*These authors equally contributed to this work.
The aims of this study were (1) to identify the useful clinical parameters of noninvasive approach for distinguishing nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD), and (2) to determine whether the levels of the identified parameters are correlated with the severity of liver injury in patients with NASH.
One hundred and eight consecutive patients with biopsy-proven NAFLD (age, 39.8±13.5 years, mean±SD; males, 67.6%) were prospectively enrolled from 10 participating centers across Korea.
According to the original criteria for NAFLD subtypes, 67 patients (62.0%) had NASH (defined as steatosis with hepatocellular ballooning and/or Mallory-Denk bodies or fibrosis ≥2). Among those with NAFLD subtype 3 or 4, none had an NAFLD histologic activity score (NAS) below 3 points, 40.3% had a score of 3 or 4 points, and 59.7% had a score >4 points. Fragmented cytokeratin-18 (CK-18) levels were positively correlated with NAS (
Serum fragmented CK-18 levels can be used to distinguish between NASH and NAFL. Further evaluation is required to determine whether the combined measurement of serum CK-18 and ferritin levels improves the diagnostic performance of this distinction.
Nonalcoholic fatty liver disease (NAFLD) is recognized as one of the most common cause of chronic liver diseases in Western countries, as well as in Korea.
Numerous biomarkers have been investigated in order to discriminate NASH from simple steatosis.
The aims of this study are (1) to identify the useful clinical parameters of a noninvasive approach to distinguish NASH from NAFL; and (2) to determine whether these levels would be related to the severity of the liver injury in patients with NASH.
All consecutive patients who underwent liver biopsy for suspected NAFLD between Jan. 2009 and Jul. 2011 were recruited prospectively at ten Korean university hospital: Soon Chun Hyang University Bucheon Hospital, Seoul St. Mary's Hospital of The Catholic University of Korea, Uijheongbu St. Mary's Hospital of The Catholic University of Korea, Yeouido St. Mary's Hospital of The Catholic University of Korea, Bucheon St. Mary's Hospital of The Catholic University of Korea, St. Paul's Hospital of The Catholic University of Korea, Bucheon St. Vincent's Hospital of The Catholic University of Korea, Incheon St. Mary's Hospital of The Catholic University of Korea, Soon Chun Hyang University Seoul Hospital, Chungbuk National University hospital of Chungbuk National University.
Elevation of aminotransferase levels for more than 3 months and/or fatty liver detected by ultrasonography were the main reasons for liver biopsy. Patients with history of significant alcoholic drinking (> 20 g/day) or hepatotoxic/herb medication were excluded. Patients with other causes of steatogenic drug (e.g. systemic steroids), viral, cholestatic, autoimmune, metabolic or hereditary disorder were also excluded. The patients who have undergone bariatric surgery with last 5 years were excluded.
All enrolled patients were Koreans, and the study protocol was approved by the review board at each participating institution. All subjects gave consents prior to the participation.
The medical history, including co-morbid illness (such as hypertension or diabetes) and drug/herb intake, anthropometric, laboratory and clinical data were collected from all patients at the same day of liver biopsy.
Body mass index (BMI) was calculated as body weight in kilograms divided by body height in square meters (kg/m2). Waist circumference was measured in a standing position at a level of the umbilicus with the tape all around the body in the horizontal position.
Venous blood samples were taken in the morning after a 12 hours overnight fasting on the day of liver biopsy. The laboratory evaluation in all patients included a blood cell count and the measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), total cholesterol, triglyceride (TG), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, albumin, glucose, C-reactive protein (CRP), immunoreactive insulin, ferritin, and fragmented CK-18. IR was evaluated according to homeostatic model assessment (HOMA),
The diagnosis of metabolic syndrome
All the patients underwent an ultrasonic guided percutaneous liver biopsy using a 16-gauge needle (Acecut®; TSK Laboratory, Tochigi, Japan) under local anesthesia. Liver biopsy specimens were fixed in 10% buffered formalin, embedded in paraffin and stained with hematoxylin and eosin, Masson-trichrome, and/or reticulin stain. The slide were reviewed in conference by both experienced hepatopathologists (ESJ and HKK) who were blinded to all clinical, demographic and laboratory information, the diagnosis of NASH were made by consensus.
Histological grading and staging of NAFLD were scored semiquantitatively according to the original criteria for NAFLD subtypes,
Serum samples were obtained from the patients on the same day of the liver biopsies and stored at -80℃ until just before analysis. The levels of the apoptosis-associated CK-18 in sera the were measured by the M30-Apoptosense enzyme-linked immunosorbent assay (ELISA) kit (PEVIVA AB, Bromma, Sweden).
Continuous variables were expressed as means±standard deviation. Categorical data analysis was performed using the Chi-square test and Fisher's exact test, as appropriate. Quantitative data analysis was performed using independent
There were 108 patients recruited in this study. The baseline clinical and laboratory characteristics of the patients are described in
The levels of fasting glucose, HOMA-IR, AST, ALT and portion of female are higher in subtype 3 and 4 of original criteria for NAFLD than subtype 1 and 2 (
According to original criteria for NAFLD subtypes, the patients were categorized into 1 patient (1.0%) of NAFLD type 1, 40 (37.0%) of NAFLD type 2, 39 (36.1%) of NAFLD type 3 and 28 (25.9%) of NAFLD type 4. Therefore, with original criteria for NAFLD subtypes, 67 (62.0%) had NASH (steatosis with hepatocellular ballooning and/or Mallory-Denk bodies or fibrosis≥2).
With NAS system, there were 9 patients (8.3%) with "NAS ≤2", 54 (50%) with "NAS: 3-4", and 45 (41.7%) with "NAS≥5", respectively (
In NASH group (NAFLD subtype 3 or 4), none had below 3 points of NAS, 27 (40.3%) had 3-4 points of NAS, and 40 (59.7%) had over 4 points of NAS. In non-NASH group (NAFLD subtype 1 or 2), 9 (22.0%) had below 3 points of NAS, 27 (65.9%) had 3-4 points of NAS, and 5 (12.2%) had over 4 points of NAS (
CK-18 level had positive correlation with systolic blood pressure, AST, ALT, HDL-cholesterol, ferritin, lobular inflammation, ballooning, fibrosis, NAS and NAFLD subtype (
The serum CK-18 level was significantly higher in NAFLD subtype 3 or 4 group than that of NAFLD subtype 1 or 2. However, the ferritin level was not significantly elevated in this group (
A fragmented CK-18 cutoff value of 235.5 U/L calculated using the receiver operating characteristic curve showed a sensitivity of 69.0%, a specificity of 64.9%, and positive and negative predict values (PPV and NPV) of 75.5% (95% confidence interval [CI] 62.4-85.1) and 57.1% (95% CI 42.2-70.9), respectively, for the diagnosis of NASH. Additional measurement of ferritin to CK-18 improved a specificity of 85.2% and a PPV of 92.6% (95% CI 82.4-97.1), respectively (
Since the progression is very different depending on NAFLD subtype, the diagnosis of NASH is important to predict prognosis and to identify candidates who require treatment. Although there has been several previous studies investigating CK-18 as a biomarker to replace biopsy, this research provides an evidence that CK-18 actually helps in the diagnosis of NASH in Koreans.
Nowadays, the activity of NAFLD is assessed by typical histologic findings with original criteria
In previous study, 16% of biopsies did not meet NASH criteria yet had a NAS ≥5,
In the present study, we assessed histology activity with original criteria along with NAS. There is no patient scored below NAS 3 in group of NAFLD subtype 3 or 4. On the other hand, thirty two patients from NAFLD subtype 1 or 2 scored 3 or greater points under NAS system. The discordance rate between the original criteria and NAS in the diagnosis of NASH was 29.6%.
Elevation of aminotransferase level can be a clue for diagnosis of NAFLD. However, it has some of its own weaknesses in the diagnosis of NAFLD. 1) It does not assess the degree of lipid accumulation, 2) It does not provide a cause of liver disease and 3) It does not discriminate between NASH and NAFL.
Apoptosis of hepatocytes plays an important role in the progression of the NASH and the liver injury. Hepatocytes containing Mallory bodies are likely to undergo apoptosis. The major components of Mallory body include CK-8 and 18, ubiquitin and heat shock proteins 70 and 90.
Markedly increased level of plasma CK-18 fragments was noted in the patients with NASH compared with the patients with simple steatosis as well as that of the normal biopsies (median [interquartile range]: 765.7 U/L [479.6-991.1], 202.4 U/L [160.4-258.2], 215.5 U/L [150.2-296.2], respectively;
The main reason for the results being different from our studies is that they classified the patients according to the consensus of the NASH CRN Pathology Committee Criteria, while we used the original NAFLD criteria. Furthermore, by discerning NAFLD subtype 1 & 2 and NAFLD subtype 3 & 4, we were able to demonstrate some different qualities from the existing studies which only distinguished simple steatosis and NASH. Also, patients with morbid obesity were included in some studies used in the meta-analysis. Though CK-18 is a very promising biomarker for the determination of NASH, cutoff level to diagnose NASH has not been confirmed and assay for CK-18 is not commercially available as yet. Therefore, there are some obstacles to be overcome in order to apply CK-18 in the clinical practice.
An elevation of serum ferritin concentrations in the absence of iron overload, can be resulted from inflammation, liver necrosis and alcohol abuse.
This study has some limitations. First, histologic findings of the liver biopsy were used as the gold standard. Sampling variability or interpretation error could be present. Nevertheless, liver biopsy is currently the only reference standard, and the slide were reviewed in conference by two experienced hepatopathologists. Second, we could not compare our cohort with non-NAFLD control cohort. Using the viral hepatitis cohort as the control is not recommended due to the variety of confounding factors that may complex the viral hepatitis pathology. Moreover, obtaining normal liver tissue without any overt problems is next to impossible and also unethical. Third, the patients were recruited in a tertiary academic hospital and all patients had liver biopsy performed. This meant patients with more severe disease activity than NAFLD patients in the general population may have been included in our cohort. Unfortunately, we didn't get HFE genotyping to exclude primary hemochromatosis and we could check fasting transferrin saturation in only a limited number of patients. Despite several limitations, as far as we know, this is the first prospective study recruiting to biopsy proven NAFLD in large scale in Korea, we are planning to have a further observational study with this cohort.
In this prospective cohort study, measurement of serum CK-18 and ferritin levels helped to distinguish NASH from simple steatosis, and to assess the fibrosis in Korean patients with biopsy proven NAFLD. We need further evaluation on whether the combined measurement of serum CK-18 and ferritin levels improves the diagnostic performance of NASH. To confirm these results, larger validation analyses and longitudinal prospective studies are needed.
The writing group would like to appreciate Dr. Seul Ki Min for providing us with statistical advice.
This study was supported by the Research Fund of the Korean Association for the Study of the Liver (KASL), Korea.
The authors have no conflicts to disclose.
alanine aminotransferase
aspartate aminotransferase
area under receiver operating characteristics
body mass index
blood pressure
cytokeratin-18
C-reactive protein
enzyme-linked immunosorbent assay
gamma-glutamyl transpeptidase
high-density lipoprotein
homeostatic model assessment-insulin resistance
insulin resistance
Korean Association for the Study of the Liver
low-density lipoprotein
positive likelihood ratio
negative likelihood ratio
confidence interval
nonalcoholic fatty liver
nonalcoholic fatty liver disease
NAFLD histologic activity score
nonalcoholic steatohepatitis
NASH Clinical Research Network
negative predictive value
positive predictive value
triglyceride
Correlation between nonalcoholic fatty liver disease subtype and nonalcoholic fatty liver disease histology activity score. NAFLD, nonalcoholic fatty liver disease; NAS, nonalcoholic fatty liver histologic activity score.
Correlation scattergrams for cytokeratin-18 levels. CK-18, cytokeratin-18; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL, high-density lipoprotein; NAS, nonalcoholic fatty liver disease activity score; NAFLD, nonalcoholic fatty liver disease.
Correlation scattergrams for ferritin levels. CK-18, cytokeratin-18; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL, high-density lipoprotein; NAFLD, nonalcoholic fatty liver disease.
Baseline characteristics of Korean patients with nonalcoholic fatty liver disease, relative to nonalcoholic fatty liver disease subtype
Parameter (mean±SD) | Total | NAFLD subtype 1 & 2 | NAFLD subtype 3 & 4 | |
---|---|---|---|---|
Sex (M: F) | 73:35 | 35:6 | 38:29 | 0.002 |
Age (yr) | 38.95±13.48 | 36.44±11.60 | 40.49±14.38 | 0.130 |
Height (m) | 1.68±0.09 | 1.69±0.08 | 1.67±0.99 | 0.213 |
Weight (kg) | 81.34±15.95 | 81.44±13.87 | 81.28±17.18 | 0.960 |
BMI (kg/m2) | 28.71±3.77 | 28.34±3.51 | 28.94±3.93 | 0.433 |
BMI ≤25 kg/m2: >25 kg/m2 | 24: 84 | 11: 30 | 13: 54 | 0.390 |
Waist (cm) | 94.22±11.85 | 95.07±7.68 | 93.72±13.76 | 0.635 |
Hip (cm) | 103.16±9.69 | 103.81±6.61 | 102.78±11.14 | 0.657 |
Metabolic syndrome (No: Yes) | 56:52 | 21:20 | 35:32 | 0.918 |
Systolic blood pressure (mmHg) | 127.20±14.27 | 124.05±14.74 | 129.31±13.66 | 0.075 |
Diastolic blood pressure (mmHg) | 78.37±10.29 | 78.03±8.76 | 78.60±11.26 | 0.788 |
Fasting glucose (mg/dL) | 107.28±41.20 | 98.24±16.03 | 112.89±50.27 | 0.031 |
Insulin (mU/L) | 23.55±33.40 | 16.01±11.10 | 28.37±41.30 | 0.102 |
HOMA-IR | 5.99±8.33 | 3.93±3.49 | 7.45±10.28 | 0.040 |
AST (U/L) | 63.54±41.62 | 43.78±19.26 | 75.82±46.88 | <0.001 |
ALT (U/L) | 108.68±82.07 | 84.22±53.76 | 123.88±92.70 | 0.006 |
Total Cholesterol (mg/dL) | 193.54±35.14 | 189.70±31.84 | 195.94±37.10 | 0.381 |
LDL Cholesterol (mg/dL) | 125.37±37.37 | 125.06±43.64 | 125.56±33.19 | 0.957 |
HDL Cholesterol (mg/dL) | 41.85±11.05 | 41.34±11.76 | 42.14±10.73 | 0.746 |
Triglyceride (mg/dL) | 201.27±131.71 | 180.29±72.95 | 213.52±155.43 | 0.162 |
CK-18 (U/L) | 417.77±517.58 | 279.58±199.20 | 512.20±636.24 | 0.010 |
Ferritin (ng/mL) | 240.13±202.03 | 189.99±146.42 | 267.98±223.55 | 0.090 |
NAFLD, nonalcoholic fatty liver disease; BMI, body mass index; HOMA-IR, homeostatic model assessment-insulin resistance; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CK-18, cytokeratin-18.
Histologic features of Korean patients with nonalcoholic fatty liver disease
Parameter | Grade, stage or score | Total | NAFLD subtype 1 & 2 | NAFLD subtype 3 & 4 | |
---|---|---|---|---|---|
NAFLD subtype | 1: 2: 3: 4 | 1: 40: 39: 28 | 1: 40: 0: 0 | 0: 0: 39: 28 | |
Steatosis | 1: 2: 3 | 69: 25: 14 | 29: 7: 5 | 40: 18: 9 | 0.457 |
Lobular inflammation | 0: 1: 2 | 12: 68: 28 | 10: 25: 6 | 2: 43: 22 | 0.001 |
Ballooning | 0: 1: 2 | 48: 47: 13 | 41: 0: 0 | 7: 47: 13 | <0.001 |
Fibrosis | 0: 1: 2: 3: 4 | 19: 54: 27: 10: 1 | 12: 25: 0: 0: 0 | 7: 29: 27: 10: 1 | <0.001 |
NAS | ≤2 | 9 | 9 | 0 | <0.001 |
3-4 | 54 | 27 | 27 | ||
≥5 | 45 | 5 | 40 |
NAFLD: nonalcoholic fatty liver disease; NAS: nonalcoholic fatty liver histologic activity score.
Correlation coefficient of cytokeratin-18 and serum ferritin levels in patients with nonalcoholic fatty liver disease
CK-18 |
Ferritin |
|||
---|---|---|---|---|
Correlation coefficient | Correlation coefficient | |||
Age (yr) | 0.025 | 0.800 | 0.012 | 0.915 |
Height (cm) | 0.045 | 0.660 | 0.128 | 0.245 |
Weight (kg) | 0.062 | 0.540 | 0.167 | 0.129 |
BMI (kg/m2) | 0.114 | 0.260 | 0.137 | 0.213 |
Waist (cm) | 0.130 | 0.280 | 0.173 | 0.172 |
Hip (cm) | 0.196 | 0.100 | 0.187 | 0.139 |
Systolic BP (mmHg) | 0.227 | 0.030 | 0.148 | 0.203 |
Diastolic BP (mmHg) | 0.155 | 0.140 | 0.134 | 0.248 |
Fasting glucose (mg/dL) | 0.055 | 0.590 | 0.099 | 0.372 |
Insulin (mU/L) | 0.033 | 0.770 | 0.503 | <0.001 |
HOMA-IR | 0.030 | 0.800 | 0.457 | <0.001 |
AST (U/L) | 0.609 | 0.000 | 0.439 | <0.001 |
ALT (U/L) | 0.588 | 0.000 | 0.216 | 0.049 |
Total Cholesterol (mg/dL) | 0.161 | 0.113 | 0.143 | 0.201 |
LDL Cholesterol (mg/dL) | 0.076 | 0.519 | 0.105 | 0.398 |
HDL Cholesterol (mg/dL) | 0.249 | 0.024 | 0.069 | 0.557 |
Triglyceride (mg/dL) | 0.130 | 0.221 | 0.100 | 0.380 |
CK-18 (U/L) | - | - | 0.228 | 0.022 |
Ferritin (ng/mL) | 0.432 | 0.000 | - | - |
Steatosis | 0.145 | 0.149 | 0.162 | 0.153 |
Lobular inflammation | 0.387 | 0.000 | 0.172 | 0.129 |
Ballooning | 0.231 | 0.020 | 0.127 | 0.266 |
Fibrosis | 0.314 | 0.002 | 0.272 | 0.015 |
NAS | 0.401 | 0.000 | 0.258 | 0.022 |
NAFLD subtype | 0.283 | 0.006 | 0.195 | 0.085 |
BMI, body mass index; HOMA-IR, homeostatic model assessment-insulin resistance; BP, blood pressure; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CK-18, cytokeratin-18; NAS, NAFLD histologic activity score, NAFLD, nonalcoholic fatty liver disease.
The use of cytokeratin-18 and serum ferritin levels for the diagnosis of significant fibrosis and nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease, using original criteria
CK-18 | Ferritin | CK-18 or ferritin | |
---|---|---|---|
Cutoff | 253.5 U/L | 160 ng/mL | |
Sensitivity (%) | 69.0 | 70.8 | 65.8 |
Specificity (%) | 64.9 | 58.1 | 85.2 |
LR+ | 1.96 | 1.69 | 4.44 |
LR- | 0.48 | 0.50 | 0.40 |
PPV (%) (95% CI) | 75.5 (62.4-85.1) | 72.3 (58.2-83.1) | 92.6 (82.5-97.1) |
NPV (%) (95% CI) | 57.1 (42.2-70.9) | 56.3 (39.3-71.8) | 46.9 (33.7-60.6) |
AUROC | 0.605 | 0.602 | 0.577 |
AUROC, area under receiver operating characteristics; LR+, positive likelihood ratio; LR-, negative likelihood ratio; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; CK-18, cytokeratin-18.