Am J Hum Genet 33:203-208, 1981
Acid Lipase Cross-Reacting Material in Wolman Disease
and Cholesterol Ester Storage Disease
BARBARA K. BURTON' AND SUZANNE P. REED
Material cross-reacting with antibodies to acid lipase was demonstrated in
fibroblasts of three patients with Wolman disease and three with choles-
terol ester storage disease. Quantitation of the immunologically cross-
reacting material (CRM) by a single radial immunodiffusion method
revealed normal levels in both mutant cell types. CRM specific activity
toward triolein and cholesteryl oleate was reduced about 200-fold in the
Wolman disease fibroblasts and 50- to 100-fold in the cholesterol ester
storage disease cells when compared to normal.
Wolman disease and cholesterol ester storage disease are genetically distinct auto-
somal recessive disorders characterized biochemically by a deficiency of lysosomal
acid lipase activity in tissues of affected individuals [1-3]. Patients with Wolman
disease exhibit failure to thrive, vomiting and diarrhea, hepatosplenomegaly, and
enlarged, calcified adrenal glands, and die in early infancy . Massive accumula-
tion of cholesterol esters and triglycerides can be demonstrated in most tissues .
Cholesterol ester storage disease, a milder disorder, is characterized clinically by
hepatomegaly and hypercholesterolemia . Cholesterol ester accumulation occurs
and is accompanied by a variable accumulation of triglycerides .
Two acid lipases of different molecular weights have recently been purified from
normal human placenta: a larger acid lipase (I) with a mol. wt. of 102,500 and a
Received May 28, 1980; revised August 12, 1980.
This work was supported by the North Carolina United Way and by a Basil O'Connor Starter
Research grant from the March ofDimes Birth Defects Foundation to B. K. B. The study was presented
in part at the meeting of the Society for Pediatric Research, San Antonio, Texas, April 30-May 2, 1980.
Both authors: Department of Pediatrics, Bowman Gray School ofMedicine, 300 S. Hawthorne Rd.,
Winston-Salem, NC 27103.
© 1981 by the American Society of Human Genetics. 0002-9297/81/3302-0004$02.00
BURTON AND REED
smaller acid lipase (II) with a mol. wt. of 30,600 . The two enzyme forms were
shown to possess similar biochemical characteristics, but the relationship between
the two was not established. To further clarify this issue and to investigate the nature
ofthe genetic mutation affecting acid lipase activity in Wolman disease and choles-
terol ester storage disease, antibodies were prepared to both acid lipases from
normal human placenta. Using these antibodies, immunologic identity was demon-
strated for the two enzyme preparations. In addition, CRM identical with the
normal was demonstrated in cultivated fibroblasts from patients with both ofthese
disorders. A sensitive immunoassay for acid lipase CRM was developed, making
possible precise quantitation of the CRM in normal and mutant cells.
MATERIALS AND METHODS
Cholesteryl [1-'4C]oleate and glycerol tri[1-'4C]oleate (triolein) were purchased from
Amersham-Searle (Arlington Heights, Ill.). Unlabeled cholesteryl oleate and triolein and
bovine serum albumin were from Sigma (St. Louis, Mo.). Eagle's minimal essential medium
(MEM), fetal calf serum, Freund's complete adjuvant, and goat antiserum to rabbit immu-
noglobulins were obtained from Gibco (Grand Island, N.Y.). Agarose was from Bio-Rad
Acid lipase activity was assayed as described  using either cholesteryl oleate or triolein as
substrate. Protein assays by the Folin method  used bovine serum albumin as a standard.
Two peaks ofacid lipase activity, designated acid lipases I and II, were purified from normal
human placenta as described .
Preparation of normal placenta homogenates was done as described . Three fibroblast
cultures from patients with Wolman disease (GM 1606, GM 2109, and GM 2211) and one
from a patient with cholesterol ester storage disease (GM 0863) were obtained from the
Human Mutant Cell Repository, Camden, N.J. An additional cell line from a patient with
cholesterol ester storage disease (K. W.) was obtained from Dr. A. L. Beaudet and a third
(P. W.) from Dr. P. M. Coates. Control fibroblast cultures were from skin biopsies from
normal individuals. Cells were cultivated in MEM supplemented with 15% fetal calf serum
and a commercial antibiotic-antimycotic solution in an atmosphere of5% CO2. At confluen-
cy, the medium was decanted, and the cells washed twice with normal saline, then harvested
using 0.25% trypsin in normal saline. The cell pellets were washed twice in normal saline and
resuspended in distilled water. Lysates were prepared by three 10-second bursts ofsonication
using a Sonic 300 dismembranator with microtip and were subsequently centrifuged for 10
min at 1,000 g prior to use in enzyme assays or immunodiffusion experiments.
New Zealand white rabbits were immunized by intradermal injections at multiple sites with
0.2 mg protein (acid lipase I or II) that was emulsified in Freund's complete adjuvant. The
animals were boosted after 2 weeks by a similar procedure using 0.1 mg protein. Beginning2
weeks after the booster injection, they were bled weekly from the marginal ear vein. Serum
was separated and the IgG fraction was prepared as described .
Immunoelectrophoresis was performed as described . Immunotitration of acid lipase
activity in placenta homogenates and fibroblast lysateswas performed usinga fixed quantity
of enzyme activity and increasing concentrations of the IgG fraction of antisera to acid
lipases I and II. Control experiments used the IgG fraction of serum from unimmunized
rabbits. Following incubation for 1 hr at 370C, the mixtures were kept at 4VC for 24 hrs.
Immune complexes were centrifuged at 12,000 g for 20 min, and acid lipase activity was
measured in the supernatant.
Double immunodiffusion was done in 1% agarose gel in phosphate buffered saline (PBS),
0.01 M phosphate, 0.14 M NaCl, pH 7.0, containing 0.02% sodium azide. Single radial
immunodiffusion was done in 1% agarose gel in PBS according to Mancini et al. ,
modified as follows: the IgG fraction ofrabbit antiserum to acid lipase I or II was incorporat-
ed in the gel (0.25 mg IgG/ml), and 15 ml ofantibody-containing agarose was poured onto a
petri dish (10 cm diameter). Wells with a 3-mm diameter were punched at 10-mm intervals,
and 50 Ml of enzyme-containing solutions (approximately 300-600 mg fibroblast protein or
0.3-1.4ttgpurified acid lipase) was applied to the wells using a microliter syringe (Hamilton,
Reno, Nev., 701 N). Diffusion proceeded for 72 hrs at 40 C. The plates were washed with PBS
for 48 hrs at 250C and were then covered with goat antiserum to rabbit immunoglobulins.
Diffusion proceeded for 48 hrs at 4°C after which the diameter of the unstained precipitin
rings was measured and ring area calculated. A standard curve for determining the quantity
of acid lipase CRM in crude fibroblast lysates was generated using the purified placental
Immunoelectrophoresis performed with antiserum to acid lipase I (al) or acid
lipase II (all) and crude placenta homogenate revealed a single precipitin arc in
each case. When double immunodiffusion was done using either antiserum, lines of
identity were observed between purified placental acid lipases I and II (fig. 1) and
between the purified preparations and crude placenta homogenate. Precipitin lines
of identity were also observed between crude preparations ofplacenta and normal
fibroblasts (fig. 2). Using either antiserum, CRM was demonstrated in lysates of
fibroblasts from three patients with Wolman disease and three with cholesterol ester
C . 2 ,..V.
FIG. 1.-Double gel diffusion of antiserum to acid lipase I (al) against acid lipases I and II and
antiserum to acid lipase 11 (aII) against acid lipases I and II Plate is stained for protein with 1%7c
Coomassie Blue G-250 (Eastman, Rochester. N.Y.) in methanol:water:acetic acid (5:5:1).
BURTON AND REED
FIG. 2.-Double gel diffusion ofantiserum to acid lipase I (ac ) against placenta homogenate (p) and
a normal fibroblast lysate (F). Unstained plate.
storage disease. Furthermore, the CRM in each case was identical with the normal
(fig. 3). Greater then 94% of acid lipase activity as assayed with either cholesterol
oleate or triolein could be precipitated from preparations of normal placenta or
fibroblasts using the IgG fraction of either antiserum.
Calibration of the single radial immunodiffusion assay for the quantitation of
acid lipaseCRM was carried out using both purified placental enzyme preparations
(acid lipases I and II) in gels containing either of the two antiplacental acid lipase
antisera (aI or aII). A linear relationship was established between ring area and
FIG. 3.-Double gel diffusion ofantiserum to acid lipase I (cI) against fibroblast lysates from normal
control (N), patient with Wolman disease (W), and patient with cholesterol ester storage disease (C).
enzyme concentration for both enzyme preparations in each ofthe antibody plates,
with similar-sized rings for the two enzyme preparations.
Fibroblast lysates from normal individuals and patients with Wolman disease
and cholesterol ester storage disease were assayed for acid lipase activity toward
cholesteryl oleate and triolein. The CRM was measured in the lysates by the single
radial immunodiffusion method, and the ratio ofacid lipase activity to CRM (CRM
specific activity) was calculated (table 1). In the cell lines of patients with Wolman
disease, normal quantities of CRM were detected while the CRM specific activity
was reduced about 200-fold. Normal quantities ofCRM were also demonstrated in
the cholesterol ester storage disease cell lines, while CRM specific activity was
reduced 50- to 100-fold.
The demonstration ofapparent antigenic identity between acid lipases I and II of
normal human placenta suggests that the two enzyme forms are very closely related.
The larger acid lipase I may simply represent an aggregate ofthe smaller acid lipase
II and, ifthis is the case, it may be an artifact ofthe purification procedure that is not
of significance in vivo. Alternatively, acid lipase I may be a multimeric form of the
smaller enzyme. Another explanation is suggested by the recent demonstration of
high molecular weight precursors for a number ofdifferent lysosomal enzymes [12,
13]: acid lipase I is synthesized as an active precursor of acid lipase II and later
processed to form the smaller enzyme. Further investigation will be necessary to
distinguish between these possibilities.
The sensitive immunoassay developed in this study has made it possible to
quantitate acid lipase CRM without dependence on assays of residual enzymatic
activity. A similar approach has recently been reported for quantitation of /8-
galactosidase CRM . The extremely low levels of residual acid lipase activity in
mutant cells would make interpretation of immunotitration experiments difficult.
The double antibody technique significantly enhances the sensitivity of the single
ACID LIPASE AciiVITY AND ANTIGENICALI Y CRM IN NORMAL AND MUTANT FIBROBLASTS
SPECIFIC ACTI VII Y*
CRM SPECIFIC ACTIVITYt
Wolman disease (no. =
(no. - 3).
NOTF: Mean values are given with the rangeinparenlhescs.
*nnol substralte cleasved/hr/mg pr0t1in.
; nmol substralte cleasved/hr/upg CRM.
BURTON AND REED
radial immunodiffusion assay  and, in this case, allows for the precise quantita-
tion of as little as 0.2mugenzyme protein. Both antisera (al and all) were found to
precipitate over94% ofthe acid lipase activity in normal cells, indicating that either
could be used in the immunoassay. The apparent antigenic identity between acid
lipases I and II on double gel diffusion and the similar ring sizes for the two in the
single radial immunodiffusion assay also suggests that either could be used as a
Normal quantities of acid lipase CRM were demonstrated in fibroblasts of
patients with Wolman disease and cholesterol ester storage disease, even though
these same cells are severely deficient in acid lipase activity. This suggests that the
primary defect in both disorders is a structural gene mutation resulting in produc-
tion of a protein with normal antigenicity but markedly diminished enzymatic
We thank Drs. Arthur L. Beaudet and Paul M. Coates for generously providing us with
fibroblasts from their patients with cholesterol ester storage disease. Mrs. Brenda Cook
provided expert secretarial assistance.
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