Background and Aim: To evaluate the importance of cholesterol and bile acid concentrations in the intestinal lumen to cholesterol homeostasis, we investigated the effect of cholesterol‐, bile salt‐ ...or cholestyramine‐administration on the regulation of intestinal mRNA related to cholesterol and bile acid metabolism.
Methods: Male Wistar rats fed on standard laboratory chow (AIN‐93) were allocated into four experimental groups: (i) control group; (ii) cholesterol group (CH), which was fed cholesterol in diet (2% w/w) for 14 days; (iii) cholic acid (CA) group, which was fed CA in diet (1% w/w) for 14 days; (iv) cholestyramine (CT) group, which was fed CT in diet (5% w/w) for 14 days. Blood, liver and small intestine were obtained after 14 days. Serum lipids and bile acids were measured by colorimetric assays, and hepatic and intestinal mRNA relating to lipid and bile acid metabolism was studied by reverse transcription–polymerase chain reaction.
Results: Intestinal ABCG8, liver X receptor α, small heterodimer partner (SHP) and sterol regulatory element binding protein 1c (SREBP‐1c) mRNA expressions were markedly increased in the CH group. Intestinal multidrug resistance associated protein (MRP) 2 and MRP3 mRNA expressions were markedly increased in the CA group. In the CT group intestinal MRP2, ABCG5, ABCG8, SHP and SREBP‐1c mRNA expressions were markedly decreased.
Conclusion: The bile acid availability in the intestinal lumen alters the expression of various intestinal mRNA relating to not only bile acid metabolism, but also lipid metabolism. Though the mechanism of the intestinal SHP elevation is unclear, cholesterol feeding may affect the intestinal bile acid metabolism via intestinal SHP expression.
Background & Aims: Multispecific organic anion–transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and ...blood–brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver.
Methods: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA–injected
Xenopus laevis oocytes.
Results: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, ~600 μmol/L; Oatp2, ~30 μmol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1.
Conclusions: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity “backup” system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma.
GASTROENTEROLOGY 1999;117:688-695
The quantification of hepatitis B virus (HBV) DNA is used to monitor antiviral treatment for HBV infection. Recently, an HBV-DNA quantification reagent and assay protocol have been developed for the ...µTASWako g1 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), a fully automated genetic analyzer that uses PCR-capillary electrophoresis. We evaluated the performance of the newly developed µTASWako HBV-DNA assay using standard and clinical samples.
The performance of µTASWako HBV-DNA was evaluated using 3rd World Health Organization International Standard for HBV. Thereafter, we evaluated the correlation between the serum HBV DNA concentrations obtained using the µTASWako HBV-DNA and the Roche Cobas AmpliPrep/COBAS TaqMan HBV test, version 2.0 (CAP/CTM HBV test v.2.0) using 190 serum samples from possible HBV carriers.
The limit of detection of the µTASWako HBV-DNA was 7.1 IU/mL and that of the CAP/CTM HBV test v.2.0 was 14.6 IU/mL. Seventy-six of the 190 samples yielded values between 1.3 - 7.8 log IU/mL from both assays. The correlation between the results of the assays for these samples was good, with a Deming regression equation of y = 0.929x + 0.041, 95% confidence intervals (CI) for the slope and intercept of 0.892 - 1.12 and -0.474 - 0.110, respectively, and a correlation coefficient r = 0.924. In the low concentration range of 1.3 - 4.0 log IU/mL (n = 64), the Deming regression equation was y = 0.893x + 0.126, and the 95% CIs for the slope and intercept were 0.915 - 1.342 and -0.930 - 0.025, respectively, and r = 0.809. There was also a close correlation for HBV DNA genotype C (n = 41), with a Deming regression equation of y = 0.975x - 0.048, 95% CIs of the slope and intercept of 0.872 - 1.183 and -0.591 - 0.188, respectively, and r = 0.950. The correlations of the four HBV DNA level categories (not detected, < LLOQ (< 1.3 log IU/mL), 1.3 - 3.2 log IU/mL, and ≥ 3.3 log IU/mL) between the two assays for the 190 samples was also compared, and the overall concordance rate was 81.6% (155/190), with a κ statistic of 0.73 (standard error, 0.040; 95% CI, 0.647 - 0.803).
The µTASWako HBV-DNA has a performance comparable with that of the CAP/CTM HBV test, v.2.0. Thus, µTASWako HBV-DNA is useful for the monitoring of HBV infection.
Background and Aim: The liver and small intestine play an important role in maintaining cholesterol and bile acid balance within the body. The purpose of this study was to evaluate the effect of ...bile duct ligation (BDL) on the expression of intestinal and hepatic genes that are important for cholesterol and bile acid metabolism.
Methods: Rats were allocated to the BDL group or the sham operation group. Blood, liver and small intestine were obtained after 24, 72 and 168 h from both groups. Serum and hepatic lipids were measured by colorimetric assays and hepatic and intestinal mRNA related to lipid metabolism was studied by reverse transcription–polymerase chain reaction (RT‐PCR).
Results: Hepatic apolipoprotein (Apo) AIV, multidrug resistant protein (Mrp)2, adenosine triphosphate (ATP) binding cassette transporter (Abc)g5 and Abcg8 expression constantly decreased after BDL. Intestinal Apo AIV, Apo CIII, Mrp2, Abcg5 and Abcg8 expression remarkably decreased 24 h after BDL and recovered 72 and 168 h after BDL. Hepatic small heterodimer partner (Shp) expression did not change after BDL. Conversely, intestinal Shp expression remarkably decreased 24 h after BDL (16% of sham operation) and slightly recovered 168 h after BDL (58% of sham operation).
Conclusion: Several intestinal mRNA expressions important for lipid (Apo AIV, Apo CIII, Abcg5 and Abcg8) and bile acid (Mrp2 and Shp) metabolism were decreased in the early phase of obstructive jaundice and the expression of these intestinal mRNA recovered in the late phase of obstructive jaundice.
The secretion of bilirubin conjugates from hepatocytes into bile represents a decisive step in the prevention of hyperbilirubinemia. The bilirubin conjugates, monoglucuronosyl bilirubin (MGB) and ...bisglucuronosyl bilirubin (BGB), were previously suggested to be endogenous substrates for the apical multidrug resistance protein (MRP2), a member of the adenosine triphosphate (ATP)‐binding cassette family of transporters (symbol ABCC2), also termed canalicular multispecific organic anion transporter. We have characterized this ATP‐dependent transport using membrane vesicles from human embryonic kidney (HEK) cells expressing recombinant rat as well as human MRP2. MGB and BGB, 3H‐labeled in the glucuronosyl moiety, were synthesized enzymatically with recombinant UDP‐glucuronosyltransferase 1A1, and stabilized with ascorbate. Rates for ATP‐dependent transport of MGB and BGB (0.5 μmol/L each) by human MRP2 were 183 and 104 pmol × mg protein−1 × min−1, respectively.
Km
values were 0.7 and 0.9 μmol/L for human MRP2, and 0.8 and 0.5 μmol/L for rat MRP2, with MGB and BGB as substrates, respectively. Leukotriene C4
and 17β‐glucuronosyl estradiol, which are both known high‐affinity substrates for human MRP2, inhibited 3HMGB transport with IC50
values of 2.3 and 30 μmol/L, respectively. Cyclosporin A competitively inhibited human and rat MRP2‐mediated transport of 3HMGB, with Ki
values of 21 and 10 μmol/L, respectively. Our results provide direct evidence that recombinant MRP2, cloned from rat as well as human liver, mediates the primary‐active ATP‐dependent transport of the bilirubin conjugates MGB and BGB.
Bilirubin is taken up from blood into hepatocytes by sinosuidal membrane transporters and then excreted into bile through the bile canalicular membrane mainly as bilirubin glucuronides. (1) Mechanism ...of bilirubin uptake into hepatocytes: many organic anions are incorporated into hepatocytes by organic anion transporting polypeptides (rat, oatp1, oatp2, oatp3; human, OATP), liver-specific transporter (rlst/HLST), and/or by organic anion transporters (OAT2, OAT3). Oatp1 and HLST transport bilirubin monoglucuronide. However, a transporter of unconjugated bilirubin in the sinusoidal membrane has not as yet been identified. Unconjugated bilirubin may also go across the hepatocyte sinusoidal membrane by a diffusion process. (2) Intrahepatic transport and conjugation of bilirubin: ligandin carries bilirubin to the endoplasmic reticulum (ER) of hepatocytes. In the ER, bilirubin is conjugated by bilirubin uridine diphosphate (UDP)-glycosyltransferase (bilirubin UGT; UGT1A1) to form mono- and diglucuronides of bilirubin. (3) Transport mechanism of bilirubin glucuronides across the hepatocyte canalicular membrane: at the canalicular membrane, bilirubin glucuronides are excreted into bile by multidrug resistance-associated protein 2 (MRP2), a member of the ATP-binding cassette transporter family. (4) Regurgitation of bilirubin glucuronides into blood: MRP3, which is located in the lateral membrane, transports bilirubin glucuronides into blood under conditions of impaired biliary bilirubin excretion.
Regulation of bilirubin glucuronide transporters during hyperbilirubinemia in hepatic and extrahepatic tissues is not completely clear. In the present study, we evaluated the regulation of the ...bilirubin glucuronide transporters, multidrug resistance-associated proteins (MRP)2 and 3, in rats with obstructive jaundice. Bile duct ligation (BDL) or sham operation was performed in Wistar rats. Liver and kidneys were removed 1, 3, and 5 days after BDL (n = 4, in each group). Serum and urine were collected to measure bilirubin levels just before animal killing. MRP2 And MRP3 mRNA expressions were determined by real-time RT-PCR. Protein expression of MRP2 and MRP3 was determined by Western blotting. Renal MRP2 function was evaluated by para-aminohippurate (PAH) clearance. The effect of conjugated bilirubin, unconjugated bilirubin, human bile, and sulfate-conjugated bile acid on MRP2 gene expression was also evaluated in renal and hepatocyte cell lines. Serum bilirubin and urinary bilirubin excretion increased significantly after BDL. In the liver, the mRNA expression of MRP2 decreased 59, 86, and 82%, and its protein expression decreased 25, 74, and 93% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. In contrast, the liver expression of MRP3 mRNA increased 138, 2,137, and 3,295%, and its protein expression increased 560, 634, and 612% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. On the other hand, in the kidneys, the mRNA expression of MRP2 increased 162, 73, and 21%, and its protein expression increased 387, 558, and 472% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. PAH clearance was significantly increased after BDL. The mRNA expression of MRP2 increased in renal proximal tubular epithelial cells after treatment with conjugated bilirubin, sulfate-conjugated bile acid or human bile. Upregulation of MRP2 in the kidneys and MRP3 in the liver may be a compensatory mechanism to improve bilirubin clearance during obstructive jaundice.
Introduction
The hematology analyzer, Celltac G (Nihon Kohden), designed to improve platelet count (Plt) accuracy, is equipped with new sheath flow control technology. Clinical evaluation of the ...Celltac G was assessed by comparability with XN‐9000 (Sysmex Corporation) and CELL‐DYN Sapphire (Abbott Diagnostics). The accuracy of all three analyzers, which use different measuring principles, was compared with the immunoplatelet reference method (FCM‐Ref).
Methods
Repeatability and within‐laboratory imprecision were assessed using 10 clinical fresh whole blood samples and three control materials with differing levels. Carryover was evaluated using 6 clinical fresh whole blood samples. For method comparison between the three analyzers, 388 samples were used. Plt accuracy among the three analyzers was evaluated using 54 blood samples, including 42 samples with a platelet count less than 50x109/L. The International Council for Standardization in Haematology method for Plt was used as the FCM‐Ref.
Results
The Celltac G showed sufficient performance with regard to imprecision, carryover, and comparability. The Analytical Measurement Interval (AMI) and linearity for all parameters of Plt were validated within 4.6 to 809.1 (×109/L). All hematology analyzers showed some disagreement in Plt when compared with the immunoplatelet reference method.
Conclusion
The Celltac G hematology analyzer is suitable for clinical use. Platelet count evaluation of the three analyzers suggests the need to determine a reportable measurement interval (RMI) in the clinical laboratory for adequate reporting of a Plt from multiple different values.
Background
The automated hematology analyzer Celltac G (Nihon Kohden) was designed to improve leukocyte differential performance. Comparison with analyzers using different leukocyte detection ...principles and differential leukocyte count on wedge film (Wedge‐Diff) shows its clinical utility, and comparison with immunophenotypic leukocyte differential reference method (FCM‐Ref) shows its accuracy performance.
Methods
For method comparison, 598 clinical samples and 46 healthy volunteer samples were selected. The two comparative hematology analyzers (CAAs) used were XN‐9000 (Sysmex) and CELL‐DYN Sapphire (Abbott). The FCM‐Ref provided by the Japanese Society for Laboratory Hematology was selected, and a flow cytometer Navios (Beckman‐Coulter) was used. In manual differential, two kinds of automated slide makers were used: SP‐10 (Sysmex) for wedge technique and SPINNER‐2000 (Lion‐Power) for spinner technique. The spinner technique avoids the issue of Wedge‐Diff smudge cells by removing the risk of breaking cells and non‐uniformity of blood cell distribution on films (Spinner‐Diff).
Results
The Celltac G showed sufficient comparability (r = 0.67–1.00) with the CAAs for each leukocyte differential counting value at 0.00–40.87(109/L), and sufficient comparability (r = 0.73–0.97) with FCM‐Ref for each leukocyte differential percentage at 0.4–78.5. The identification ratio of the FCM‐Ref in CD45‐positive cells was 99.7% (99.4% to 99.8%). Differences were found between FCM‐Ref/Celltac G/XN‐9000/Spinner‐Diff and Wedge‐Diff for monocytes and neutrophils. The appearance ratio of smudge cells on wedge and spinner film was 12.5% and 0.5%.
Conclusion
The Celltac G hematology analyzer's leukocyte differential showed adequate accuracy compared with the CAAs, FCM‐Ref, and two manual methods and was considered suitable for clinical use.
Leukocyte differential in two hematology analyzers and a manual leukocyte differential method on spinner film showed consistency compared with the reference flow cytometry method provided by an immunophenotypic leukocyte differential reference method, whereas in a manual leukocyte differential method on wedge film for percentages of monocytes and neutrophils showed inconsistency.
Abbreviatons: TAA, Test automated analyzer; CAA: Comparative automated analyzer; BA, basophil; EO, eosinophil; LY, lymphocyte; MO, monocyte; NE, neutrophil; 5diff, number of identified normal five‐part leukocyte WBC cell differential events; CD45+, number of CD45‐positive cell events.
New Delhi metallo-β-lactamase (NDM)-producing gram-negative rods, including Acinetobacter species, are a global problem but have rarely been isolated in Japan. To our knowledge, this is the first ...study to isolate an NDM-1-producing Acinetobacter soli strain, KUH106, in Japan. We analyzed this strain using next-generation sequencing to examine the plasmid carrying NDM-1. This plasmid, named pKUH106_NDM1, is 41,135 bp in length and contains genetic contexts with the structure ISAba14-aph(3′)-VI-ISAba125-blaNDM-1ble-MBL. Comparative analysis of the plasmid revealed that it resembled the plasmids of Acinetobacter detected in various countries, such as the A. soli isolate from Taiwan and the Acinetobacter baumannii isolate from a healthcare facility in Osaka Prefecture, Japan. These results suggest that blaNDM-1 may spread via this plasmid in Acinetobacter species. This phenomenon needs to be confirmed through the genetic analysis of A. baumannii and other carbapenem-resistant Acinetobacter species. In particular, blaNDM-1 and other resistance genes must be investigated, and the spread of these genes in the community must be cautioned.
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