Long-Term Follow-up of Patients With NAFLD and
Elevated Liver Enzymes
Mattias Ekstedt,1 Lennart E. Franzén,2 Ulrik L. Mathiesen,3 Lars Thorelius,4 Marika Holmqvist,5
Göran Bodemar,1 and Stergios Kechagias6
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of elevated liver enzymes in
patients of developed countries. We determined the long-term clinical and histological courses of
such patients. In a cohort study, 129 consecutively enrolled patients diagnosed with biopsyproven NAFLD were reevaluated. Survival and causes of death were compared with a matched
reference population. Living NAFLD patients were offered repeat liver biopsy and clinical and
biochemical investigation. Mean follow-up (SD) was 13.7 (1.3) years. Mortality was not increased in patients with steatosis. Survival of patients with nonalcoholic steatohepatitis (NASH)
was reduced (P ⴝ .01). These subjects more often died from cardiovascular (P ⴝ .04) and
liver-related (P ⴝ .04) causes. Seven patients (5.4%) developed end-stage liver disease, including
3 patients with hepatocellular carcinoma. The absence of periportal fibrosis at baseline had a
negative predictive value of 100% in predicting liver-related complications. At follow-up, 69 of
88 patients had diabetes or impaired glucose tolerance. Progression of liver fibrosis occurred in
41%. These subjects more often had a weight gain exceeding 5 kg (P ⴝ .02), they were more
insulin resistant (P ⴝ .04), and they exhibited more pronounced hepatic fatty infiltration (P ⴝ
.03) at follow-up. In conclusion, NAFLD with elevated liver enzymes is associated with a clinically significant risk of developing end-stage liver disease. Survival is lower in patients with
NASH. Most NAFLD patients will develop diabetes or impaired glucose tolerance in the long
term. Progression of liver fibrosis is associated with more pronounced insulin resistance and
significant weight gain. (HEPATOLOGY 2006;44:865-873.)
See Editorial on Page 802
H
epatic steatosis, the accumulation of lipids in
hepatocytes, can occur in association with a
wide range of toxins, drugs, and diseases.1 In
routine clinical practice most cases have previously been
Abbreviations: NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic
steatohepatitis.
From the 1Division of Gastroenterology and Hepatology, Department of Molecular and Clinical Medicine, University Hospital, Linköping, Sweden; 2Department
of Pathology, Clinical Research Centre, University Hospital, Örebro, Sweden; 3Department of Internal Medicine, County Hospital, Oskarshamn, Sweden; 4Division
of Radiology, Department of Medicine and Care, University Hospital, Linköping,
Sweden; 5Division of Social Medicine and Public Health Science, Department of
Health and Society, University Hospital, Linköping, Sweden; and 6Division of
Internal Medicine, Department of Medicine and Care, University Hospital, Linköping, Sweden.
Received May 1, 2006; accepted June 28, 2006.
Supported by the Research Council of Southeast Sweden, grant F2004-303.
Address reprint requests to: Stergios Kechagias, M.D., Ph.D., Division of Internal Medicine, Department of Medicine and Care, University Hospital, SE-581 85
Linköping, Sweden. E-mail: [email protected]; fax: (46) 13-14-99-91.
Copyright © 2006 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.21327
Potential conflict of interest: Nothing to report.
associated with excessive alcohol consumption, but during recent years hepatic steatosis has most commonly
been associated with obesity, insulin resistance, and hyperlipidemia, all components of metabolic syndrome.2
Nonalcoholic fatty liver disease (NAFLD) is now recognized as one of the most common causes of chronic liver
disease worldwide. Patients with NAFLD are often identified by asymptomatic elevation of liver enzymes, most
frequently of serum alanine aminotransferase (ALT), and
nonalcoholic hypertransaminasemia, in which viral or
other causes of liver disease are excluded, has been used as
a surrogate marker for NAFLD.3,4
Analysis of data from the Third National Health and
Nutrition Examination Survey (NHANES III) shows the
prevalence of elevated serum aminotransferases to be
7.9% in the United States, with most instances unexplained and thus attributed to NAFLD.5 Obesity has
been established as a major risk factor, albeit not a prerequisite, for NAFLD. Because obesity is reaching epidemic
proportions worldwide, the population at risk for developing chronic liver disease and its complications from
NAFLD will most likely increase over time. Thus, there is
a critical need to better understand the natural history of
865
866
EKSTEDT ET AL.
NAFLD. Published follow-up studies where repeat liver
biopsies were performed have either included relatively
small numbers of patients, and/or repeat biopsies were
obtained after a rather modest length of follow-up.6-11
Our aim was to describe the long-term clinical and
histological development of NAFLD in patients originally referred because of chronically elevated liver enzymes.
Patients and Methods
Subjects. We studied in detail all patients (n ⫽ 212)
referred consecutively between 1988 and 1993 to the Department of Gastroenterology and Hepatology, University Hospital in Linköping, or the Department of Internal
Medicine, Oskarshamn County Hospital for evaluation
of persistently (⬎6 months) elevated serum ALT and/or
aspartate aminotransferase (AST), defined as ⬎41 U/L
(0.70 ␮kat/L), and/or elevated serum alkaline phosphatase, ⬎106 U/L (1.80 ␮kat/L). A diagnostic workup was
performed including physical examination, laboratory investigations, and liver biopsy.12
One hundred and forty-four patients were diagnosed
with hepatic steatosis without any other concomitant liver
disease or medication associated with fatty infiltration of
the liver. Seven of these subjects reported at baseline current or previous average weekly alcohol consumption ⱖ
140 g and were excluded from follow-up. The remaining
137 patients originally diagnosed with NAFLD constituted the cohort of this follow-up study.
Data Collection. Each subject in the study cohort was
currently identified by linking his or her unique personal
identification number to the National Registry of Population. All medical records from primary care health centers and hospitals were reviewed. Special attention was
given to development of chronic diseases and signs of
alcohol abuse. Subjects who had died during follow-up
were identified and their causes of death were obtained by
reviewing their medical records and the information obtained from the Registry of Causes of Death.
All participants in the study cohort who were still living were asked to participate in this follow-up study.
Those who accepted were offered clinical and biochemical
investigation and ultrasonography and a biopsy of the
liver.
Subjects had blood drawn for a complete blood count
and analysis of prothrombin, thyroid-stimulating hormone, transferrin, iron, transferrin saturation, ferritin,
AST, ALT, alkaline phosphatase, gamma glutamyl transferase, bilirubin, fasting total cholesterol, low-density lipoprotein, high-density lipoprotein, triglycerides, fasting
plasma glucose, fasting serum insulin, and plasma protein
HEPATOLOGY, October 2006
electrophoresis including albumin, ␣1-antitrypsin, ceruloplasmin, and immunoglobulins. In addition, blood was
obtained for detection of hepatitis B surface antigen, anti–
hepatitis C virus (HCV) antibodies, hepatitis B virus
DNA, HCV RNA, transglutaminase antibodies, antinuclear antibodies, smooth muscle antibodies, and mitochondrial antibodies. Moreover, genomic DNA isolated
from anticoagulated venous blood was used to identify
the C282Y, H63D, and S65C mutations in the HFE gene
as well as the Z and S mutations in the Pi gene. Subjects
were considered to have diabetes mellitus if they were
receiving dietary or drug treatment for this disease. The
remaining subjects had a 75-g oral glucose tolerance test
after an overnight fast.
The prevalence of liver-related complications of those
in the general population in the same age range (36-80
years) as the NAFLD cohort was estimated by obtaining
data from the Swedish Hospital Discharge Register. Included were all individuals living in the same geographical
area as the NAFLD cohort who had been hospitalized in
2004 with primary or secondary diagnoses (according to
the International Classification of Diseases, Tenth Revision) of cirrhosis, chronic hepatic failure, portal hypertension, hepatorenal syndrome, ascites, esophageal varices, or
hepatocellular carcinoma.
Liver Biopsy and Histopathological Evaluation.
Liver biopsies of all subjects were performed percutaneously with ultrasound guidance and 1.6-mm Biopince
needles on an outpatient basis. All biopsies at baseline and
at follow-up were read by the same liver pathologist
(L.E.F.), who was blinded to patient details. Liver histology was scored according to the system developed by
Brunt et al.,13 except that acidophil bodies and glycogenated nuclei were not assessed and that PAS-D Kupffer
cells were scored as present or absent. Moreover, quantitative steatosis was measured as the percentage of the liver
biopsy containing fat.14 Nonalcoholic steatohepatitis
(NASH) was defined as steatosis plus any stage of fibrosis
or as steatosis plus lobular inflammation plus ballooning
degeneration.15
Statistical Analysis. Statistical analyses were performed using SPSS software (version 11.5 for Windows).
For continuous variables, differences between 2 groups
were evaluated with the Student t test when data were
normally distributed and with the Mann-Whitney U test
when the assumption of normality was not met. For dichotomous variables, differences were tested using the ␹2
test corrected for continuity or Fisher’s exact test. Observed survival and causes of death of NAFLD patients
until September 30, 2005, were compared with those of a
reference population, which was obtained from Statistics
Sweden. The reference population comprised all those
HEPATOLOGY, Vol. 44, No. 4, 2006
(n ⫽ 44,745) of the same age and sex living in the same
county as each patient with NAFLD at baseline. Survival
curves were constructed according to the Kaplan-Meier
method. One-sample log-rank tests were used for comparison with the reference population. Causes of death
were compared using the z test with Bonferroni correction. A P value ⬍ .05 was considered statistically significant.
Ethical Considerations. Written informed consent
was obtained from all participating subjects. The study
design was approved by the ethics committee at the University Hospital in Linköping.
Results
Study Population. Eight subjects who denied previous or current alcohol consumption ⱖ 140 g/wk at baseline were reclassified as having alcoholic liver disease at
follow-up based on information in their medical records
or self-reported alcohol consumption ⬎ 140 g/wk. Thus,
of the 137 patients originally diagnosed with NAFLD,
129 patients were considered to have had NAFLD when
included in the study. Baseline clinical and biochemical
characteristics of this cohort are shown in Table 1.
Of these 129 patients, 104 were alive at follow-up, and
88 agreed to participate in this study (Fig. 1). Follow-up
started March 10, 2003, and was completed September
30, 2005. Mean follow-up time ⫾ SD was 13.7 ⫾ 1.3
years from time of diagnosis of NAFLD, with a total of
1,202 person-years. Clinical and biochemical characteristics of the 88 patients who went to a consultation at follow-up are shown in Table 1. Sixteen patients were alive
but did not return for follow-up. The baseline clinical,
biochemical, and histological parameters of this group
were not significantly different than those of the participatants in the follow-up study.
Histology at Baseline. At baseline 71 patients (55%)
fulfilled criteria for NASH. Four of these patients had
cirrhosis. Twelve patients (9%) had steatosis with unspecific inflammation, and 46 (36%) had simple steatosis.
Patients with NASH were significantly older than patients with steatosis with or without unspecific inflammation (54.5 ⫾ 12.4 vs. 46.7 ⫾ 12.3 years, respectively, P ⫽
.001). There were no other significant differences in baseline clinical and biochemical parameters between the 2
histological groups.
Survival. A total of 26 subjects with NAFLD died
during the follow-up period (including 1 patient who
died shortly after diagnostic workup at follow up). Of
these, 19 had NASH at baseline, including 1 patient with
cirrhosis, and 7 had steatosis with or without unspecific
inflammation. At the end of the follow-up period, sur-
EKSTEDT ET AL.
867
vival of NAFLD patients was significantly lower than that
of the reference population (78% vs. 84%, respectively;
P ⫽ .006; Fig. 2A). Subgroup analysis showed that survival among NASH patients was significantly lower than
the corresponding reference population (70% vs. 80%,
respectively, P ⫽ .01; Fig. 2B), whereas survival did not
differ significantly between patients with steatosis with or
without unspecific inflammation and the corresponding
reference population (Fig. 2C).
Causes of Death and Liver-Related Morbidity and
Mortality Among Subjects With NAFLD. Eleven
(15.5%) patients with NASH died from cardiovascular
disease, 4 (5.6%) from extrahepatic malignancy, 2 (2.8%)
from liver-related causes (metastatic hepatocellular carcinoma and variceal hemorrhage), 1 (1.4%) from neurological disease, and 1 (1.4%) from respiratory disease. A
comparison of the causes of death of patients with NASH
with those of the corresponding reference population
showed it was significantly more common for patients
with NASH to die from liver-related causes (2.8% vs.
0.2%, respectively, P ⫽ .04) and from cardiovascular disease (15.5% vs. 7.5%, respectively, P ⫽ .04). Five nonNASH patients (8.6%) died from cardiovascular disease,
1 (1.7%) from extrahepatic malignancy, and 1 (1.7%)
from renal disease. No significant differences in causes of
death were found between non-NASH patients and the
corresponding reference population.
Although 25 patients died during follow-up and 16
patients with NAFLD chose not to participate in the follow-up study, their medical records from hospitals and
primary care health centers were available. A review of
those records showed that no NAFLD patient who was
alive but did not participate in the follow-up study had
developed liver-related complications. Of the NAFLD
patients who died during follow-up, 2 had developed cirrhosis-related complications prior to death. One female
patient subsequently died from variceal hemorrhage, and
one male patient who died from acute myocardial infarction had previously developed ascites, and cirrhosis was
diagnosed with postmortem biopsy. Among the 88
NAFLD patients who participated in the follow-up study,
5 had developed cirrhosis-related complications (ascites
in 2 patients, ascites and hepatocellular carcinoma in 2
patients, of whom 1 died shortly after diagnostic workup
at follow-up, and hepatocellular carcinoma in 1 patient
who had undergone successful orthoptic liver transplantation during follow-up). Thus, of 129 NAFLD patients,
7 (5.4%) developed cirrhosis-related complications during follow-up. There were no significant differences in
baseline clinical, biochemical, and histological parameters
between the 7 patients who developed end-stage liver disease during follow-up and the 15 patients who died from
868
EKSTEDT ET AL.
HEPATOLOGY, October 2006
Table 1. Clinical and Biochemical Features of Cohort at Baseline and at Follow-Up [Mean ⴞ SD or n (%)]
Age (years)
Sex (male)
BMI (kg/m2)
Overweight
Obese
Previously diagnosed diabetes
Diabetes diagnosed at consultation visit
IGT diagnosed at consultation visit
Hypertensive
Manifest cardiovascular disease
Hypertriglyceridemia
Metabolic syndrome
ALT (U/L)
AST (U/L)
AST/ALT ratio
ALP (U/L)
Bilirubin (mg/dL)
Albumin (g/dL)
Platelet count (⫻ 109/L)
Prothrombin (INR)
Ferritin (␮g/L)
Glucose (mg/dL)
IRHOMA
Triglycerides (mg/dL)
Cholesterol (mg/dL)
HDL (mg/dL)
LDL (mg/dL)
Negative for HBsAg/anti-HCV
Negative for HBV DNA/HCV RNA
Positive for ANA/SMA/AMA
Positive for transglutaminase antibodies
Mutation in the HFE gene* (C282Y homozygosity/C282Y compound heterozygosity)
Mutation in the Pi gene† (ZZ/SZ/MZ/MS)
Ceruloplasmin ⬍ 0.20 g/L
At Baseline (n ⴝ 129)
At Follow-Up (n ⴝ 88)
51.0 ⫾ 12.9
87 (67%)
28.3 ⫾ 3.8
72 (56%)
37 (29%)
11 (8.5%)
NA
NA
93 (72%)
14 (11%)
74 (57%)
NA
76 ⫾ 43
45 ⫾ 23
0.6 ⫾ 0.2
61 ⫾ 33
0.64 ⫾ 0.30
4.1 ⫾ 0.3
235 ⫾ 67
1.0 ⫾ 0.1
232 ⫾ 317
NA
NA
190 ⫾ 134
236 ⫾ 59
NA
NA
129/129
NA/NA
37/23/0
NA
NA
NA
0
61.0 ⫾ 11.0
62 (70%)
29.1 ⫾ 4.7
49 (56%)
29 (33%)
37 (42%)
14 (16%)
18 (20%)
83 (94%)
16 (18%)
35 (40%)
52 (59%)
60 ⫾ 35
35 ⫾ 15
0.7 ⫾ 0.3
65 ⫾ 37
0.78 ⫾ 0.33
4.2 ⫾ 0.4
194 ⫾ 94
1.0 ⫾ 0.2
192 ⫾ 159
125 ⫾ 38
3.8 ⫾ 3.5
157 ⫾ 89
202 ⫾ 43
51 ⫾ 19
123 ⫾ 37
88/88
88/88
12/8/0
0
2/0
0/0/10/3
0
NOTE. Conversions: ALT, AST, and ALP (U/L) ⫻ 0.017 ⫽ ␮kat/L; bilirubin (mg/dL) ⫻ 17.1 ⫽ ␮mol/L; albumin (g/dL) ⫻ 10 ⫽ g/L; glucose (mg/dL) ⫻ 0.055 ⫽
mmol/L; triglycerides (mg/dL) ⫻ 0.011 ⫽ mmol/L; cholesterol, HDL, and LDL (mg/dL) ⫻ .026 ⫽ mmol/L.
Abbreviations: NS, not significant; NA, not available; BMI, body mass index; IGT, impaired glucose tolerance; ALT, alanine aminotransferase; AST, aspartate
aminotransferase; ALP, alkaline phosphatase; INR, international normalized ratio; IRHOMA, insulin resistance according to homeostasis model assessment16; HDL,
high-density lipoprotein; LDL, low-density lipoprotein; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV; hepatitis C virus; ANA, antinuclear antibodies,
SMA, smooth muscle antibodies; AMA, mitochondrial antibodies. Overweight was defined as BMI ⬎ 25 kg/m2 but ⱕ 30 kg/m2, obesity as BMI ⬎ 30 kg/m2, diabetes
as fasting plasma glucose ⱖ 126 mg/dL requiring treatment or plasma glucose ⬎ 199 mg/dL 2 h after oral administration of 75 g of glucose, impaired glucose
tolerance as plasma glucose ⬎ 140 mg/dL but ⱕ 199 mg/dL 2 h after oral administration of 75 g of glucose, hypertension as blood pressure ⱖ 130/85 or requiring
treatment, and hypertriglyceridemia as fasting triglycerides ⱖ 150 mg/dL. Metabolic syndrome was defined as having at least 3 of the following17: (1) waist
circumference ⬎ 102 cm in men or ⬎ 88 cm in women; (2) fasting triglycerides ⬎ 150 mg/dL; (3) fasting HDL ⬍ 40 mg/dL in men or ⬍ 50 mg/dL in women; (4)
blood pressure ⬎ 130/85 mm Hg or a diagnosis of hypertension; (5) fasting glucose ⬎ 110 mg/dL or a diagnosis of diabetes mellitus.
*C282Y, H63D, and S65C mutations in the HFE gene were identified from genomic DNA.
†Z and S mutations in the Pi gene were identified from genomic DNA.
cardiovascular disease without previous development of
end-stage liver disease.
During 2004, 62 of every 100,000 inhabitants of the
same geographical area were hospitalized with diagnoses
of liver-related complications. Of these, 57% lacked etiologic diagnosis. The 2 most common etiologic diagnoses
were alcoholic liver disease and hepatitis C, accounting
for 37% and 3%, respectively, of the diagnoses.
Metabolic and Cardiovascular Characteristics of
Study Cohort. At baseline most patients were overweight or obese. Fasting plasma glucose was not measured
at baseline, and thus the prevalence of diabetes at onset of
the study cannot be reported (Table 1). The corresponding clinical parameters at follow-up are shown in Table 1.
Histology at Baseline Versus Clinical Outcome. Of
the 71 patients with NASH at baseline, 7 (10%) developed end-stage liver disease during follow-up, whereas
none of the 58 patients with steatosis with or without
unspecific inflammation developed complications related
to chronic liver disease during follow-up. Subgroup analysis showed no patient who had NAFLD without fibrosis
(n ⫽ 60) or stage 1 fibrosis (n ⫽ 31) at baseline had
HEPATOLOGY, Vol. 44, No. 4, 2006
EKSTEDT ET AL.
869
Fig. 1. Details about patients studied, showing reasons for exclusions. aBoth patients were diagnosed with hepatocellular carcinoma at follow-up.
One patient died shortly after diagnostic work-up at follow-up. bOne patient developed hepatocellular carcinoma and underwent orthoptic liver
transplantation during follow-up. PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; AAT, ␣1-antitrypsin.
developed complications related to chronic liver disease
during follow-up. One of the 4 patients (25%) with cirrhosis at baseline, 3 of the 22 patients (14%) with stage 2
fibrosis at baseline, and 3 of the 12 patients (25%) with
stage 3 fibrosis at baseline were found to have developed
end-stage liver disease during follow-up.
Forty-two patients with NASH at baseline returned for
follow-up. Of these, 30 patients (71%) had diabetes. Of
the patients with steatosis with or without unspecific inflammation, 21 (46%) had diabetes. This difference was
statistically significant (P ⫽ .01). Moreover, manifest cardiovascular disease at follow-up was significantly more
common among patients with NASH at baseline than
among those without NASH (29% vs. 9%, respectively,
P ⫽ .02).
Histological Development. Follow-up biopsies were
obtained from 68 patients after 13.8 ⫾ 1.2 years (range
10.3-16.3 years). Three patients, one of whom had un-
dergone orthoptic liver transplantation, did not undergo a
repeat liver biopsy because they had cirrhosis at baseline.
Moreover, 2 patients without cirrhosis at baseline did not
undergo a liver biopsy at follow-up because they were
diagnosed with ascites and hepatocellular carcinoma at
follow-up. These 2 patients were considered as having
fibrosis that had progressed in stage.
Fifteen patients who returned for follow-up refused
a repeat liver biopsy (Fig. 1). The clinical and biochemical parameters of these patients were not significantly
different than those of the patients who agreed to repeat liver biopsies, nor were the baseline histological
features.
Two patients had normal liver histology at follow-up.
Overall, quantitative steatosis was significantly lower at
follow-up (12.5% ⫾ 9.7% vs. 8.8% ⫾ 7.4%, P ⫽ .004).
Between the first and second biopsies, fibrosis stage progressed in 29 patients (41%), was unchanged in 30 pa-
870
EKSTEDT ET AL.
HEPATOLOGY, October 2006
tients (43%), and regressed in 11 patients (16%); see
Table 2.
The presence of necroinflammatory changes at baseline was not associated with progression in fibrosis stage at
follow-up (Table 2). A separate analysis of the 36 patients
without fibrosis at baseline who underwent liver biopsy at
follow-up showed only a small number exhibited histological features associated with hepatic necroinflammation at baseline. Despite this, 17 patients (47%) had
developed fibrosis at follow-up (Table 2).
Those patients whose fibrosis had progressed in stage at
follow-up had significantly higher ALT levels (P ⫽ .005),
significantly higher AST levels (P ⫽ .003), and significantly lower platelet counts (P ⫽ .003). Moreover, at
follow-up subjects with progressive fibrosis had a weight
gain exceeding 5 kg significantly more often (P ⫽ .02),
were significantly more insulin resistant according to homeostasis model assessment16 (P ⫽ .04), and had significantly more pronounced hepatic fatty infiltration (P ⫽
.03; Table 3).
We were not able to find any association between baseline clinical and biochemical parameters with future progression of fibrosis.
Discussion
Fig. 2. (A) Overall survival of NAFLD patients originally referred
because of elevated liver enzymes. (B) Survival of NASH patients. (C)
Survival of patients with nonalcoholic liver steatosis with and without
unspecific inflammation. Survival of patients was compared with that of
a reference population comprising all subjects of the same age and sex
living in the same county as each patient at baseline.
In person-years, this is the largest reported study of a
follow-up series of biopsy-proven NAFLD patients originally referred because of elevated liver enzymes. Although we cannot rule out unknown biases in referral for
hepatological exploration, we believe that our study has
several methodological strengths. First, all patients referred because of elevated liver enzymes were consecutively enrolled. Second, all patients underwent liver
biopsy at baseline, and thus the diagnoses of NAFLD
were based on histological criteria. Third, clinical and
histological follow-up exceeded 10 years for all patients, and time to follow-up did not vary considerably
between patients.
The natural history of patients with biopsy-proven
NAFLD and elevated liver enzymes previously has not
been well defined. One of the main findings of the present
study was that survival among these patients was lower
than that of the matched reference population. A previous
study18 reported an association between NAFLD without
fibrosis or inflammation with a benign clinical course
without excess mortality. We confirmed this finding; but
in addition, we have shown that NASH, which was more
common in the older patients in our NAFLD cohort, is
associated with increased overall mortality.
Recently, Adams et al.19 reported that survival of
NAFLD patients was lower than the expected survival in
HEPATOLOGY, Vol. 44, No. 4, 2006
EKSTEDT ET AL.
871
Table 2. Changes in Fibrosis Stage Between First and Second Biopsies; Comparison of Baseline Necroinflammatory
Parameters Between Patients With Progressive Fibrosis and Patients With Nonprogressive Fibrosis (n ⴝ 70); and Baseline
Necroinflammatory Parameters of Patients Without Fibrosis at Baseline (n ⴝ 36)
Fibrosis Stage at Follow-Up [n (%)]
Fibrosis Stage at Baseline
F0 (n
F1 (n
F2 (n
F3 (n
F4†
⫽
⫽
⫽
⫽
36)
19)
11)
4)
F0
F1
F2
F3
F4
19 (53%)
5 (26%)
0
0
8 (22%)
9 (47%)
5 (45%)
0
6 (17%)
3 (16%)
1 (9%)
1 (25%)
3 (8%)
1 (5%)
2 (18%)
1 (25%)
0
1 (5%)
3* (27%)
2* (50%)
Necroinflammation
Progressive Fibrosis
(n ⴝ 29)
Nonprogressive Fibrosis
(n ⴝ 41)
P
Lobular inflammation
Portal inflammation
Periportal inflammation
Hepatocellular ballooning
Mallory’s hyaline
4 (14%)
9 (31%)
4 (14%)
3 (10%)
1 (3%)
1 (3%)
7 (17%)
3 (7%)
2 (5%)
1 (2%)
NS
NS
NS
NS
NS
Steatosis Without Fibrosis
(n ⴝ 36)
Lobular inflammation
Portal inflammation
Periportal inflammation
Hepatocellular ballooning
Mallory’s hyaline
Fibrosis Present at Follow-Up
(n ⴝ 17)
Fibrosis Absent at Follow-Up
(n ⴝ 19)
1 (6%)
2 (12%)
1 (6%)
1 (6%)
0
0
1 (5%)
1 (5%)
1 (5%)
0
NS
NS
NS
NS
NS
Abbreviations: F0, no fibrosis; F1, expanded portal tracts and/or focally or extensively present zone 3 perisinusoidal/pericellular fibrosis; F2, focal or extensive
periportal fibrosis and zone 3 perisinusoidal/pericellular fibrosis; F3, portal fibrosis with focal or extensive bridging fibrosis and zone 3 perisinusoidal/pericellular fibrosis;
F4, cirrhosis; NS; not significant.
*Two of these 5 patients were diagnosed with ascites and hepatocellular carcinoma and did not undergo liver biopsy at follow up. †Three of 4 patients with cirrhosis
at baseline were alive, all of whom returned for follow-up. Repeat liver biopsy was not performed in these patients. Two of the patients had not developed complications
related to chronic liver disease during follow-up, and 1 had undergone orthoptic liver transplantation because of hepatocellular carcinoma. Dichotomous variables were
tested using Fisher’s exact test.
the general population. In their study the NAFLD diagnosis of most patients was confirmed by imaging studies
rather than histologically, and follow-up time was highly
variable. In the present study we have extended their findings, showing increased mortality of patients with NASH
but not of patients with steatosis. Moreover, we were able
to show that the higher mortality of NASH patients was
primarily a result of cardiovascular disease and, to a lesser
extent, liver-related causes.
Another main finding of our study, consistent with the
increased cardiovascular mortality among NAFLD patients, is that most NAFLD patients (78%) were diagnosed with diabetes or impaired glucose tolerance (IGT)
at follow-up. Although we do not know with certainty
how many NAFLD patients had diabetes or IGT at baseline, these findings indicate liver enzyme elevation due to
NAFLD is strongly associated with future onset of type 2
diabetes or IGT. Given the strong association between
insulin resistance and NAFLD, it is reasonable to recommend lifestyle modifications to all patients with NAFLD.
Not only do such modifications reduce the risk of developing type 2 diabetes,20,21 but an intense dietary interven-
tion, in particular, may also improve liver histology in
those with NAFLD.22
Interestingly, 3 of the 7 patients who developed end-stage
liver disease during follow-up were diagnosed with hepatocellular carcinoma. All 3 had previously been diagnosed with
diabetes. These findings are in accordance with those of a
previous report that identified diabetes as an independent
risk factor for hepatocellular carcinoma.23 Our data primarily indicate that the association between diabetes and hepatocellular carcinoma is a result of the high prevalence of
NAFLD in patients with diabetes. With the increasing prevalence of NAFLD, particularly at younger ages, the modestly
increased relative risk of mortality and the low absolute risk
of end-stage liver disease that these patients have may be of
considerable public health significance in the near future.
In the present study, 29 NAFLD patients (41%)
showed progression in the stage of their fibrosis. It is difficult to compare these results with those of other studies
because previous investigations of fibrosis change in
NAFLD over time were limited to small numbers of patients and the patients generally had undergone sequential
biopsies because of clinical indications, potentially biasing
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EKSTEDT ET AL.
HEPATOLOGY, October 2006
Table 3. Clinical, Biochemical, and Histological Features at
Follow-Up of Patients With Progressive Fibrosis and Patients
With Nonprogressive Fibrosis [Mean ⴞ SD or n (%)]
Age (years)
Follow-up time (years)
Sex (male)
BMI (kg/m2)
BMI ⬎ 25
Weight gain ⬎ 5 kg
IGT
Diabetes
Hypertension
Metabolic syndrome
Alcohol consumption (g/week)
ALT (U/L)
AST (U/L)
AST/ALT ratio
ALP (U/L)
Bilirubin (mg/dL)
Albumin (g/dL)
Platelet count (⫻ 109/L)
Prothrombin (INR)
Ferritin (␮g/L)
Glucose (mg/dL)
IRHOMA
Triglycerides (mg/dL)
Cholesterol (mg/dL)
HDL (mg/dL)
LDL (mg/dL)
Mutation in the HFE gene*
Mutation in the Pi gene†
Quantitative steatosis (%)
Progressive
Fibrosis
(n ⴝ 29)
Nonprogressive
Fibrosis
(n ⴝ 41)
P
61.1 ⫾ 11.0
14.0 ⫾ 1.0
21 (72%)
29.6 ⫾ 3.3
28 (97%)
16 (55%)
7 (24%)
15 (52%)
28 (97%)
18 (62%)
46 ⫾ 44
75 ⫾ 44
42 ⫾ 17
0.6 ⫾ 0.2
61 ⫾ 21
0.8 ⫾ 0.4
4.2 ⫾ 0.4
205 ⫾ 59
1.0 ⫾ 0.1
207 ⫾ 193
124 ⫾ 32
5.2 ⫾ 5.3
167 ⫾ 96
201 ⫾ 44
48 ⫾ 11
123 ⫾ 38
6 (21%)
5 (17%)
11.3 ⫾ 8.3
60.1 ⫾ 11.1
13.7 ⫾ 1.3
29 (71%)
28.3 ⫾ 5.3
34 (83%)
10 (24%)
7 (17%)
24 (58%)
38 (93%)
21 (51%)
28 ⫾ 36
51 ⫾ 25
31 ⫾ 13
0.7 ⫾ 0.4
67 ⫾ 51
0.7 ⫾ 0.2
4.1 ⫾ 0.4
252 ⫾ 62
1.0 ⫾ 0.09
174 ⫾ 125
127 ⫾ 44
2.9 ⫾ 1.5
144 ⫾ 84
205 ⫾ 43
55 ⫾ 25
127 ⫾ 35
16 (39%)
4 (10%)
7.3 ⫾ 6.6
NS
NS
NS
NS
NS
.02
NS
NS
NS
NS
NS
.005
.003
NS
NS
NS
NS
.003
NS
NS
NS
.04
NS
NS
NS
NS
NS
NS
0.03
NOTE. Conversions: ALT, AST, and ALP (U/L) ⫻ 0.017 ⫽ ␮kat/L; bilirubin
(mg/dL) ⫻ 17.1 ⫽ ␮mol/L; albumin (g/dL) ⫻ 10 ⫽ g/L; glucose (mg/dL) ⫻
0.055 ⫽ mmol/L; triglycerides (mg/dL) ⫻ 0.011 ⫽ mmol/L; cholesterol, HDL,
and LDL (mg/dL) ⫻ 0.026 ⫽ mmol/L. Abbreviations: NS, not significant; BMI,
body mass index; IGT, impaired glucose tolerance; ALT, alanine aminotransferase;
AST, aspartate aminotransferase; ALP, alkaline phosphatase; INR, international
normalized ratio; IRHOMA, insulin resistance according to homeostasis model
assessment16; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Metabolic syndrome was defined as having at least 3 of the following17: (1) waist
circumference ⬎ 102 cm in men or ⬎ 88 cm in women; (2) fasting triglycerides ⬎ 150 mg/dL; (3) fasting HDL ⬍ 40 mg/dL in men or ⬍ 50 mg/dL in
women; (4) blood pressure ⬎ 130/85 mm Hg or a diagnosis of hypertension; (5)
fasting glucose ⬎ 110 mg/dL or a diagnosis of diabetes mellitus. *The C282Y,
H63D, and S65C mutations in the HFE gene were identified from genomic DNA.
††Z and S mutations in the Pi gene were identified from genomic DNA. Continuous
variables were normally distributed (except IRHOMA) and thus were analyzed with
the unpaired Student t test (IRHOMA was analyzed with the Mann-Whitney U test).
Dichotomous variables were analyzed using the ␹2 test corrected for continuity.
results toward patients with more severe or atypical disease.6-10 In a recently published study,11 progression of
fibrosis was found in 37% of 103 NAFLD patients. However, follow-up was short (mean 3.2 years). Moreover, this
study included patients who were markedly obese and few
had simple steatosis, making it likely that the participants
were not reflective of the general NAFLD population
with elevated liver enzymes.
Although most patients in the present study were
nonobese, more pronounced insulin resistance at follow-up was associated with progression of liver fibrosis.
This is consistent with the findings of previous studies.8,11,24 We have shown that NAFLD patients with progressive fibrosis were, in addition to being more insulin
resistant, significantly more likely to have a weight gain
exceeding 5 kg and more severe hepatic fatty infiltration at
follow-up.
A previous study7 reported a relatively benign histological course among NAFLD patients without fibrosis. According to the results of the present study, this view
should be modified. Although none of the 60 NAFLD
patients without fibrosis at baseline developed clinically
manifest liver disease during follow-up, 17 (47%) of the
36 patients who underwent repeat liver biopsy at follow-up had developed fibrosis, including 3 who had developed bridging fibrosis.
A limitation of all NAFLD studies investigating hepatic histology is the significant sampling variability of
routine liver biopsy.25 We cannot with certainty quantify
the effect of sampling error on our results.
A total of 31 subjects without cirrhosis at baseline who
did not have end-stage liver disease at follow-up did not
undergo repeat liver biopsy. Their clinical, biochemical,
and baseline histological parameters were not different,
and thus it is unlikely this group had a different histological course than the group of patients who underwent liver
biopsy at follow-up.
Although a clinicobiological score for the selection of
candidates for liver biopsy in NAFLD has been proposed
in a cross-sectional study,8 in the present long-term follow-up study we were not able to find parameters useful in
the clinical setting that could predict the progression of
fibrosis in a patient with NAFLD. The results of liver
biopsy at baseline was the best predictor of developing
cirrhosis-related complications at follow-up, with a positive predictive value of 18% in patients with periportal
fibrosis and a negative predictive value of 100% in patients without established periportal fibrosis.
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