LIVER DISEASE
NAFLD in Children: A Prospective Clinical-Pathological
Study and Effect of Lifestyle Advice
Valerio Nobili,1 Matilde Marcellini,1 Rita Devito,2 Paolo Ciampalini,3 Fiorella Piemonte,4 Donatella Comparcola,1
Maria Rita Sartorelli,1 and Paul Angulo5
Nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease in adults,
is incompletely characterized in children. We conducted a prospective study to better char-
acterize the clinical presentation of NAFLD in children and to determine the effect of
lifestyle advice in the management of pediatric NAFLD. From June 2001 to April 2003, 84
children (age 3-18.8 yr) who had elevated aminotransferases and the diagnosis of NAFLD
confirmed via liver biopsy underwent a 2-hour oral glucose tolerance test and a 12-month
program of lifestyle advice consisting of diet and physical exercise. Thirty-four (40.5%)
patients were obese (body mass index [BMI] >97th percentile), and 43 (51.2%) were over-
weight (BMI 85th-97th percentile). Ten (12%) had abnormal glucose tolerance; 10 (12%) had
elevated triglycerides, cholesterol, or both; and all had normal blood pressure. Most children
(67/84, 80%) were insulin-resistant, including the 7 children with normal BMI (<85th
percentile). Increased liver fibrosis was present in 49 (58.1%) patients and was indepen-
dently associated with obesity (OR 2.7, 95% CI 1.2-6.2) and age (1-year increase; OR 1.2,
95% CI 1.04-1.5). A 12-month program with diet and physical exercise resulted in a signif-
icant decrease in BMI, and levels of fasting glucose, insulin, lipids, and liver enzymes, as well
as liver echogenicity on ultrasonography. In conclusion, children with NAFLD are almost
always insulin-resistant regardless of BMI. Obesity and older age are independently associ-
ated with increased liver fibrosis. A simple lifestyle advice program significantly improves
insulin resistance, and the liver disease in pediatric NAFLD. (HEPATOLOGY 2006;44:458-465.)
With the increasing prevalence of obesity anddiabetes mellitus in the general population,1nonalcoholic fatty liver disease (NAFLD) has
become a common cause of chronic liver disease.2-5
NAFLD often clusters with conditions related to central
obesity, including high triglyceride levels, low high-den-
sity lipoprotein cholesterol levels, high blood pressure,
and hyperglycemia.6 NAFLD may present as a spectrum
of disease from asymptomatic steatosis with or without
elevated aminotransferases to cirrhosis with complica-
tions of liver failure and hepatocellular carcinoma.7 Re-
cent studies on natural history have demonstrated that liver-
related morbidity and mortality usually occur when the
disease has progressed to advanced fibrosis and cirrhosis.8,9
Over the last 4 decades, the prevalence of overweight
and obesity in children has increased dramatically.10 In
the United States, the prevalence of overweight and obe-
sity among children aged 6 to 19 years was 31% and 16%,
respectively, in 2001-2002 compared with a 5% obesity
prevalence reported in 1963-1965.10 Given the strong
association of NAFLD with increased body mass index
(BMI), NAFLD affects a substantial proportion of chil-
dren. The reported prevalence of NAFLD among chil-
dren is 2.6%, but this increases to as much as 53% among
obese children.11,12 Paralleling the increasing prevalence
of obesity in the pediatric population, NAFLD is ex-
pected to become one of the most common causes of
end-stage liver disease in both children and young adults.
Although NAFLD is common and potentially serious,
few series of pediatric NAFLD have been reported.13-18
Further data on the clinical presentation of NAFLD and
the prevalence of underlying metabolic abnormalities in
children are necessary. Additionally, there are few studies
on the effect of therapeutic intervention with lifestyle
Abbreviations: NAFLD, nonalcoholic fatty liver disease; BMI, body mass index;
OGTT, oral glucose tolerance test; HOMA, homeostatic model assessment; ISI,
insulin sensitivity index; NAS, NAFLD activity score; NASH, nonalcoholic steato-
hepatitis; ALT, alanine aminotransferase.
From the 1Liver Unit and the Departments of 2Pathology, 3Endocrinology, and
4Molecular Medicine, Bambino Gesu` Children’s Hospital, Rome, Italy; and the
5Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine,
Rochester, MN.
Received November 9, 2005; accepted May 2, 2006.
Address reprint requests to: Valerio Nobili, M.D., Liver Unit, Research Institute,
Bambino Gesu` Children’s Hospital, S. Onofrio 4 Square, 00165 Rome, Italy.
E-mail: nobili66@yahoo.it; fax: (39) 668592192.
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.21262
Potential conflict of interest: Nothing to report.
458

modifications on liver disease in children. We report a
prospective study of 84 children with biopsy-proven
NAFLD. We sought to better characterize the presenta-
tion of NAFLD in children and to determine the benefi-
cial effects of lifestyle intervention with diet and physical
exercise in children with NAFLD.
Patients and Methods
The study met the ethical guidelines of the 1975 Dec-
laration of Helsinki and was approved by the Ethics Com-
mittee of the Bambino Gesu` Children’s Hospital and
Research Institute. Informed consent was obtained from
all patients or their guardians.
Patients. Eighty-four consecutive, untreated patients
(59 male, 25 female) with a mean age of 11.7
3.3 years
(range, 3-18.8) who were seen in our institution from
June 2001 to April 2003 were included in the study. All
patients were evaluated in our unit due to elevated serum
aminotransferase levels. Aminotransferase levels were per-
sistently elevated in 69 patients but fluctuated between
normal and elevated in 15 patients. Patients with fluctu-
ating levels had at least 2 measurements of elevated ami-
notransferases during medical follow-up before liver
biopsy was performed. In all 84 patients, liver ultrasonog-
raphy revealed increased echogenicity suggestive of steato-
sis, and liver biopsy confirmed NAFLD as the only
explanation for the elevated aminotransferase levels. Poly-
merase chain reaction tests revealed that all patients were
HCV RNA–negative. Secondary causes of steatosis, in-
cluding alcohol abuse (
140 g/wk), total parenteral nu-
trition, and the use of medications known to precipitate
steatosis were excluded in all cases. Hepatitis A, B, C, D,
E, and G; cytomegalovirus; and Epstein-Barr virus infec-
tions were ruled out with appropriate tests. In all cases,
autoimmune liver disease, metabolic liver disease, Wil-
son’s disease, and -1-antitrypsin were ruled out using
standard clinical and laboratory evaluation as well as liver
biopsy features. To compare BMI among different ages
and in both boys and girls, the BMI Z score was calcu-
lated. The Z score represents the number of standard
deviations above or below the considered population
mean value based on standardized tables for children.19
Obesity was defined as a BMI above 2 standard deviations
that corresponds to a percentile above the 97th percentile
adjusted for age and sex.Overweight was defined as a BMI
within the 85th to the 97th percentile. Hypertriglyceri-
demia was defined as fasting triglyceride level 160 mg/
dL, and hypercholesterolemia when fasting total
cholesterol levels were200 mg/dL.20-22
Evaluation of Glucose Metabolism and Insulin
Sensitivity. All patients underwent a 2-hour oral glucose
tolerance test (OGTT) with the standard 1.75 g/kg of
glucose, or a maximum of 75 g. Glucose tolerance status
was determined according to the recently revised Ameri-
can Diabetes Association classification, in which fasting
plasma glucose levels up to 99 mg/dL are considered nor-
mal; impaired fasting glucose is defined by a fasting
plasma glucose level of 100 to 125 mg/dL; impaired glu-
cose tolerance is defined by a 2-hour plasma glucose level
of 140 to 199 mg/dL; and diabetes mellitus is defined by
a fasting plasma glucose level
126 mg/dL or a 2-hour
plasma glucose level
200 mg/dL.23
The degree of insulin sensitivity/resistance was deter-
mined via the homeostatic model assessment (HOMA)
using the formula insulin resistance  (insulin  glu-
cose)/22.524 and by the OGTT-derived insulin sensitivity
index (ISI) using the formula ISI  (10,000/square root
of (fasting glucose fasting insulin) (mean glucose
mean insulin during OGTT).25 Both the HOMA and the
OGTT-derived ISI have a significant correlation with the
gold standard euglycemic hyperinsulinemic glucose
clamp technique. A HOMA value 2 or ISI value 6
were considered an indication of insulin resistance.
Liver Histology. Biopsies were performed in all chil-
dren using an automatic core biopsy device (Biopince,
Amedic, Sweden) with a 150-mm-long 18-gauge needle
able to cut tissue with extreme precision in lengths up to
33 mm.26 Liver biopsies were at least 15 mm long and
were read by a single blinded liver pathologist. Biopsies
were routinely processed (i.e., formalin-fixed and paraf-
fin-embedded) and analyzed in sections stained with he-
matoxylin-eosin, Van Gieson, PAS-D, and Prussian blue
stain. Iron storage was scored from 0 to 4 according to
Searle et al.27 Immunohistochemical staining with -1-
antitrypsin was used to exclude -1-antitrypsin defi-
ciency–associated liver disease.
Histological features including steatosis, inflammation
(portal and lobular), hepatocyte ballooning, and fibrosis
were scored according to the scoring system for NAFLD
recently developed by the NIH-sponsored NASH Clini-
cal Research Network.28 The features of steatosis (0-3),
lobular inflammation (0-3), and hepatocyte ballooning
(0-2) were combined into a score that ranged from 0 to 8
known as the NAFLD activity score (NAS). Cases with
NAS
5 have the diagnosis of nonalcoholic steatotohepa-
titis (NASH), cases with NAS 2 are considered “non-
NASH,” and cases with a NAS of 3 or 4 are considered
“borderline.”
Furthermore, the pattern of liver injury on liver biopsy
was recorded to determine the proportion of children
meeting criteria for type 1 and type 2 steatohepatitis as
described recently by Schwimmer at al.18 Briefly, type 1
NASH refers to the presence of steatosis with hepatocyte
ballooning and/or perisinusoidal fibrosis in the absence of
HEPATOLOGY, Vol. 44, No. 2, 2006 NOBILI ET AL. 459