“What is abnormal ALT?” Prof Seng Gee Lim Co-Chairman Singapore Hepatitis Conference ALT: Biologic Variability • Intra-individual variation • Diurnal variation: up to 45% • Highest in the afternoon • Lowest at night • Day-to-day variation: 10-30% • Variability other than liver disease • Gender • Age • Exercise: Moderate exercise↓ • BMI- correlation not clearly understood, but may be due to: • ALT isoenzyme made in adipose tissue • NAFLD Dufour DR, et al. Clin Chem. 2000;46:2027-2049. Yang RZ, et al. Genomics. 2002;79: 445-450. Gender and Age Affect Upper Reference Limits for ALT Males Females Dufour DR, et al. Clin Chem. 2000;46:2027-2049. ALT Measurement Limitations •ALT correlated with numerous clinical factors • 1033 blood donors with negative serology Variable Pearson Correlation P Value Age 0.126 .0001 BMI 0.345 .0001 Triglycerides 0.223 .0001 Cholesterol 0.167 .0001 Bilirubin 0.115 .0002 Iron 0.084 .006 Uric Acid 0.355 .0001 GGT 0.549 .0001 Piton A, et al. Hepatology.1998;27:1213-1219. ALT Measurement Limitations • ALT associated with several demographic factors Variable Regression Coefficient P Value Male 0.118 .0001 BMI 0.177 .0001 Age 0.012 .02 Smoker -0.242 .03 Piton A, et al. Hepatology.1998;27:1213-1219. ALT Measurement Limitations • Definition of normal values • Statistical definition • Standard practice to define normal lab values • Middle 95% of healthy volunteers • Influenced by reference population • Abnormal: >97.5th percentile • Biological definition • Based on risk of developing disease/complication • Cholesterol, blood sugar • Abnormal: ALT level associated with disease • Requires standardization of laboratory assays ALT Measurement Limitations: What Type of Variability Exists Between Labs? • In 1997 College of American Pathologists survey, average variation in aminotransferase values between laboratories using the same methods,1 4–9% • Between laboratories using different assays, range was 39–85 U/L for the same specimen • Multi-enzyme control sera (eg, Seraclear-HE) designed as calibrator to be used in different labs2 • Study in Japan demonstrated <10% inter-laboratory variation for ALT3 • Majority of Japanese laboratories use same reference method (Japanese Society of Clinical Chemistry method) 1. Dufour DR, et al. Clin Chem. 2000;46:2027-2049; 2. Eto A, et al. Clin Chem. 1995;41:872-880; 3. Tatsumi N, et al. Southeast Asian J Trop Med Public Health. 1999;30(suppl 3):99-104. ALT Measurement Limitations: Conclusions • Intra-individual variability in ALT • Inter-individual variability other than liver disease • Age, gender, BMI • Debate about normal ALT • Statistical approach • Prati (bottom 95%): 30 (M) and 19 (F) U/L • Piton (bottom 95%): 42–66 (M) and 31–44 (F) U/L • Biological approach • Increased liver mortality ? • Problem of laboratory standardization • Best evidence • Prati method most reasonable to define absence of liver disease How good is ALT in diagnosing liver damage? Other Markers Author Hasanjani et al. Int J Clin Pract. 2005; 59:791–794. Myers et al. J Hepatol. 2003; 39:222–230. ter Borg et al. N= Test HBeAg status 91 AST Negative 223 AST GGT actitest Mixed AST Mixed 233 Lancet. 1998; 351:1914–1918. AURO C 0.82 p-value NA 0.005 AST≈ALT <0.0001 predicting activity but NS between tests AST>ALT <0.005 0.82 0.807 ROC curves (ter Borg et al.) ROC curves (Myers et al.) 1.0 1.0 0.8 0.75 0.6 0.4 ALT (AUC=0.757) AST (AUC=0.807) AST, HBV DNA (AUC=0.832) 0.2 0 Sensitivity Sensitivity comparison 0.50 Actitest (0.82 ± 0.04) AST (0.82 ± 0.04) ALT (0.81 ± 0.04) 0.25 0 0 0.2 0.4 0.6 1-specificity 0.8 1.0 0 0.2 5 0.50 0.75 1-specificity 1.0 ALT and Prognosis Baseline ALT and Liver Mortality • n=141,108 (men=94,533, women=46,575) Korean Insurance clients • Baseline variables: demographics, BP, glucose, BMI, smoking, alcohol, self reported chronic illnesses and FHx Baseline ALT (IU/L) Number Death from liver disease (n) RR (95% CI) <20 37,425 45 1 (0.7–1.4) 20-29 36,589 113 2.9 (2.4–3.5) 30-39 11,975 110 9.5 (7.9–11.5) 40-49 4,068 74 19.2 (15.3–24.2) 50-99 3,887 117 30.0 (25.0–36.1) 100 589 42 59.0 (43.4–80.1) Multivariate Analysis: Men •ALT 8.93 (7.49 to 10.64) •Hypertension 1.91 (1.46 to 2.49) •F. Glucose≥7 3.18 (2.29 to 4.42) •Alcohol≥200g/d 3.33 (1.64 to 6.76) •FHx liver disease 2.62 (1.93 to 3.56) Women 27.32 (11.24 to 66.40) 3.63 (0.75 to 17.45) NA NA Kim HC, et al. BMJ. 2004;328:983-988. Is this a good study? • ALT is not a disease, it’s a surrogate marker • What is the cause of raised ALT? • Is raised ALT a predictor of prognosis in those with known liver disease? Concerns with study: • Limited baseline variables • No disease or cause of raised ALT • No multivariate analysis of important (missing) baseline variables US National Health and Nutrition Examination Survey (NHANES III) • n=14,950 adults was examined 1988-1994 prospectively and mortality determined by death certificate • HBV and HCV were excluded serologically • Abn ALT defined as M>30 U/L, F>19 U/L Liver Disease Mortality Normal ALT (n=12,794) Elevated ALT (n=2156) n HR (age adj) HR (multivariate adj) 18 1.0 1.0 16 11.2 (3.1–40.5) 8.2 (2.1–319) Ruhl, Gastroenterol 2009 Issues with NHANES III • Small numbers of liver disease mortality • Strong HR after multivariate adjustment but wide confidence intervals • The cause of liver death not ascertained • However, abn ALT is a signal to investigate further Natural History of ALT for Patient Management: The R.E.V.E.A.L. Study • The R.E.V.E.A.L. study was a large prospective cohort study that examined the risk of cirrhosis and HCC • Elevated ALT (≥45 IU/L) at study entry was a significant univariate risk factor for HCC development (HR 4.1 (2.8-6.0); P < 0.001) • Elevated ALT levels were not an independent risk factor, unlike HBV DNA levels, which were the strongest predictor of HCC risk • ALT U/L ≥45 adjusted HR = 1.1 (0.7-1.7) No. (%) of Participants (N=3653) PersonYears of Follow-up No. of HCC cases Incidence Rate Per 100,000 Person-Years Crude HR (95% CI)* 3435 (94) 39,469 133 337 1.0 218 (6) 2310 31 1342 4.1 (2.8–6.0) 873 (24) 10,154 11 108 1.0 300–9999 1161 (32) 13,518 15 111 1.0 (0.5–2.2) 0.96 10,000–99,999 643 (18) 7404 22 297 2.7 (1.3–5.6) 0.006 100,000–999,999 349 (9) 3845 37 962 8.9 (4.6–17.5) <0.001 ≥1 million 627 (17) 6858 79 1152 10.7 (5.7–20.1) <0.001 Level of ALT,U/L <45 ≥45 HBV DNA, copies/mL <300 (Undetectable) * Cox proportional hazard models were used Chen CJ, et al. JAMA 2006; 295:65–73. P-value <0.001 Natural History of ALT for Patient Management: The R.E.V.E.A.L. Study • Elevated ALT (≥45 IU/L) levels at study entry were an independent predictor of risk of cirrhosis • Multivariate adjusted RR = 1.5 (P < 0.05) • HBV DNA levels >106 copies/mL at study entry were associated with the greatest risk of cirrhosis Multivariate-adjusted relative risk (95% CI)* All patients (N=3582) HBeAg-negative patients (N=3037) 1.0 (ref) 1.0 (ref) 1.5 (1.1–2.1)† 1.6 (1.0–2.6) 1.0 (ref) 1.0 (ref) 300–9999 1.4 (0.9–2.2) 1.4 (0.9–2.1) 10,000–99,999 2.5 (1.6–3.8)‡ 2.4 (1.5–3.7)‡ 100,000–999,999 5.6 (3.7–8.5)‡ 5.4 (3.5–8.3)‡ ≥1 million 6.5 (4.1–10.2)‡ 6.7 (4.1–11.0)‡ Level of ALT,U/L <45 ≥45 HBV DNA, copies/mL <300 (Undetectable) * Multiple Cox proportional hazard regression analysis - adjusted for age, sex, cigarette smoking, alcohol consumption, HBeAg status † P<0.05; ‡ P<0.001 Iloeje UH, et al. Gastroenterol. 2006; 130:678–686. Natural History of ALT for Patient Management: The R.E.V.E.A.L. Study • ALT status at baseline is a stronger predictor of liver disease mortality in HBsAg(-) participants than HBsAg(+) participants ALT level, IU/L Adjusted RR (95% CI)* Among HBsAg(-) subjects (N=19,665) Among HBsAg(+) subjects (N=3,653) 1–19 1.0 1.0 20–29 2.5 (1.4–4.4)† 1.2 (0.7–1.9) 30–39 3.4 (1.6–7.0)† 2.3 (1.4–4.0)‡ 40–49 9.8 (4.9–19.6)† 1.6 (0.8–3.5) 50–99 8.5 (4.3–16.4)† 3.0 (1.8–5.1)† ≥100 31.4 (14.7–66.9)† 1.3 (0.5–3.6) * In the HBsAg(-) group, gender, age, cigarette smoking, alcohol consumption and waist:hip ratio were adjusted. In the HBsAg(+) group, gender, age, cigarette smoking, alcohol consumption, HBeAg status, liver cirrhosis at cohort entry and family history of liver disease were adjusted † P<0.001; ‡ P<0.01 • In HBsAg(+) participants HBV DNA and liver cirrhosis were stronger predictors of liver disease Jen CL, et al. J Hepatol. 2006; 44(S2):S173. Abstract 465. Critique of the R.E.V.E.A.L. study • Analysis of data and prediction based on baseline values - only a single time point • ALT can fluctuate especially in HBeAg-negative disease • ALT as a risk factor for HCC development • Univariate HR= 4.1 (95% CI, 2.8–6.0) • Multivariate HR=HR = 1.1 (95% CI, 0.7-1.7) • This means that after adjusting for other important variables eg HBV DNA, ALT is no longer significant • ALT seems to be a more important risk factor in non-HBV cases…..but runs into same problems: no multivariate analysis of other important risk factors • Eg if we suspect NASH then we should evaluate severity of DM, lipid levels, etc and include these ALT and Hepatic Complications Stratified: ALT <0.5 x ULN ALT >0.5-1 x ULN ALT >1-2 x ULN ALT >2-6 x ULN ALT >6 x ULN 30 (%) Risk of Complications 3,233 Chinese CHB patients Cox proportional hazards model: • 20 • 10 For complications – Male gender – Stigmata of liver disease – Increasing age – Low albumin at baseline – High AFP at baseline For survival – Male gender – Increasing age – Hepatitis symptoms – Low albumin at baseline 0 0 30 60 90 120 150 180 Months of Follow-up Yuen MF, et al. Gut. 2005; 54:1610–1614. ALT and Hepatic Complications Stratified: ALT <0.5 x ULN ALT >0.5-1 x ULN ALT >1-2 x ULN ALT >2-6 x ULN ALT >6 x ULN 30 (%) Risk of Complications 3,233 Chinese CHB patients Concerns about conclusions of this study: • • • 20 • 10 No baseline ultrasound except in those with AFP Liver complications based on single ALT value at baseline Not data on whether ALT was persistently normal or intermittently Multivariate analysis does not show ALT as an independent risk factor 0 0 30 60 90 120 150 180 Months of Follow-up Yuen MF, et al. Gut. 2005; 54:1610–1614. REVEAL – HCV • 89,293 community based persons participated in a prospective study for HCV. • n=925 HCV+ were followed for >10y for HCC Lee, J Clin Oncol 2010 Conclusions • The normal range for ALT is the subject of considerable debate • Normal ranges can be calculated by large cross sectional population studies and generally has been around 30 U/L for men and 19 U/L for women • However, it has not been established that those falling outside the normal range are at risk in a properly conducted multivariate analysis • In those with HBV, ALT≥45 were not found to be associated with increased riskof HCC in 2 large cohort studies, but there is a small risk of cirrhosis • In HCV only levels≥45 are associated with HR~4.5 of HCC development • ALT is not a disease, and as a surrogate marker, it needs to be validated within that disease
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