum aminotransferase levels as markers of HCV for primary screening. Therefore, at present, an anti‐HCV antibody test is required for the efficient screening of HCV carriers in all health examinations.
Aim: We have previously reported in mice the hepatic inflammatory in graft versus host response (GVHR) model due to the disparity of major histocompatibility complex class‐II. The regulatory T ...(Treg) cells have been reported to control excessive immune response and prevent immune‐related diseases. This study aimed to investigate the pathogenesis profiles of chronic GVHR progression, focusing on the Treg cells.
Methods: GVHR mice induced by parental spleen CD4+ T cell injection were sacrificed after 0, 2, 4, and 8 weeks (G0, G2, G4, G8). Further, one GVHR group received anti‐IL‐10 antibody in advance and were maintained for 2 weeks. Pathologic profiles of hepatic infiltrating inflammatory cells were evaluated by haematoxylin and eosin and immunohistochemistry staining with surface markers including Treg cell markers.
Results: Remarkable hepatic inflammatory in G2 significantly and gradually improved over time up to G8. In immunohistochemical staining, the increased IL‐10 receptor β+ Tr1 cells in G2 were maintained through to G8; although other inflammatory cells decreased from G2 to G8. By contrast, in the anti‐IL‐10 antibody received‐GVHR mice, the Tr1 cells were not detectable with significant inflammatory aggravation, while FoxP3+ Treg cells significantly enhanced.
Conclusions: These findings in the GVHR mice suggest that the expression and activity of Treg cells, especially the Tr1 cells, might be key factors for pathologic alteration in immune‐related liver disease.
The inhabitants living in a specific region of Kizaki area in Kamisu-town, Ibaraki Prefecture exhibited uncommon clinical central nervous system symptoms. A graphite furnace atomic absorption ...spectrophotometer detected markedly elevated concentration of arsenic (4.5 ppm) in their drinking well water. Further investigation using HPLC, GC/MS and HPLC/ICP/MS demonstrated that the structures of the arsenic were bis(diphenylarsine)oxide (BDPAO), diphenylarsinic acid (DPAA) and phenylarsonic acid (PAA), compounds that can be derived from the chemical warfare agents, diphenylchloroarsine (DA) and diphenylcyanoarsine (DC). The predominant form of the arsenic compound in the well water was DPAA (maximum 15 ppm), so that it was calculated that the inhabitants ingested 11-30 mg of DPAA daily. This is the first report of inhabitants that were injured by drinking well water contaminated with organic arsenic compounds that were likely derived from chemical weapons.
Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in ...formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.
Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X ...receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 ± 6.2 nmol/g liver). Cholesterol 7α-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor α (LXRα) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4α (HNF4α), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4α target genes, CYP7A1, 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase, CYP27A1, and NTCP.
In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4α caused by reduced bile acids in CTX liver.
PDF
