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Contents lists available at ScienceDirect

Digestive and Liver Disease

journal homepage: www.elsevier.com/locate/dld

Liver, Pancreas and Biliary Tract

Reduced lysosomal acid lipase activity – A potential role in the

pathogenesis of non alcoholic fatty liver disease in pediatric patients
Praveen Kumar Conjeevaram Selvakumar a , Mohammad Nasser Kabbany a , Rocio Lopez b ,
Giulia Tozzi d , Anna Alisi c , Naim Alkhouri a,b,∗ , Valerio Nobili c,∗∗
a
Department of Pediatric Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, USA
b
Digestive Disease Institute, Cleveland Clinic, Cleveland, OH, USA
c
Liver Unit of IRCCS Bambino Gesù Children’s Hospital – IRCCS, Rome, Italy
d
Neurologic and Muscle Disease Unit of IRCCS Bambino Gesù Children’s Hospital – IRCCS, Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Background: Within the spectrum of nonalcoholic fatty liver disease (NAFLD), recent evidence suggests
Received 23 February 2016 that adult patients with nonalcoholic steatohepatitis (NASH) have significantly lower blood lysosomal
Accepted 21 April 2016 acid lipase (LAL) activity than those with steatosis. This has not been studied in pediatric patients with
Available online xxx
NAFLD.
Aim: Investigate blood LAL activity in pediatric patients with NAFLD and assess its correlation with
Keywords:
histological severity.
Liver fibrosis
Methods: We collected data on consecutive children with biopsy-proven NAFLD including demographics,
Lysosomal acid lipase activity
Nonalcoholic fatty liver disease
anthropometrics, and routine laboratory tests. The histological features were graded according to the
Nonalcoholic steatohepatitis NAFLD activity scoring proposed by Kleiner et al. Blood LAL activity was measured prospectively using
Lalistat 2.
Results: A total of 168 children were included for analysis. Mean age was 12.6 ± 8.5 years, 60.1% were
males and 52.4% had NASH. Children with significant fibrosis (stage 2–3, n = 64) had a significantly lower
LAL activity compared to those with mild fibrosis (stage 0–1, n = 104). There was no significant difference
in LAL activity between children with NASH compared to those without NASH.
Conclusion: Reduced blood LAL activity correlates with severity of liver fibrosis in children with NAFLD
indicating a potential role of reduced LAL activity in the pathogenesis of NAFLD-induced fibrosis.
© 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

1. Introduction in the general pediatric population and 50–70% in obese children

Nonalcoholic fatty liver disease (NAFLD), characterized by accu-
mulation of triglyceride (TG) and free cholesterol in hepatocytes, is
considered the hepatic manifestation of metabolic syndrome [1].
NAFLD encompasses a spectrum of diseases ranging from sim-
ple steatosis to nonalcoholic steatohepatitis (NASH) which can
progress to fibrosis and eventually cirrhosis. There has been a
substantial increase in the prevalence of NAFLD in children and ado-
lescents in Western countries with a reported prevalence of 3–10%
∗ Corresponding author at: Chief of Hepato-metabolic Diseases Unit, Head of Liver
Research Unit, Bambino Gesù Children Hospital, Rome, Italy. Tel.: +39 0668592192.
∗∗ Corresponding author at: Cleveland Clinic Main Campus, Mail Code
A111, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel.: +1 216 445 7126;
fax: +1 216 444 2974.
E-mail addresses: alkhoun@ccf.org (N. Alkhouri), nobili66@yahoo.it (V. Nobili).
[2,3].
Lysosomal acid lipase (LAL) is a hydrolase which plays a key role
in lipid metabolism through the hydrolysis of TG and cholesterol
esters in lysosomes. LAL is also shown to help in degradation of
intracellular lipids through a lysosomal autophagy pathway [4,5].
Therefore LAL deficiency (LAL-D), an autosomal recessive disease,
leads to progressive accumulation of cholesteryl esters and TG in
the liver, spleen, dyslipidemia and premature atherosclerosis [6].
LAL-D is a heterogeneous disease that can present in childhood or
adolescence with hepatomegaly, elevated transaminases and fatty
infiltration of the liver, all of which are characteristic features of
the more common NAFLD [7].
A recent study reported reduced blood LAL activity in adult
patients with evidence of NAFLD on ultrasonography (US) com-
pared to normal weight healthy controls [8]. In the NAFLD
cohort, patients with reduced LAL activity also had higher serum
total cholesterol and liver enzymes [8]. More importantly, in a

http://dx.doi.org/10.1016/j.dld.2016.04.014
1590-8658/© 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Selvakumar PKC, et al. Reduced lysosomal acid lipase activity – A potential role in the pathogenesis
of non alcoholic fatty liver disease in pediatric patients. Dig Liver Dis (2016), http://dx.doi.org/10.1016/j.dld.2016.04.014
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sub-analysis of 35 patients with biopsy-proven NAFLD, the authors
observed a progressive decrease of LAL activity from patients with
simple steatosis to those with NASH. Furthermore, LAL activity
below the median value had a positive association with ALT val-
ues and the presence of NASH leading the authors to speculate that
reduced LAL activity may contribute to liver damage and disease
progression. However, LAL activity in children with NAFLD has not
been investigated. The aim of this study was to examine blood LAL
activity in pediatric patients with NAFLD and assess its correlation
with the histological severity of NAFLD and the presence of NASH.
2. Materials and methods
2.1. Patient population
Consecutive children with evidence of hepatic steatosis on liver
ultrasonography who were evaluated at the Liver Unit of the Bam-
bino Gesù Children’s Hospital from July 2012 to July 2014 were
included after approval by the Ethics Committee of the Bam-
bino Gesù Children’s Hospital and Research Institute, Rome, Italy.
Informed consent was obtained from each patient or responsible
guardian. Patients were excluded if they had any of the following:
(1) alcohol consumption; (2) hepatic viral infections (such as hep-
atitis A, B, C, D, and E; cytomegalovirus; and Epstein–Barr virus); (3)
history of parenteral nutrition; (4) use of drugs that are known to
induce steatosis (e.g., prednisone, valproate, or amiodarone); and
(5) known liver disease, like autoimmune hepatitis and metabolic
liver diseases such as alpha-1-antitrypsin deficiency, glycogen stor-
age diseases, organic acidemias, urea cycle defect, tyrosinemia,
mitochondrial hepatopathies or Wilson’s disease were excluded.
These were ruled out using standard clinical, laboratory, and histo-
logical criteria.
2.2. Anthropometrical measurements
Clinical variables were recorded, which included standard pro-
cedures for height and weight [9]. The body mass index (BMI) and
its standard deviation score (Z score) were calculated [10]. Obe-
sity was defined by a BMI greater than or equal to 95th percentile,
adjusted for age and sex according to CDC growth charts. Waist cir-
cumference (WC) was also measured, which was evaluated at the
highest point of the iliac crest with a standing subject [11].
2.3. Laboratory assessment
Laboratory data collection included total cholesterol, high
density lipoprotein (HDL) cholesterol, low density lipoprotein
(LDL) cholesterol, TG, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), serum ␥-glutamyltransferase (GGT), fas-
ting glucose and insulin. These were obtained and measured by
standard laboratory methods. The homeostasis model assessment
of insulin resistance (HOMA-IR) [12] was calculated as a measure
for insulin resistance.
2.4. Liver biopsy
The clinical indication for biopsy was either to assess the
presence of NASH and degree of fibrosis or other likely inde-
pendent or competing liver diseases. Liver biopsy was performed
in all children, after an overnight fast, using an automatic core
biopsy 18 Gauge needle (Biopince, Amedic, Sweden) under general
anaesthesia and ultrasound guidance. A Sonoline Omnia Ultra-
sound machine (Siemens, Germany) equipped with a 5-MHz probe
(5.0 C 50, Siemens) and a biopsy adaptor was employed. Two
biopsy passes within different liver segments were performed
for each subject. The length of liver specimen (in millimetres)
Please cite this article in press as: Selvakumar PKC, et al. Reduced lysosomal acid lipase activity – A potential role in the pathogenesis
of non alcoholic fatty liver disease in pediatric patients. Dig Liver Dis (2016), http://dx.doi.org/10.1016/j.dld.2016.04.014
was recorded. Only samples with a length ≥15 mm and includ-
ing at least 6 complete portal tracts were considered adequate
for the purpose of the study. Biopsies were evaluated by a sin-
gle pathologist who was blinded to clinical and laboratory data.
Biopsies were routinely processed (i.e., formalin-fixed and paraffin-
embedded) and sections of liver tissue, 5 ␮m thick, were stained
with hematoxylin–eosin, Van Gieson, PAS-D, and Prussian blue
stain. Liver biopsy features were graded according to the NAFLD
activity scoring (NAS) system proposed by Kleiner et al. [13].
2.5. DBS preparation
The dried blood spot (DBS) samples were prepared using What-
man paper #903, according to the requirements of the National
Committee for Clinical Laboratory Standard Protocol (NCCLS).
Blood was spotted on to filter paper on the day of venipuncture
and allowed to dry overnight at room temperature. Samples were
analyzed within 1 week of collection.
2.6. LAL activity
A new method for determining LAL activity in dried blood spot
(DBS) has been developed [14] by using the fluorimetric substrate
4-methylumbelliferyl palmitate (4MU palmitate) with cardiolipin
present as an activator of LAL. Other lipases in whole blood may
interfere with the measurement of LAL in DBS, so a LAL inhibitor
is used. Lalistat 2 (Chemicals Tools, South Bend, IN, USA) is a spe-
cific inhibitor of LAL [15,16] and by measuring total lipase activity
and lipase activity in presence of Lalistat 2, LASL activity can be
determined in DBS.
A reference range was established using blood from sam-
ples submitted to our laboratory for routine diagnostic testing.
In healthy individuals, mean LAL activity is approximately
1.00 nmol/spot/h with a range of 0.73–3.03 nmol/spot/h. LAL
activity less than or equal to 0.03 nmol/spot/h (<3% of nor-
mal) is considered consistent with LAL-D. LAL activity less than
0.73 nmol/spot/h was considered reduced [14].
2.7. Statistical analysis
Statistical analysis was performed using STATISTICA soft-
ware version 2010 (Chicago, IL, USA). Continuous variables were
normally distributed and were expressed as mean ± standard devi-
ation (SD). To compare the means of continuous variables between
groups the Student’s t-test was performed. Chi-squared test was
used to determine the distribution of categorical variables between
groups. To determine differences between groups for continuous
non-normally distributed variables, medians were compared using
the Mann–Whitney U or Kruskal–Wallis test. Pearson’s chi-square
test was used for categorical factors. Spearman rank correla-
tion coefficients (rho) were calculated to assess the relationship
between LAL activity and anthropometrical and biochemical fea-
tures.
3. Results
3.1. Correlation of LAL activity with anthropometric and
laboratory characteristics
A total of 168 children with liver biopsy were included for
analysis. Mean age was 12.6 ± 8.5 years and 60.1% were males.
Demographic, anthropometric and laboratory characteristics are
listed in Table 1. In our population, mean BMI was found to be 27.1
with a mean BMI z-score of 3. Mean LAL activity for 168 children
was 1.3 nmol/spot/h. Spearman rank correlation showed a weak
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Fig. 1. Box and whisker plot showing association between lysosomal acid lipase activity and individual histology features. LAL, lysosomal acid lipase activity.

Table 1 3.2. Correlation of LAL activity and liver histology

Demographic, anthropometric and laboratory data of study population (N = 168).

Factor Summary Total (N = 168) Table 2 presents a summary of the histological features of
Data available (n) the liver biopsy specimens in 168 children. NASH was diagnosed
Age (years) 12.6 ± 8.5 168 in 52.4% of the patients by an experienced pathologist. None of
Male 101 (60.1) 168 the liver biopsies showed liver cirrhosis. There was no signif-
Body mass index (kg/m2 ) 27.1 ± 5.4 168 icant association between LAL activity and different grades of
Body mass index z-score 3.0 ± 1.7 168
steatosis, lobular inflammation, portal inflammation, or balloon-
Waist circumference (cm) 86.3 ± 11.8 127
Waist circumference height ratio 0.56 ± 0.08 127 ing (Tables 3 and 4, Fig. 1). Similarly there was also no significant
AST (UI/L) 26.0 (22.0, 32.0) 168 difference in LAL activity between children with NASH (1.2 ± 0.80)
ALT (UI/L) 26.5 (20.0, 38.0) 168 compared to those without NASH (1.3 ± 0.57) as shown in Fig. 2.
GGT (UI/L) 15.0 (12.0, 22.0) 167
However children with significant fibrosis (stage 2–3, n = 64) had
Total cholesterol (mg/dl) 160.6 ± 37.4 168
LDL-cholesterol (mg/dl) 98.6 ± 35.0 168 a significantly lower LAL activity (1.10 ± 0.45) compared to those
HDL-cholesterol (mg/dl) 44.0 (38.0, 50.0) 165 with mild fibrosis (stage 0–1, n = 104, LAL activity 1.4 ± 0.80).
Triglycerides (mg/dl) 86.0 (62.0, 124.0) 168 Similar results were also obtained after adjusting for age, BMI,
Fasting glucose (mg/dl) 81.7 ± 12.3 166 triglycerides and cholesterol. Of note there was no significant dif-
Fasting insulin (mg/dl) 14.8 (9.7, 22.6) 150
ference in ALT and AST levels between mild and significant fibrosis
HOMA-IRa 3.8 ± 2.2 150
LALb activity (nmol/spot/h) 1.3 ± 0.70 168 groups (ALT: 25.5 [20.0, 36.0] vs. 28.0 [18.0, 49.0]; p value = 0.94,
AST: 26.0 [22.0, 31.0] vs. 28.0 [23.0, 37.0]; p value = 0.16 respec-
Values presented as mean ± SD, median (P25, P75) or N (%).
a
Homeostasis model assessment of insulin resistance.
tively).
b
Lysosomal acid lipase.
4. Discussion

but significant negative correlation between LAL activity and BMI z-
score (rho = −0.28), total cholesterol (rho = −0.22), LDL cholesterol
(rho = −0.21), and HOMA-IR (rho = −0.31); p-value <0.05 for all of
them. There was no significant correlation of LAL activity with WC,
WC/height ratio, HDL cholesterol or TG.
Reduced LAL activity has been shown to be associated with
intrahepatic lipid accumulation and dyslipidemia [4,17]. Reduced
LAL activity may also impair lipid degradation and affect the
cholesterol export pathway from the liver contributing to the devel-
opment of NAFLD which was supported by recent evidence from

Please cite this article in press as: Selvakumar PKC, et al. Reduced lysosomal acid lipase activity – A potential role in the pathogenesis
of non alcoholic fatty liver disease in pediatric patients. Dig Liver Dis (2016), http://dx.doi.org/10.1016/j.dld.2016.04.014
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Table 2
Liver histology features.

Histology features Total N = 168

Summary N (%)

Steatosis grade
0: <5% 4 (2.4)
1: 5–33% 41 (24.4)
2: 34–65% 90 (53.6)
3: ≥66% 33 (19.6)
Lobular inflammation grade
0: No foci 27 (16.1)
1: <2 foci per 200× field 112 (66.7)
2: 2–4 foci per 200× field 29 (17.3)
Portal inflammation grade
0: None 54 (32.1)
1: Mild 89 (53.0)
2: More than mild 25 (14.9)
Ballooning grade
0: None 84 (50.0)
1: Few 62 (36.9)
2: Many 22 (13.1)
NASa 3.5 ± 1.5
Pathologist diagnosis
Not NASHb 80 (47.6)
NASH 88 (52.4)
Fibrosis stage Fig. 2. Box and whisker plot showing association between lysosomal acid lipase
0: None 46 (27.4) activity and nonalcoholic steatohepatitis. LAL, lysosomal acid lipase activity; NASH,
1: Perisinusoidal or periportal 58 (34.5) nonalcoholic steatohepatitis.
2: Perisinusoidal and periportal 52 (31.0)
3: Bridging 12 (7.1)
a
Nonalcoholic fatty liver disease activity scoring. although many uncertainties remain regarding the enzymes
b
Nonalcoholic steatohepatitis. responsible for the intracellular lipid degradation. More recently,
the central role of the lysosomal pathway in the metabolism and
storage of lipids in the hepatocytes has been discovered. LAL
adults [8]. Therefore, we investigated the activity of LAL in pedi- belongs to a group of acid hydrolases that require a very low pH
atric patients with biopsy-proven NAFLD. The main findings of our (4.5–5) for optimal function therefore it is sequestered in lysosomes
study are: (1) There was a negative correlation between LAL activ- [21]. Autophagy makes it possible for lipid droplets to reach the
ity and BMI z-score, total cholesterol, LDL cholesterol and HOMA-IR lysosomes via the formation of autophagosomes that can fuse with
suggesting a worse metabolic profile in children with NAFLD and lysosomes. Reduced LAL activity may impair lipid degradation and
reduced LAL activity; (2) Among children with NAFLD, those with affect the cholesterol export pathway from the liver contributing to
significant fibrosis had lower LAL activity than children with mild hypercholesterolemia, insulin resistance and potentially the devel-
fibrosis. In contrast to recent adult study where lower LAL activity opment of NAFLD and its progression. This is further supported by
was reported in patients with NASH compared to those with sim- evidence from study by Singh et al. which underlines the impor-
ple steatosis, we found no association between LAL activity and tance of autophagy and lysosomal pathway in the degradation of
the presence of NASH or its individual histological features [8]. intracellular lipid storage in liver [4].
Our study included a significantly larger number of patients with The presence and stage of liver fibrosis are predictors of long
biopsy-proven NAFLD (n = 168) in comparison to the adult study term overall and liver-related prognosis in patients with NAFLD
(n = 35). independent of other histologic features including the presence of
Several studies have discussed the role of hepatic lipid storage NASH [22,23]. In spite of the high prevalence of NAFLD in children,
in the development of the metabolic syndrome and NAFLD [18–20] only a small proportion progress to fibrosis leading to cirrhosis. In a

Table 3
Associations between lysosomal acid lipase activity and histological features.

Feature Presence NASHa Fibrosis stages 2–3 Steatosis grades 2–3 Lobular inflammation grades 1–2 Portal inflammation grade 2 Ballooning grade 2

No 1.3 ± 0.57 1.4 ± 0.80 1.2 ± 0.54 1.3 ± 0.73 1.3 ± 0.72 1.2 ± 0.63
Yes 1.2 ± 0.80 1.10 ± 0.45 1.3 ± 0.75 1.1 ± 0.47 1.05 ± 0.51 1.4 ± 1.08
p-Value 0.57 0.013 0.85 0.18 0.097 0.41

Values presented as mean ± SD.

p-Values correspond to Student’s t-tests.
a
Nonalcoholic steatohepatitis.

Table 4
Associations between lysosomal acid lipase activity and histological features after adjusting for age, body mass index, triglycerides and cholesterol.

Feature presence NASH a Fibrosis stages 2–3 Steatosis grades 2–3 Lobular inflammation grades 1–2 Portal inflammation grade 2 Ballooning grade 2

No 1.3 (1.1, 1.4) 1.4 (1.2, 1.5) 1.2 (1.02, 1.4) 1.3 (1.2, 1.4) 1.3 (1.2, 1.4) 1.2 (1.1, 1.4)
Yes 1.3 (1.1, 1.4) 1.10 (0.93, 1.3) 1.3 (1.2, 1.4) 1.07 (0.82, 1.3) 1.07 (0.80, 1.3) 1.4 (1.09, 1.7)
p-Value 0.79 0.015 0.65 0.097 0.13 0.42

Values presented as mean (95% CI).

p-Values correspond to ANCOVA.
a
Nonalcoholic steatohepatitis.

Please cite this article in press as: Selvakumar PKC, et al. Reduced lysosomal acid lipase activity – A potential role in the pathogenesis
of non alcoholic fatty liver disease in pediatric patients. Dig Liver Dis (2016), http://dx.doi.org/10.1016/j.dld.2016.04.014
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study by Alkhouri et al., only 15% of 67 children with biopsy proven
NAFLD were found to have significant fibrosis (stage 2–3) [24].
Genetic polymorphisms such as PNPLA3 I148M and TM6SF2 E167K
have also been shown to predispose certain patients to higher rates
of NAFLD progression to fibrosis [25].
Evidence of low LAL activity in patients with significant liver
fibrosis in our study suggest that partial LAL deficiency may be
present in a subset of children with NAFLD leading to faster disease
progression to fibrosis and potentially cirrhosis. Whether LAL could
become a potential therapeutic target for advanced NAFLD in these
patients with low activity remains to be determined. Interestingly,
a recent phase 3 randomized controlled trial that included adults
and children with LALD showed that enzyme replacement therapy
with Sebelipase Alfa, a recombinant LAL, significantly decreased
hepatic lipid content and improved ALT [26]. Enzyme replacement
therapy with recombinant human LAL in Lipa knock out lal−/−
mice with advanced disease diminished liver fibrosis implicating a
potential role for LAL in the progression of liver fibrosis [27]. Long-
term prospective follow up studies are needed to understand the
cause–effect relationship of reduced LAL activity with pathogenesis
and progression of NAFLD.
Our study is the first study to include a cohort of children with
biopsy-proven NAFLD to assess the correlation between LAL activ-
ity and histologic features of NAFLD. The limitations of the study
include the fact that our patients were referred to a large tertiary
care medical center, with a high prevalence of NAFLD and NASH.
Thus, these findings may not be generalizable to the pediatric popu-
lation. Another limitation is the lack of healthy controls to compare
LAL activity between healthy subjects and patients with NAFLD.
Small sample size may have limited our power to detect the true
association between LAL activity and histologic features of NAFLD
other than fibrosis.
In summary, our study demonstrated low LAL activity in chil-
dren with NAFLD and significant liver fibrosis which might point
towards a potential role of LAL activity in pathogenesis of hepatic
fibrosis in NAFLD. However, long-term prospective follow up stud-
ies are needed to understand the cause–effect relationship of
reduced LAL activity with disease progression. Further insight in to
the role of LAL activity in NAFLD might help to develop novel treat-
ment strategies such as enzyme replacement therapy for pediatric
NAFLD.
Conflict of interest
None declared.
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