Precise evaluation of liver histology by computerized morphometry shows that steatosis influences liver stiffness measured by transient elastography in chronic hepatitis C.

BACKGROUND: Liver stiffness evaluation (LSE) by Fibroscan is now widely used to assess liver fibrosis in chronic hepatitis C. Liver steatosis is a common lesion in chronic hepatitis C as in other chronic liver diseases, but its influence on LSE remains unclear. We aimed to precisely determine the influence of steatosis on LSE by using quantitative and precise morphometric measurements of liver histology. METHODS: 650 patients with chronic hepatitis C, liver biopsy, and LSE were included. Liver specimens were evaluated by optical analysis (Metavir F and A, steatosis grading) and by computerized morphometry to determine the area (%, reflecting quantity) and fractal dimension (FD, reflecting architecture) of liver fibrosis and steatosis. RESULTS: The relationships between LSE and liver histology were better described using morphometry. LSE median was independently linked to fibrosis (area or FD), steatosis (area or FD), activity (serum AST), and IQR/LSE median. Steatosis area ≥4.0 % induced a 50 % increase in LSE result in patients with fibrosis area <9 %. In patients with IQR/LSE median ≤0.30, the rate of F0/1 patients misclassified as F ≥ 2 by Fibroscan was, respectively for steatosis area <4.0 and ≥4.0 %: 12.6 vs 32.4 % (p = 0.003). Steatosis level did not influence LSE median when fibrosis area was ≥9 %, and consequently did not increase the rate of F ≤ 3 patients misclassified as cirrhotic. CONCLUSION: A precise evaluation of liver histology by computerized morphometry shows that liver stiffness measured by Fibroscan is linked to liver fibrosis, activity, and also steatosis. High level of steatosis induces misevaluation of liver fibrosis by Fibroscan.

Glucocorticoids reduce alveolar and trabecular bone in mice.

UNLABELLED: Glucocorticoid (GC) treatment is the main cause of secondary osteoporosis. There are some controversies about the relationships between alveolar bone loss and bone loss at the appendicular and axial skeleton. OBJECTIVE: To assess, in parallel, the effects of GCs on alveolar bone and on the tibia in a mice model. METHODS: Five-month-old male Swiss-Webster mice were randomized into two groups. Pellets releasing 5 mg/kg/day of prednisolone or control pellets were subcutaneously implanted for 28 days. After euthanasia, the right tibia and the right hemimandible of each mouse were analyzed by histomorphometry and microcomputed tomography. Alveolar bone consists of a thin slab between the incisor and the molar roots connected with the alveolar processes. A 2D-frontal section was done through the pulp chamber of the first molar and was used to measure the thickness of the alveolar bone slab. A 2D-sagittal section was done through the pulp chamber of the three molars and was used to measure bone volume in the alveolar processes. RESULTS: At day 28, thickness and bone volume of alveolar bone were significantly decreased in the GC group (P<0.05). At the tibia, GCs decreased bone formation with a reduced mineral apposition rate and bone formation rate and a significant decrease in BV/TV and Tb.Th (P<0.05). CONCLUSION: Although the amount of alveolar bone is very low in the mouse, this study shows that GCs can induce an alveolar bone loss in long-term treated animals.

Determination of reliability criteria for liver stiffness evaluation by transient elastography.

UNLABELLED: Liver stiffness evaluation (LSE) is usually considered as reliable when it fulfills all the following criteria: ≥10 valid measurements, ≥60% success rate, and interquartile range / median ratio (IQR/M) ≤0.30. However, such reliable LSE have never been shown to be more accurate than unreliable LSE. Thus, we aimed to evaluate the relevance of the usual definition for LSE reliability, and to improve reliability by using diagnostic accuracy as a primary outcome in a large population. 1,165 patients with chronic liver disease from 19 French centers were included. All patients had liver biopsy and LSE. 75.7% of LSE were reliable according to the usual definition. However, these reliable LSE were not significantly more accurate than unreliable LSE with, respectively: 85.8% versus 81.5% well-classified patients for the diagnosis of cirrhosis (P = 0.082). In multivariate analyses with different diagnostic targets, LSE median and IQR/M were independent predictors of fibrosis staging, with no significant influence of ≥10 valid measurements or LSE success rate. These two reliability criteria determined three LSE groups: "very reliable" (IQR/M ≤0.10), "reliable" (0.10< IQR/M ≤0.30, or IQR/M >0.30 with LSE median <7.1 kPa), and "poorly reliable" (IQR/M >0.30 with LSE median ≥7.1 kPa). The rates of well-classified patients for the diagnosis of cirrhosis were, respectively: 90.4%, 85.8%, and 69.5% (P < 10(-3) ). According to these new reliability criteria, 9.1% of LSE were poorly reliable (versus 24.3% unreliable LSE with the usual definition, P < 10(-3) ), 74.3% were reliable, and 16.6% were very reliable. CONCLUSION: The usual definition for LSE reliability is not relevant. LSE reliability depends on IQR/M according to liver stiffness median level, defining thus three reliability categories: very reliable, reliable, and poorly reliable LSE. (HEPATOLOGY 2013).

Comparison of eight diagnostic algorithms for liver fibrosis in hepatitis C: new algorithms are more precise and entirely noninvasive.

UNLABELLED: The sequential algorithm for fibrosis evaluation (SAFE) and the Bordeaux algorithm (BA), which cross-check FibroTest with the aspartate aminotransferase-to-platelet ratio index (APRI) or FibroScan, are very accurate but provide only a binary diagnosis of significant fibrosis (SAFE or BA for Metavir F ≥ 2) or cirrhosis (SAFE or BA for F4). Therefore, in clinical practice, physicians have to apply the algorithm for F ≥ 2, and then, when needed, the algorithm for F4 ("successive algorithms"). We aimed to evaluate successive SAFE, successive BA, and a new, noninvasive, detailed classification of fibrosis. The study included 1785 patients with chronic hepatitis C, liver biopsy, blood fibrosis tests, and FibroScan (the latter in 729 patients). The most accurate synchronous combination of FibroScan with a blood test (FibroMeter) provided a new detailed (six classes) classification (FM+FS). Successive SAFE had a significantly (P < 10(-3) ) lower diagnostic accuracy (87.3%) than individual SAFE for F ≥ 2 (94.6%) or SAFE for F4 (89.5%), and required significantly more biopsies (70.8% versus 64.0% or 6.4%, respectively, P < 10(-3) ). Similarly, successive BA had significantly (P ≤ 10(-3) ) lower diagnostic accuracy (84.7%) than individual BA for F ≥ 2 (88.3%) or BA for F4 (94.2%), and required significantly more biopsies (49.8% versus 34.6% or 24.6%, respectively, P < 10(-3) ). The diagnostic accuracy of the FM+FS classification (86.7%) was not significantly different from those of successive SAFE or BA. However, this new classification required no biopsy. CONCLUSION: SAFE and BA for significant fibrosis or cirrhosis are very accurate. However, their successive use induces a significant decrease in diagnostic accuracy and a significant increase in required liver biopsy. A new fibrosis classification that synchronously combines two fibrosis tests was as accurate as successive SAFE or BA, while providing an entirely noninvasive (0% liver biopsy) and more precise (six versus two or three fibrosis classes) fibrosis diagnosis.

Comparison of accuracy of fibrosis degree classifications by liver biopsy and non-invasive tests in chronic hepatitis C.

BACKGROUND: Non-invasive tests have been constructed and evaluated mainly for binary diagnoses such as significant fibrosis. Recently, detailed fibrosis classifications for several non-invasive tests have been developed, but their accuracy has not been thoroughly evaluated in comparison to liver biopsy, especially in clinical practice and for Fibroscan. Therefore, the main aim of the present study was to evaluate the accuracy of detailed fibrosis classifications available for non-invasive tests and liver biopsy. The secondary aim was to validate these accuracies in independent populations. METHODS: Four HCV populations provided 2,068 patients with liver biopsy, four different pathologist skill-levels and non-invasive tests. Results were expressed as percentages of correctly classified patients. RESULTS: In population #1 including 205 patients and comparing liver biopsy (reference: consensus reading by two experts) and blood tests, Metavir fibrosis (FM) stage accuracy was 64.4% in local pathologists vs. 82.2% (p < 10-3) in single expert pathologist. Significant discrepancy (≥ 2FM vs reference histological result) rates were: Fibrotest: 17.2%, FibroMeter2G: 5.6%, local pathologists: 4.9%, FibroMeter3G: 0.5%, expert pathologist: 0% (p < 10-3). In population #2 including 1,056 patients and comparing blood tests, the discrepancy scores, taking into account the error magnitude, of detailed fibrosis classification were significantly different between FibroMeter2G (0.30 ± 0.55) and FibroMeter3G (0.14 ± 0.37, p < 10-3) or Fibrotest (0.84 ± 0.80, p < 10-3). In population #3 (and #4) including 458 (359) patients and comparing blood tests and Fibroscan, accuracies of detailed fibrosis classification were, respectively: Fibrotest: 42.5% (33.5%), Fibroscan: 64.9% (50.7%), FibroMeter2G: 68.7% (68.2%), FibroMeter3G: 77.1% (83.4%), p < 10-3 (p < 10-3). Significant discrepancy (≥ 2 FM) rates were, respectively: Fibrotest: 21.3% (22.2%), Fibroscan: 12.9% (12.3%), FibroMeter2G: 5.7% (6.0%), FibroMeter3G: 0.9% (0.9%), p < 10-3 (p < 10-3). CONCLUSIONS: The accuracy in detailed fibrosis classification of the best-performing blood test outperforms liver biopsy read by a local pathologist, i.e., in clinical practice; however, the classification precision is apparently lesser. This detailed classification accuracy is much lower than that of significant fibrosis with Fibroscan and even Fibrotest but higher with FibroMeter3G. FibroMeter classification accuracy was significantly higher than those of other non-invasive tests. Finally, for hepatitis C evaluation in clinical practice, fibrosis degree can be evaluated using an accurate blood test.

Steatosis degree, measured by morphometry, is linked to other liver lesions and metabolic syndrome components in patients with NAFLD.

BACKGROUND AND AIM: We carried out morphometric measurements of steatosis to evaluate relationships between steatosis degree and other liver lesions or metabolic syndrome components in nonalcoholic fatty liver disease (NAFLD). PATIENTS AND METHODS: We developed an algorithm to measure steatosis area. Two hundred and fourteen patients with NAFLD were included in derivation (10) and validation (204) groups. Controls consisted of patients who were steatosis-free (12), patients with chronic hepatitis C (188), and patients with alcoholic chronic liver disease (94). RESULTS: Accuracy of steatosis area was considered as good or very good in at least 72% of cases by three pathologists. Steatosis areas were as follows: NAFLD = 10.3 ± 9.7%, virus = 2.4 ± 3.1%, alcohol = 7.8 ± 8.2% (P<0.0001). Steatosis area was closely related to steatosis grades in NAFLD (P<0.0001 for linear trend). Steatosis area increased from the fibrosis stage F0 to the fibrosis state F2, then decreased in the stages F3 and F4 (cirrhosis) (P<0.0001 for quadratic trend). Fibrosis was present in an average steatosis area of approximately 4% (defining significant steatosis) and in nonalcoholic steatohepatitis by approximately 8% (defining severe steatosis). Steatosis and fibrosis area increased symmetrically until approximately 10%, then steatosis area decreased to null as average fibrosis area reached 32%. Average fasting glycemia (approximately 92 mg/dl) or triglycerides and BMI plateaued before a steatosis area of approximately 4%, then increased thereafter. Significant steatosis was present in 61.3% of NAFLD versus 20.2% of viral hepatitis (P<0.0001) and in 58.7% of alcoholic liver diseases (P=0.674). CONCLUSIONS: The average threshold of steatosis area is 4% for the development of fibrosis or metabolic syndrome components and 8% for nonalcoholic steatohepatitis. Steatosis area may contribute to defining the normal range and clinical course of metabolic components.

A new combination of blood test and fibroscan for accurate non-invasive diagnosis of liver fibrosis stages in chronic hepatitis C.

OBJECTIVES: Precise evaluation of the level of liver fibrosis is recommended in patients with chronic hepatitis C (CHC). Blood fibrosis tests and Fibroscan are now widely used for the non-invasive diagnosis of liver fibrosis. Detailed fibrosis stage classifications have been developed to provide an estimation of the liver fibrosis stage from the results of these non-invasive tests. Our aim was to develop a new and more accurate fibrosis stage classification by using new scores combining non-invasive fibrosis tests. METHODS: In all, 729 patients with CHC (exploratory set: 349; validation set: 380) had liver biopsy for Metavir fibrosis (F) staging, and 6 fibrosis tests: Fibroscan, Fibrotest, FibroMeter, Hepascore, FIB-4, APRI. RESULTS: Exploratory set: Fibroscan and FibroMeter were the independent predictors of different diagnostic targets of liver fibrosis. New fibrosis indexes combining FibroMeter and Fibroscan were thus developed for the diagnosis of clinically significant fibrosis (CSF-index) or severe fibrosis (SF-index). The association of CSF- and SF-indexes provided a new fibrosis stage classification (CSF/SF classification): F0/1, F1/2, F2 ± 1, F2/3, F3 ± 1, F4. Validation set: CSF/SF classification had a high diagnostic accuracy (85.8% well-classified patients), significantly higher than the diagnostic accuracies of FibroMeter (69.7%, P<0.001), Fibroscan (63.3%, P<0.001), or Fibrotest (43.9%, P<0.001) classifications. CONCLUSIONS: The association of new fibrosis indexes combining FibroMeter and Fibroscan provides a new fibrosis stage classification. This classification is significantly more accurate than Fibrotest, FibroMeter, or Fibroscan classifications, and improves the accuracy of the non-invasive diagnosis of liver fibrosis stages to 86% without any liver biopsy.

Human serum albumin improves endothelial dysfunction and survival during experimental endotoxemia: concentration-dependent properties.

OBJECTIVES: The mechanisms by which human serum albumin might protect against sepsis-induced organ dysfunction and improve survival are not elucidated. The present study was designed to assess the effects of two concentrations of human serum albumin on endotoxin-induced mortality as well as on endothelial and organ dysfunctions in both mouse and cell models. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratories. SUBJECTS: Swiss mice (n = 10-15/group) were injected with either lipopolysaccharide or vehicle. Four and 12 hrs later, mice were infused or not with human serum albumin HSA (4% or 20%, 10 mL/kg) or normal saline (0.9% NaCl, 30 mL/kg). Human uterine vein endothelial cells were exposed to both lipopolysaccharide and tumor necrosis factor-α during 8 hrs in the presence or absence of human serum albumin (4% or 20%). MEASUREMENTS AND MAIN RESULTS: Mice survival, reactivity of mesenteric arteries, and Western blot protein analysis were assessed. Circulating endothelin-1, gluthatione, gluthatione disulfide, and creatinine plasma levels were measured. Nitric oxide production, oxidative, and nitrosative stresses were also measured in situ in endothelial cells. Human serum albumin 4%, but not human serum albumin 20% or normal saline solution, improved survival time of endotoxemic mice. Furthermore, human serum albumin 4% activated endothelial nitric oxide synthase and restored lipopolysaccharide-impaired flow-dependent endothelial dilation in mesenteric arteries. This was associated with a downregulation of nuclear factor κB and an upregulation of nuclear respiratory factor-2 and heme oxygenase-1. Human serum albumin 4% reduced lipopolysaccharide-induced renal dysfunction, enhanced endothelin-1 production and glutathione plasmatic levels, whereas human serum albumin 20% increased gluthatione disulfide. Furthermore, human serum albumin 4% but not 20% blunted lipopolysaccharide-tumor necrosis factor-α-induced oxidative and nitrosative stresses in endothelial cells and increased their gluthatione levels. CONCLUSIONS: The present data confirm a protective effect of 4% human serum albumin treatment both on mice survival and endothelial dysfunction by inhibiting inflammatory and oxidative stress pathways induced by endotoxins. Conversely, higher concentrations of human serum albumin were detrimental suggesting a dose-dependent effect.

NT-ProBNP levels, water and sodium homeostasis in healthy men: effects of 7 days of dry immersion.

Immersion is a useful tool for studying fluid-volume homeostasis. Natriuretic peptides play a vital role in renal, humoral, and cardiovascular regulation under changing environmental conditions. We hypothesized that dry immersion would rapidly induce a new steady state for water and sodium metabolism, and that serum NT-proBNP levels, a proxy measure for brain natriuretic peptide (BNP), would decrease during long-term dry immersion and increase during recovery. Eight healthy young men were studied before, during, and after 7 days of dry immersion. Body weight, water balance, and plasma volume changes were evaluated. Plasma and serum samples were analyzed for active renin, NT-proBNP, aldosterone, electrolytes, osmolality, total protein, and creatinine. Urine samples were analyzed to determine levels of electrolytes, osmolality, creatinine, and free cortisol. A stand test was performed before and after dry immersion to evaluate cardiovascular deconditioning. Long-term dry immersion induced acute changes in water and sodium homeostasis on day 1, followed by a new steady state. Plasma volume decreased significantly during dry immersion. The serum levels of NT-proBNP increased significantly in recovery (10 ± 3 ng/L before dry immersion vs. 26 ± 5 ng/L on the fourth recovery day). Heart rate in the standing position was significantly greater after immersion. Results suggest that chronic dry immersion rapidly induced a new level of water-electrolyte homeostasis. The increase in NT-proBNP levels during the recovery period may be related to greater cardiac work and might reflect the degree of cardiovascular deconditioning.

Assessment of new hyaluronic acid assays and their impact on FibroMeter scores.

BACKGROUND: We compared three hyaluronic acid (HA) assays and analyzed the impact of their variations on FibroMeter scores. METHODS: In a test group of 165 patients, HA levels were assessed with the commonly used ELISA assay from Corgenix, a new ELISA assay from Teco and an immunoturbidimetry assay from Wako, this latter tested across three different instruments. Five different FibroMeter scores were calculated. RESULTS: Correlation across the three assays (r(s) between 0.969 and 0.995) was very good. Means of differences (d) were lower when the immunoturbidimetry assay was compared on different instruments: d between -3.4 and 2.0 µg/L. However, a higher value for HA measurement was observed with Corgenix assay, compared to the other two assays (Teco and Wako): d between 27.1 and 36.4 µg/L. The assessment also demonstrated that HA variations had very little impact on FibroMeter scores: 0.0117 for virus and 0.0416 for alcoholic fibrosis scores, and between 0.58 and 1.71 for the area of fibrosis (expressed in percentage). CONCLUSIONS: The two new assays found lower values of HA, as compared to the Corgenix assay. However, these differences had very little impact on FibroMeter scores and had no impact on clinical evaluation of liver fibrosis.

Optimization and robustness of blood tests for liver fibrosis and cirrhosis.

OBJECTIVES: To optimize the performance and feasibility of fibrosis blood tests and evaluate their robustness. DESIGN AND METHODS: The derivation population included 1056 HCV patients with liver biopsy and blood markers. Validation populations included 984 patients with various viral hepatitis causes, and Fibroscan and/or liver biopsy and/or blood markers. RESULTS: The bootstrap method validated the markers of the original FibroMeter(2G), but not those of Fibrotest and Hepascore, and provided a hyaluronate-free FibroMeter(3G). AUROCs for significant fibrosis were: FibroMeter(2G): 0.853 vs. FibroMeter(3G): 0.851, p=0.489. Compared to FibroMeter(2G), FibroMeter(3G) had a significantly higher patient rate with predictive values ≥90% for significant fibrosis. Accuracy for fibrosis stage classification was: Fibrotest: 37.9%, FibroMeter(2G): 74.9%, and FibroMeter(3G): 86.9% (p<10(-3)). CONCLUSION: The bootstrap method validated FibroMeter(2G) and provided a cheaper and more feasible hyaluronate-free FibroMeter(3G) with comparable performance. Compared to binary diagnosis, fibrosis stage classification increased discrimination, with an increased accuracy to 87% for FibroMeter(3G).

Diagnosis of different liver fibrosis characteristics by blood tests in non-alcoholic fatty liver disease.

AIMS: Our aim was to develop an accurate, non-invasive, blood-test-based method for identifying the main characteristics of liver fibrosis in non-alcoholic fatty liver disease (NAFLD). METHODS: Fibrosis was staged according to NASH-CRN and Metavir systems in 226 patients with NAFLD. A fully automated algorithm measured the fractal dimension (FD) and the area of fibrosis (AOF). Independent predictors of diagnostic targets were determined using bootstrap methods. RESULTS: (i) Development. Significant fibrosis defined by NASH-CRN F ≥2 was diagnosed by weight, glycaemia, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and prothrombin index [area under the receiver operating characteristic (AUROC)=0.867]; significant fibrosis defined by Metavir F ≥2 was diagnosed by weight, age, glycaemia, AST, ALT, ferritin and platelets (FibroMeter AUROC=0.941, P<0.005). AOF was estimated by the combination of hyaluronic acid, glycaemia, AST, ALT, platelets and prothrombin index ((a) R(2) =0.530), while FD was estimated by hyaluronic acid, glycaemia, AST/ALT, weight and platelets ((a) R(2) =0.529). (ii) Evaluation. Although NASH-CRN was a better system for fibrosis staging, Metavir staging was a better reference for blood test. Thus, the patient rate with predictive values ≥90% by tests was 97.3% with Metavir reference vs. 66.5% with NASH-CRN reference (P<10(-3)). FibroMeter showed a significantly higher AUROC than the NAFLD fibrosis score for significant fibrosis, but not for severe fibrosis or cirrhosis, with both staging systems. Relationships between fibrosis lesions were well reflected by blood tests, e.g., the correlation between histological area and FD of fibrosis (r(s) =0.971, P<10(-3)) was well reflected by the relationship between respective blood tests (r(s) =0.852, P<10(-3)). CONCLUSIONS: Different characteristics of fibrosis in NAFLD can be diagnosed and quantified by blood tests with excellent accuracy.

Acoustic radiation force impulse: a new ultrasonographic technology for the widespread noninvasive diagnosis of liver fibrosis.

BACKGROUND/AIMS: As a module of a standard ultrasound imaging device, acoustic radiation force impulse (ARFI) is a new technology for liver stiffness evaluation (LSE). We aimed to evaluate accuracy, feasibility, reproducibility, and training effect of ARFI for liver fibrosis evaluation. METHODS: One hundred and one patients with chronic liver disease had LSE by Fibroscan and ARFI. LSE by ARFI was performed in the two liver lobes by two operators: an expert and a novice. Correlation and agreement were evaluated by the Pearson (Rp) and intraclass (Ric) correlation coefficients. The independent reference for liver fibrosis was fibrosis blood tests. RESULTS: ARFI results, ranging from 0.7 to 4.6 m/s, were well correlated with Fibroscan results (Rp=0.76). Fibroscan had a significantly higher area under the receiver operating characteristic curve (AUROC) than ARFI for the perprotocol diagnosis of significant fibrosis: 0.890+/-0.034 versus 0.795+/-0.047 (P=0.04). However, LSE failure occurred in zero patients using ARFI versus six patients using Fibroscan (P=0.03). Thus, on an intention-to-diagnose basis, Fibroscan and ARFI AUROCs for the diagnosis of significant fibrosis were not different: 0.791+/-0.049 versus 0.793+/-0.046 (P=0.98). Interobserver agreement was very good (Ric=0.84) and excellent for ARFI interquartile range (IQR)< or =0.30 (Ric=0.91). Indeed, agreement was independently predicted only by ARFI IQR, but not by LSE result as earlier observed for Fibroscan. ARFI AUROC was 0.876+/-0.057 in patients with ARFI IQR ratio< or =0.30, and Fibroscan AUROC was 0.912+/-0.034 in patients with Fibroscan IQR ratio less than 0.21 (P=0.59). Intersite ARFI agreement between the two liver lobes was fair (Ric=0.60). There was no training effect for LSE by ARFI. CONCLUSION: ARFI is highly feasible and reproducible, and provides diagnostic accuracy similar to Fibroscan. This new device seems noteworthy for the widespread noninvasive diagnosis of liver fibrosis.

The combination of a blood test and Fibroscan improves the non-invasive diagnosis of liver fibrosis.

BACKGROUND AND AIMS: Blood tests and liver stiffness evaluation (LSE) by ultrasonographic elastometry are accurate tools for diagnosing liver fibrosis. We evaluated whether their synchronous combination in new scores could improve the diagnostic accuracy and reduce liver biopsy requirement in algorithm. METHODS: Three hundred and ninety patients with chronic liver disease of miscellaneous causes were included. Five blood fibrosis tests were evaluated: APRI, FIB-4, Hepascore, Fibrotest and FibroMeter. The reference was fibrosis Metavir staging. RESULTS: Diagnosis of significant fibrosis (Metavir F>or=2). The most accurate synchronous combination was FibroMeter+LSE, which provided a significantly higher area under the receiver operating characteristic curve (0.892) than LSE alone (0.867, P=0.011) or Fibrometer (0.834, P<10(-3)). An algorithm using the FibroMeter+LSE combination and then a liver biopsy in indeterminate cases had 91.9% diagnostic accuracy and required significantly fewer biopsies (20.2%) than previously published Bordeaux algorithm (28.6%, P=0.02) or sequential algorithm for fibrosis evaluation (SAFE) (55.7%, P<10(-3)). The Angers algorithm performance was not significantly different between viral hepatitis and other causes. Diagnosis of cirrhosis. The most accurate synchronous combination was LSE+FibroMeter, which provided >or=90% predictive values for cirrhosis in 90.6% of patients vs 87.4% for LSE (P=0.02) and 57.9% for FibroMeter (P<10(-3)). An algorithm including the LSE+FibroMeter combination, and then a liver biopsy in indeterminate cases, had a significantly higher diagnostic accuracy than the SAFE algorithm (91.0 vs 79.8%, P<10(-3)), and required significantly fewer biopsies than the Bordeaux algorithm (9.3 vs 25.3%, P<10(-3)). CONCLUSION: The synchronous combination of a blood test plus LSE improves the accuracy of the non-invasive diagnosis of liver fibrosis and, consequently, markedly decreases the biopsy requirement in the diagnostic algorithm, notably to <10% in cirrhosis diagnosis.

Effect of alpha tocopherol acetate in Walker 256/B cells-induced oxidative damage in a rat model of breast cancer skeletal metastases.

The pathophysiological changes and the oxidative-antioxidative status were evaluated in the bone microenvironment of rat inoculated with Walker 256/B mammary gland carcinoma cells, and used alpha-tocopherol acetate (ATA) as a countermeasure. Walker 256/B cells were injected into the right femora of aged male rats. Animals were randomized into three groups: 12 rats were injected with saline (control group); 14 rats were injected with Walker 256/B cells (5x10(4)) in the medullar cavity (W256 group); 14 rats were inoculated with Walker 256/B cells and treated with ATA (45mg/kg BW) (W256+ATA group). After 20 days, rats were euthanized and the femurs were radiographed. Micro architectural parameters were measured by microcomputed tomography and histology. Serum, bone and bone marrow were evaluated for oxidative damage. In parallel, cell cultures were done in the presence of ATA and ROS were measured by fluorescence; apoptotic cells were determined in parallel. W256 groups had osteolytic damages with marked resorption of cortical and trabecular bone. W256+ATA animals presented marked osteosclerotic areas associated with tumor necrosis areas inside the bone cavity. Levels of lipid peroxidation and protein oxidation were found to increase in W256 rats; a significant reduction in SOD and GSH-p activities was also observed. W256+ATA group had significantly reduced oxidative damage, but not reversed back to the control levels. The present study shows that Walker 256/B cells induce skeletal metastases associated with oxidative damage in the bone microenvironment. ATA reduced the oxidative stress damage, enhanced osteosclerosis and tumor cell apoptosis both in vitro and in vivo.

Genetic variant in HK1 is associated with a proanemic state and A1C but not other glycemic control-related traits.

OBJECTIVE: A1C is widely considered the gold standard for monitoring effective blood glucose levels. Recently, a genome-wide association study reported an association between A1C and rs7072268 within HK1 (encoding hexokinase 1), which catalyzes the first step of glycolysis. HK1 deficiency in erythrocytes (red blood cells [RBCs]) causes severe nonspherocytic hemolytic anemia in both humans and mice. RESEARCH DESIGN AND METHODS: The contribution of rs7072268 to A1C and the RBC-related traits was assessed in 6,953 nondiabetic European participants. We additionally analyzed the association with hematologic traits in 5,229 nondiabetic European individuals (in whom A1C was not measured) and 1,924 diabetic patients. Glucose control-related markers other than A1C were analyzed in 18,694 nondiabetic European individuals. A type 2 diabetes case-control study included 7,447 French diabetic patients. RESULTS: Our study confirms a strong association between the rs7072268-T allele and increased A1C (beta = 0.029%; P = 2.22 x 10(-7)). Surprisingly, despite adequate study power, rs7072268 showed no association with any other markers of glucose control (fasting- and 2-h post-OGTT-related parameters, n = 18,694). In contrast, rs7072268-T allele decreases hemoglobin levels (n = 13,416; beta = -0.054 g/dl; P = 3.74 x 10(-6)) and hematocrit (n = 11,492; beta = -0.13%; P = 2.26 x 10(-4)), suggesting a proanemic effect. The T allele also increases risk for anemia (836 cases; odds ratio 1.13; P = 0.018). CONCLUSIONS: HK1 variation, although strongly associated with A1C, does not seem to be involved in blood glucose control. Since HK1 rs7072268 is associated with reduced hemoglobin levels and favors anemia, we propose that HK1 may influence A1C levels through its anemic effect or its effect on glucose metabolism in RBCs. These findings may have implications for type 2 diabetes diagnosis and clinical management because anemia is a frequent complication of the diabetes state.

Fatty liver and insulin resistance in obese Zucker rats: no role for mitochondrial dysfunction.

The relationship between insulin resistance and mitochondrial function is of increasing interest. Studies looking for such interactions are usually made in muscle and only a few studies have been done in liver, which is known to be a crucial partner in whole body insulin action. Recent studies have revealed a similar mechanism to that of muscle for fat-induced insulin resistance in liver. However, the exact mechanism of lipid metabolites accumulation in liver leading to insulin resistance is far from being elucidated. One of the hypothetical mechanisms for liver steatosis development is an impairment of mitochondrial function. We examined mitochondrial function in fatty liver and insulin resistance state using isolated mitochondria from obese Zucker rats. We determined the relationship between ATP synthesis and oxygen consumption as well as the relationship between mitochondrial membrane potential and oxygen consumption. In order to evaluate the quantity of mitochondria and the oxidative capacity we measured citrate synthase and cytochrome c oxidase activities. Results showed that despite significant fatty liver and hyperinsulinemia, isolated liver mitochondria from obese Zucker rats display no difference in oxygen consumption, ATP synthesis, and membrane potential compared with lean Zucker rats. There was no difference in citrate synthase and cytochrome c oxidase activities between obese and lean Zucker rats in isolated mitochondria as well as in liver homogenate, indicating a similar relative amount of hepatic mitochondria and a similar oxidative capacity. Adiponectin, which is involved in bioenergetic homeostasis, was increased two-fold in obese Zucker rats despite insulin resistance. In conclusion, isolated liver mitochondria from lean and obese insulin-resistant Zucker rats showed strictly the same mitochondrial function. It remains to be elucidated whether adiponectin increase is involved in these results.

Prognostic value of the insertion/deletion polymorphism of the ACE gene in type 2 diabetic subjects: results from the Non-insulin-dependent Diabetes, Hypertension, Microalbuminuria or Proteinuria, Cardiovascular Events, and Ramipril (DIABHYCAR), Diabete de type 2, Nephropathie et Genetique (DIAB2NEPHROGENE), and Survie, Diabete de type 2 et Genetique (SURDIAGENE) studies.

OBJECTIVE: We tested whether determination of the ACE insertion/deletion polymorphism is useful for renal and cardiovascular prognoses of type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: The French participants (3,126 of 4,912) in the Non-Insulin-Dependent Diabetes, Hypertension, Microalbuminuria or Proteinuria, Cardiovascular Events, and Ramipril (DIABHYCAR) trial were studied for their prognosis over 4 years according to their ACE insertion/deletion polymorphism. We used two cohorts of French type 2 diabetic patients for replication: a 3-year follow-up study (n = 917; Survie, Diabete de type 2 et Genetique [SURDIAGENE] study) and a case-control study on diabetic nephropathy (n = 1,277; Diabete de type 2, Nephropathie et Genetique [DIAB2NEPHROGENE] study). We investigated the effect of the insertion/deletion polymorphism on the primary outcome in the DIABHYCAR trial (defined as the first of the following events to occur: cardiovascular death, nonfatal myocardial infarction, stroke, heart failure leading to hospital admission, or end-stage renal failure) and its components. RESULTS: In DIABHYCAR, the primary outcome and most of its components were not affected by the ACE insertion/deletion genotype. Only renal outcome was favored by the I allele (P = 0.03). The risk of myocardial infarction was not affected by ACE genotype, but the probability of fatal outcome increased with the number of D alleles (P < 0.03). In SURDIAGENE, the association between the ACE I allele and renal outcome was not replicated. In DIAB2NEPHROGENE, no association was found with nephropathy. CONCLUSIONS: We were not able to demonstrate the manifest usefulness of the ACE insertion/deletion polymorphism for the prognosis of type 2 diabetic subjects.

Hemodynamic effects of the early and long-term administration of propranolol in rats with intrahepatic portal hypertension.

Background and aims The aims of this study were to evaluate a preventive effect on collateral venous circulation of long-term administration of propranolol in intrahepatic portal hypertensive rats. Methods Eighty-six Sprague-Dawley rats were allocated to two models of hepatic fibrosis, bile duct-ligated (BDL) induced and carbon tetrachloride (CCl(4)) induced. Each model was divided into two groups: one receiving placebo and the other propranolol (75 mg kg(-1) d(-1)). Mean arterial pressure (MAP), heart rate (HR), portal pressure (PP), cardiac index (CI), vascular systemic resistance, and splenorenal shunt blood flow (SRS-BF) were measured in anesthetized rats. Results In the BDL model, no significant hemodynamic changes were observed in the propranolol group compared with the placebo group. In CCl(4)-induced rats, HR (390 +/- 50 vs. 329 +/- 51 beats/min, P = .001), CI (44 +/- 11 vs. 34 +/- 10 ml/min, P = .004), PP (15.4 +/- 3.0 vs. 13.4 +/- 1.9 mmHg, P = .045), and SRS-BF (1.4 +/- 1.1 vs. 1.0 +/- 1.0 ml/min, P = .047) were significantly lower in the propranolol group. Conclusions This study showed that propranolol has a significant hemodynamic effect only in the CCl(4) model and suggested a model-dependent effect of propranolol.

Reproducibility of blood tests of liver fibrosis in clinical practice.

OBJECTIVES: To evaluate the inter-laboratory reproducibility of blood test for liver fibrosis: FibroMeter, Fibrotest, APRI and their composites variables. DESIGN AND METHODS: Four studies, including 147 patients, were performed: study #1 included 2 metachronous blood samples and 2 laboratories; studies #2, #3 and #4 included synchronous samples with assays delayed at day 1 in 12 laboratories, at day 0 in 10 laboratories and at day 0 or 1 in 2 laboratories, respectively. Agreement was evaluated by the intraclass correlation coefficient (r(ic)). RESULTS: In studies #1, #2 and #4, r(ic) for FibroMeter was 0.893, 0.942 and 0.991, respectively. In study #3, the r(ic) were: FibroMeter: 0.963, Fibrotest: 0.984, APRI: 0.949. Large simulated variations in composite variables had a weak impact on FibroMeter. CONCLUSIONS: When blood marker limits are controlled, inter-laboratory agreement of blood tests is excellent in clinical practice conditions. Blood tests are robust against the variability of composite blood variables.