American Journal of Medical Genetics 42549-554 (1992)
Evidence for Lipase Abnormality: High Levels of Free and Triacylglycerol Forms of Unsaturated Fatty Acids in Neuronal Ceroid-Lipofuscinosis Tissue Probal Banerjee, Amitava Dasgupta, Aristotle N. Siakotos, and Glyn Dawson Departments of Pediatrics (P.B., G.D.),Biochemistry and Molecular Biology (G.D.), and Pathology (A.D.), University of Chicago, Chicago, Illinois; Department of Pathology, Indiana University of School of Medicine, Indianapolis (A.N.S.) Total lipid obtained from normal and different forms of neuronal ceroid-lipofuscinoses (NCL) tissues was analyzed by high performance thin layer chromatography (HPTLC). We observed a large (>Sfold) increase in a lipid band corresponding to triolein for NCL dog pancreas and spleen and juvenile human NCL brain and infantile NCL spleen. The accumulation was less pronounced for the brain samples but apart from increased dolicholmonophosphate levels, other lipids appeared normal. Normal dog, goat, or human spleen contained virtually no triacylglycerol, and of the pathological controls, p-mannosidosisgoat spleen showed no triacylglycerol band at all. A sample of human spleen from a patient with lymphoma-associated splenomegaly displayed a strong triacylglycerol band, but gas chromatography-mass spectrometry (GC/MS) of the bands showed an equal increase in both saturated and unsaturated fatty acid containing triacylglycerols in the splenomegaly sample, in keeping with the notion of non-specific fat deposition in damaged tissue. In contrast, in all the NCL samples (spleen, pancreas, and brain) a prominent increase in the proportion of unsaturated fatty acids was observed in both free fatty acid and/or triacylglycerol bands following GUMS. The NCL-English setter dog pancreas showed a major presence of oleic acid (18: 1) (twofold increase) as compared to normal, while dog and infantile human NCL spleen samples and juvenile Batten brain (human) displayed a robust increase in linoleic acid (18:2) and sometimesin oleic acid and arachidonic acid (20 :4) (for infantile hu-
man NCL spleen). For the infantile human NCL spleen sample an increase in linoleic acid in both free fatty acid (3.2-fold)and triacylglycerol(10-fold)was observed. This suggests a new mechanism by which polyunsaturated fatty acids can be transported to various parts of an NCL-affected mammalian body in packages such as triacylglycerol and/or serum a1bumin:free fatty acid complex. Such polyunsaturated fatty acids could eventually lead to the ceroid-lipofuscin pigment characteristic of NCl tissues through peroxidation to aldehyde with subsequent Schiff base formation. This could possibly explain the deposition of ceroid-lipofuscin in widely different and non-proximal tissues such as the pancreas and the brain.
KEY WORDS: Batten, ceroid, triacylglycerol, NCL
INTRODUCTION The neuronal ceroid-lipofuscinoses (NCL) (Batten disease) are a group of inherited autosomal recessive disorders characterized clinically by dementia, seizures, and visual loss and neuropathologically by phenomenal loss of neurons, gliosis [Zeman, 1976; Armstrong et al., 1982;Lenn and Dawson, 19731, and neuronal accumulation of a characteristic autofluorescent pigment called ceroid or lipofuscin. Four forms of NCL have been described according t o the age of onset: infantile, late infantile, juvenile, and adult. Batten disease is the most common neurovisceral storage disease with incidence as high as one in 12,500 live births with about 440,000 carriers in the USA. Despite extensive investigation, the underlying biochemical defect has eluded detection. Initially, attempts Received for publication May 15, 1991; revision received Au- were made to locate changes in total lipid composition of NCL tissue [Anderson and Sperling, 1971; Hagberg et gust 21, 1991. Address reprint requests to Dr.Probal Banejee, Kennedy Cen- al., 1974; Svennerholm, 19751, but soon afterwards the ter, University of Chicago, Department of Pediatrics, 5841 major focus was laid on the autofluorescent pigment. Since then, a most exhaustive effort has been made by S. Maryland Avenue, Box 413,Chicago, IL 60637. 0 1992 Wiley-Liss, Inc.
550
Banerjee et al.
several groups over 15 years to elucidate the composition of the fluorescent deposit, and this in tu rn has led to the knowledge that lipids such as dolichol, dolichol phosphate [Kitani et al., 19901, and dolichol-linked oligosaccharides [Hall et al., 19901 and proteins such a s the dicyclohexylcarbodiimide (DCCD) reactive, lipid binding subunit (c) of ATP synthase [Palmer et al., 19891, non-oxidative modification products of proteins by cysteine methylation [Katz, 19901, and 4-hydroxynonenal [Esterbauer et al., 19841are constituents of the lipopigment. There have also been reports of increased proportions of unsaturated fatty acids in phospholipids obtained from fractions enriched in the autofluorescent deposit [Pullarkat et al., 19821 and also other reports of no change in ovine NCL pigment phospholipids [Palmer et al., 19891.It has been observed that phospholipase A, activity is diminished in NCL samples and also that 4-hydroxynonenal, which is reportedly produced in excess in NCL, strongly inhibits lysosomal enzymes such as cathepsins and thiol-dependent phospholipase A2 [Dawsin and Glaser, 1988; Dawson et al., 19901.Because of the disappearance of lipid enriched gray matter from NCL brain we expected this tissue to be of limited value unless obtained before total loss of all gray matter. With this idea in mind we looked a t the total lipid extracts from dog pancreas and spleen, and human spleen and brain directly without any fractionation by column chromatography, and we report the presence of abnormally high levels of unsaturated fatty acids in neutral lipids of NCL tissue. MATERIALS AND METHODS High performance thin layer chromatography (HPTLC) plates (10 x 10 cm) were of the LHP-K type (lot No. 004559) with 2 cm wide preadsorbent layer were obtained from Whatman Chemical Separation Inc. (Clifton, NJ). The standard lipids (triolein, methyloleate, diolein, dolichol, dolicholmonophosphate, oleic acid, galactosylceramide, phosphatidylethanolamine and phosphatidylserine, phosphatidylcholine and phosphatidylinositol) were obtained from Sigma Chemical Co. (St. Louis, MO). All reagents and solvents were of analytical grade, and the HPTLC plates were routinely prewashed by developing with chloroform : methanol : water (60:40:10, vlv) and activated for 1 h at 150°C before use. Gas chromatography-mass spectrometry (GCMS) analysis was performed on a Hewlett-Packard model 5890 A gas chromatograph coupled to a model 5970 mass-selective detector (Hewlett-Packard Corp., Palo Alto, CAI. The capillary column (0.20 mm x 25 m) in the gas chromatograph contained fused silica crosslinked with 5% phenyl methyl silicone with a 0.33 nm film thickness. Dog and human autopsy samples were obtained from Dr. A. Siakotos, Indiana University Medical Center (Indianapolis, IN 46202), and mannosidosis goat tissue was obtained from Dr. M. Z. Jones (Michigan State University, East Lansing, MI 48824).
Storage of Lipids Each standard lipid or extracted lipid mixture was weighed and dissolved in a specific volume of chloro-
form :methanol (2 : 1, v/v) (C/M) and stored under nitrogen in a Teflon seal-screw cap vial a t - 70°C until use.
Extraction of Lipids and High Performance Thin Layer Chromatography Normal or affected tissue (1g) was homogenized in ice in a mixture of water (2 ml) and chloroform :methanol (1: 1) (12 ml) using first a Polytron and then a PotterElvehjem homogenizer. The fine suspension thus obtained was diluted with chloroform: methanol (1:1) (12 ml), and the total mixture was transferred to a Teflon seal-screw cap vial; the air above the mixture displaced by blowing in dry, oxygen-free nitrogen; the tube sealed using the cap, covered from light using aluminum foil, and then gently rocked using a nutator a t room temperature (23°C) for 4 h. Following extraction, the undissolved matter (mostly protein) was separated by centrifugation at 2,OOOg for 15 min and the clear organic extract was quickly flash evaporated in dimly lit room (-10-15 min per sample) to a syrup or semi-solid. The residue thus obtained was dissolved in chloroform : methanol (2: 1) (5 ml) by bath sonication and centrifuged again to remove a small amount of undissolved matter left in the extract. The supernatant thus obtained was evaporated to 1 ml by blowing in nitrogen, quantitatively transferred to a pre-weighed Teflon sealscrew cap vial, and evaporated by blowing in nitrogen (dried in the end by evaporation in the presence of a few drops of acetone). The lipid mixture thus obtained was weighed and dissolved in chloroform :methanol (2 : 1)to a concentration of 30 mg/ml. HPTLC was carried out using solvent combinations described in the figure legends, the plate dried and partially exposed (only the standard or reference lane) to iodine vapor (2 min) by covering the sample lanes by a glass plate. The bands of interest were thus marked, scraped, and used for GUMS as described in the following section. Identical plates were dried, sprayed with 10%CuSOp in 8%H3P04,and charred at 190°C for 10 min [Banerjee et al., 19901. Gas Chromatography-MassSpectrometry Silica gel scrapings obtained from a band of interest was transferred to a 5 ml screw-cap vial and mixed with 14% (w/w) boron trifluoride in methanol (3.5 ml). After capping under nitrogen, the vial was heated in a boiling water bath for 15 min, cooled to room temperature and then water (4 ml) was added to the product. The fatty acid methyl esters thus obtained by esterification (for free fatty acid) or trans-esterification (for triacylglycerol (TG)were extracted twice with 15ml hexane, the extract neutralized with Na2C0, and dried on anhydrous Na2S0,. Finally the esters were purified by silicic acid chromatography using hexane :ether (80 :20, v/v) as the eluting agent. The solution obtained was injected (2 ~ 1 ) into the GUMS system. Densitometry Densitometric analysis of the HPTLC-lipid-bands was carried out using a Hoefer GS 300 densitometer as detailed by Banerjee et al. [19901.
Triacylglycerols in NCL Tissue
551
RESULTS Dog Pancreas Comparison of the lipids from NCL pancreas (dog 28) with normal (dog 107),by HPTLC and charring, showed a decrease in cholesterol and phospholipids (the phospholipids are not shown here) and a large increase (sixfold peak area by densitometry) in a band just above the FFA band (Fig. 1, lanes 1, 2; Fig. 3a). This band was later identified as mixed TG by comparisonwith triolein (lanes 7 and 8). A prominent increase in the intensity of the free fatty acid band was also observed for the NCL sample (lanes 1and 2). GUMS analysis of these bands revealed a twofold increase in the proportion of oleic acid (18: 1)in the TG and FFA bands and a 50%decrease in palmitic acid (16 :0) in the free fatty acid band in NCL pancreas (Table I). The previously observed elevation of dolicholmonophosphate can be clearly seen (Fig. 1, lane 2). Dog Spleen HPTLC-charring showed a prominent increase in the intensity of the TG band (twofold peak area by densitometry) for the NCL dog (No. 28) as compared to normal 2. a: Total lipid obtained from normal dog (No. 107) spleen (age (No. 107) even though there was no appreciable change at Fig. autopsy, 18months) (lane1,150 pg), NCL dog (No. 28)spleen (age at in the free acid band (Fig. 2a, lanes 1,2). GC/MS anal- autopsy, 18 months) (lanes 2,7, 150 pg each), 0-mannosidosis goat ysis revealed similar results with a 70% increase in spleen (lane 3, 150 Fg), normal goat spleen (lane 41, human infantile spleen (H.R., age at autopsy, 9 yrs) (lane 5, 150 pg), human linoleic (18: 2) acid and a 38% decrease in stearic acid NCL splenomegaly spleen (age at autopsy, 8 yrs) (lane 6, 150 pg), normal (Table I) in the TG band. A repeat experiment (Fig. 2b) human brain (age a t autopsy, 20 yrs) (lane 8,300 pg), and human juvenile NCL brain (B.P., age at autopsy, 18 yr) (lane 9, 300 pg) were showed that the HPTLC profiles were reproducible. compared with triolein (TrOle, lane lo), methyloleate (MeOle, lane l l ) , and dolichol (Dol, lane 11). The fastest moving band in all the Human Brain plates was due to cholesteryl esters (note the increase in mannosidosis An increase in the TG band (twofold peak area by goat spleen, lane 3,as compared to normal, lane 4). b A second experidensitometry) was observed for the juuenile NCL brain ment to compare lipids (300pg/lane) obtained from Batten and normal normal dog (No. 107) pancreas (lane 1) and NCL dog (No. 28) sample (B.P.) as compared to normal (Fig. 2a, lanes 8,9; tissues: pancreas (lane 2); normal dog (No. 107) spleen (lane 3)and NCL dog Fig. 3b) after HPTLC-charring. GUMS results showed a (No. 28) spleen (lane 4); normal human spleen (lane 5) and human 2.5-fold increase in linoleic acid (18:2) in the TG band infantile NCL (H.R.) spleen (lane6);normal human brain (lane7)and juuenile NCL (B.P.) brain (lane 8); standard lipids (lane 9). and appreciable increase in both linoleic (95%)and oleic human Both plates developed in hexane: ether: acetic acid, 75 : 21 :4 (vlv). acid (73%)as well as a decrease in palmitic (16 :0 ) (42%) and stearic (18: 0) (44%) acids in the free fatty acid band Human Spleen (Table I). Other lipids were essentially as normal apart from an elevation of dolichol (Fig. 2, lanes 8, 91, which In this case an infantile NCL (INCL)spleen (H.R.)was has been reported before [Ivy et al., 19841. compared with one normal and 2 pathological controls which were a patient with splenomegaly associated with lymphoma and one with Gaucher disease (M.H.).While the TG band for the normal was essentially undetectable even by GUMS (not shown here), a strong triacylglycerol band (15-fold increase in peak area by densitometry as compared to normal, Fig. 3c)was observed in the NCL, splenomegaly, and Gaucher spleen samples (Fig. 2a, lanes 5,6). GUMS analysis revealed a 10-fold increase in linoleic acid, a 60% increase in palmitoleic acid (16: 1)and a 50% decrease in both palmitic and stearic acids in the NCL sample as compared to splenomegaly spleen. A 25% decrease in oleic acid was also observed in the TG band of the INCL sample, and GCI Fig. 1. Total lipid extractsfrom normal dog (No. 107) pancreas (age a t autopsy, 18 months) (lanes 1,6,150 pg each) and NCL dog (No. 28) MS of the FFA bands revealed a 3.2-fold increase in pancreas (age at autopsy, 18 months) (lane 2, 150 pg; lane 7,30 pg) were compared with standard lipids: dolichol (Dol, lane 3), dol- linoleic acid and a 24% loss of palmitic acid accompanied icholmonophosphate (DolP, lane 4), methyloleate (MeOle,lanes 5, lo), by a striking appearance of 6.5% of arachidonic acid free fatty acid (oleicacid was used) (FFA, lane 5)triolein (TrOle,lane 8), (Table I). Comparison with a normal spleen sample, and diolein (DiOle, lane 9). Note the large increase in the band correwhich contained saturated and unsaturated fatty acids sponding to triolein in the NCL samples in lanes 2 and 7. Developed in in the ratio saturated: unsaturated :-6 :4, showed an hexane :ether: acetic acid 70 : 15 : 2 (v/v).
Banerjee et al.
552 255,
portions of free oleic acid (22.6%) and linoleic acid (10.9%) were lower than or similar to splenomegaly spleen oleic acid (35.6%) and linoleic acid (8.2%). Mannosidosis Goat Spleen as a Pathological Control As can be seen from Figure 2a (lanes 3,4) there was essentially no triacylglycerol band at all in either the normal (lane 4) or the mannosidosis goat spleen (lane 3) samples confirming that TG elevation is not just a nonspecific accompaniment to lysosomal storage. The only prominent change observed in the affected goat spleen sample was the appearance of the prominent cholesteryl ester band. The free fatty acid bands were of the same intensity, and it was difficult to detect any TG in either spleen, even by GUMS.
1
TO HCL Weon (Human H R )
C
CE
Fig. 3. Densitometric analysis of HFTLC bands of lipids: a) NCL dog (No. 28) pancreas lipids compared with normal dog (No. 107) pancreas; b)juvenile NCL brain (human, B.P) lipids compared with normal human brain lipids; c) infanfile NCL spleen (human, H.R) lipids compared with normal human spleen lipids.
even greater increase in unsaturated fatty acids in INCL spleen. The triacylglycerol-fatty acids were practically undetectable by GUMS for normal spleen, while in Gaucher spleen 19.9% of triacylglycerol-fatty acids and 15.3% of FFA was arachidonic acid (Table I). The presence of less commonly observed FFA like 20:3 (2.3%), 20 :2 (3.3%),and 20: 0 (0.6%) (not shown in Table I) was also observed in Gaucher triacylglycerol. However, unlike the human NCL spleen sample, the levels of other triacylglycerol-unsaturated fatty acids in the Gaucher spleen sample such as oleic acid (18: 1) (12.6%) and linoleic acid (18:2) (not detected) were lower than those in splenomegaly spleen (Table I). Pro-
DISCUSSION A general increase in free and TG forms of oleic and/or linoleic acids and in one case arachidonic acid at the expense of saturated fatty acids was observed in all the NCL samples as compared to normal. Sometimes the accumulation was large (up to 15-foldpeak area by densitometry following HPTLC) and such increase in oleic and linoleic acids was not observed in the pathological controls like mannosidosis goat spleen-, myeloma associated splenomegaly spleen-, and Gaucher spleenderived triacylglycerol and free fatty acids. An added feature distinguishing the NCL samples from the other storage diseases is that Batten disease is almost never known to be accompanied by splenomegaly. The lipid-pattern in the NCL samples were clearly distinguishable from that in Wolman disease (which involves splenomegaly) in which mainly cholesteryl esters and, sometimes, TGs are stored [Fredrickson and Ferrans, 19781and the fatty acid composition is normal. Wolman disease is an acid lipase deficiency, suggesting that NCL could be a more specificunsaturated fatty acid lipase deficiency. In current literature on Batten disease, the major focus has been on characterizing the constituents of the isolated ceroid pigment which is characteristic of NCL tissue. This is a lipid-protein complex of which over half the protein is claimed to be the strongly hydrophobic c-subunit of mitochondria1 ATP synthase [Hall et al., 1990;Palmer et al., 19891,but this could be an artifact of the isolation procedure. In addition the presence of P-amyloid precursor protein [Wisniewski and Maslinska, 19891,S-methylcysteine- and methylated lysinecontaining proteins [Katz and Gerhardt, 19901and abnormal lipids such as conjugated dolichols have been reported. However, the structure of the autofluorescent material remains controversial and seems most likely to be lipid derived. It has been reported by Esterbauer et al. [19841, van Kujik et al. [19861, and Siakotos et al. L19881 that linoleic acid can undergo hydroperoxidation followed by breakdown to produce 4-hydroxynonenal (HNE). This could lead to the formation of autofluorescent ceroid pigment via the formation of Schiff bases. The failure to normally metabolize glycerides containing such fatty acids could be the source of this HNE [Ivy et al., 19841.
Triacylglycerols in NCL Tissue
553
TABLE I. Relative Abundance (Weight %) of Fatty Acids in the Free Fatty Acid and Triacylglycerol Bands* ~
Source of tissue Triacylglycerols Normal dog pancreas NCL dog pancreas Normal dog spleen NCL dog spleen Normal human brain NCL human Guvenile) brain Human splenomegaly spleen NCL human (infantile) spleen Gaucher human spleen Free fatty acids Normal dog pancreas NCL dog pancreas Normal dog spleen NCL dog spleen Normal human brain NCL human Guvenile) brain Human splenomegaly spleen NCL human (infantile) spleen Gaucher human spleen
~
~~~~~
14:O
16:l
16:O
18:2
18: 1
18:O
20:4
4.8 3.8 4.4 3.4 6.8 5.0 2.3 1.5 5.5
11.9 7.8 7.0 6.2 13.7 10.3 5.7 8.9
32.6 21.0 27.2 25.6 35.1 28.3 34.0 19.8 31.3
9.1 2.3 8.2 13.9 4.8 11.6 3.0 32.1
N.D.
22.3 51.6 31.8 37.6 23.0 26.2 43.2 32.8 12.6
16.1 13.4 21.4 13.4 12.5 16.1 11.0 4.9 30.7
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
34.2 16.1 29.5 35.3 42.4 24.9 30.6 20.3 20.4
17.4 12.9 7.7 6.9 4.4 8.6 8.2 26.2 10.9
25.4 47.7 23.9 24.5 22.0 38.1 35.6 22.6 22.6
22.9 14.8 25.6 26.2 24.1 13.4 16.1 16.5 16.9
N.D.
N.D. N.D. 4.2
N.D. N.D. 6.5 2.9 2.0 2.0
N.D. N.D. 2.6 6.8 7.0 7.1 8.5 5.1 5.8 4.4
19.9 2.3
N.D. N.D. N.D. N.D. N.D.
6.5 15.3 * N.D. = not detected. When the same tissue homogenate was extracted, variability of results among several extractions in our hands was
Evidence for Lipase Abnormality: High Levels of Free and Triacylglycerol Forms of Unsaturated Fatty Acids in Neuronal Ceroid-Lipofuscinosis Tissue Probal Banerjee, Amitava Dasgupta, Aristotle N. Siakotos, and Glyn Dawson Departments of Pediatrics (P.B., G.D.),Biochemistry and Molecular Biology (G.D.), and Pathology (A.D.), University of Chicago, Chicago, Illinois; Department of Pathology, Indiana University of School of Medicine, Indianapolis (A.N.S.) Total lipid obtained from normal and different forms of neuronal ceroid-lipofuscinoses (NCL) tissues was analyzed by high performance thin layer chromatography (HPTLC). We observed a large (>Sfold) increase in a lipid band corresponding to triolein for NCL dog pancreas and spleen and juvenile human NCL brain and infantile NCL spleen. The accumulation was less pronounced for the brain samples but apart from increased dolicholmonophosphate levels, other lipids appeared normal. Normal dog, goat, or human spleen contained virtually no triacylglycerol, and of the pathological controls, p-mannosidosisgoat spleen showed no triacylglycerol band at all. A sample of human spleen from a patient with lymphoma-associated splenomegaly displayed a strong triacylglycerol band, but gas chromatography-mass spectrometry (GC/MS) of the bands showed an equal increase in both saturated and unsaturated fatty acid containing triacylglycerols in the splenomegaly sample, in keeping with the notion of non-specific fat deposition in damaged tissue. In contrast, in all the NCL samples (spleen, pancreas, and brain) a prominent increase in the proportion of unsaturated fatty acids was observed in both free fatty acid and/or triacylglycerol bands following GUMS. The NCL-English setter dog pancreas showed a major presence of oleic acid (18: 1) (twofold increase) as compared to normal, while dog and infantile human NCL spleen samples and juvenile Batten brain (human) displayed a robust increase in linoleic acid (18:2) and sometimesin oleic acid and arachidonic acid (20 :4) (for infantile hu-
man NCL spleen). For the infantile human NCL spleen sample an increase in linoleic acid in both free fatty acid (3.2-fold)and triacylglycerol(10-fold)was observed. This suggests a new mechanism by which polyunsaturated fatty acids can be transported to various parts of an NCL-affected mammalian body in packages such as triacylglycerol and/or serum a1bumin:free fatty acid complex. Such polyunsaturated fatty acids could eventually lead to the ceroid-lipofuscin pigment characteristic of NCl tissues through peroxidation to aldehyde with subsequent Schiff base formation. This could possibly explain the deposition of ceroid-lipofuscin in widely different and non-proximal tissues such as the pancreas and the brain.
KEY WORDS: Batten, ceroid, triacylglycerol, NCL
INTRODUCTION The neuronal ceroid-lipofuscinoses (NCL) (Batten disease) are a group of inherited autosomal recessive disorders characterized clinically by dementia, seizures, and visual loss and neuropathologically by phenomenal loss of neurons, gliosis [Zeman, 1976; Armstrong et al., 1982;Lenn and Dawson, 19731, and neuronal accumulation of a characteristic autofluorescent pigment called ceroid or lipofuscin. Four forms of NCL have been described according t o the age of onset: infantile, late infantile, juvenile, and adult. Batten disease is the most common neurovisceral storage disease with incidence as high as one in 12,500 live births with about 440,000 carriers in the USA. Despite extensive investigation, the underlying biochemical defect has eluded detection. Initially, attempts Received for publication May 15, 1991; revision received Au- were made to locate changes in total lipid composition of NCL tissue [Anderson and Sperling, 1971; Hagberg et gust 21, 1991. Address reprint requests to Dr.Probal Banejee, Kennedy Cen- al., 1974; Svennerholm, 19751, but soon afterwards the ter, University of Chicago, Department of Pediatrics, 5841 major focus was laid on the autofluorescent pigment. Since then, a most exhaustive effort has been made by S. Maryland Avenue, Box 413,Chicago, IL 60637. 0 1992 Wiley-Liss, Inc.
550
Banerjee et al.
several groups over 15 years to elucidate the composition of the fluorescent deposit, and this in tu rn has led to the knowledge that lipids such as dolichol, dolichol phosphate [Kitani et al., 19901, and dolichol-linked oligosaccharides [Hall et al., 19901 and proteins such a s the dicyclohexylcarbodiimide (DCCD) reactive, lipid binding subunit (c) of ATP synthase [Palmer et al., 19891, non-oxidative modification products of proteins by cysteine methylation [Katz, 19901, and 4-hydroxynonenal [Esterbauer et al., 19841are constituents of the lipopigment. There have also been reports of increased proportions of unsaturated fatty acids in phospholipids obtained from fractions enriched in the autofluorescent deposit [Pullarkat et al., 19821 and also other reports of no change in ovine NCL pigment phospholipids [Palmer et al., 19891.It has been observed that phospholipase A, activity is diminished in NCL samples and also that 4-hydroxynonenal, which is reportedly produced in excess in NCL, strongly inhibits lysosomal enzymes such as cathepsins and thiol-dependent phospholipase A2 [Dawsin and Glaser, 1988; Dawson et al., 19901.Because of the disappearance of lipid enriched gray matter from NCL brain we expected this tissue to be of limited value unless obtained before total loss of all gray matter. With this idea in mind we looked a t the total lipid extracts from dog pancreas and spleen, and human spleen and brain directly without any fractionation by column chromatography, and we report the presence of abnormally high levels of unsaturated fatty acids in neutral lipids of NCL tissue. MATERIALS AND METHODS High performance thin layer chromatography (HPTLC) plates (10 x 10 cm) were of the LHP-K type (lot No. 004559) with 2 cm wide preadsorbent layer were obtained from Whatman Chemical Separation Inc. (Clifton, NJ). The standard lipids (triolein, methyloleate, diolein, dolichol, dolicholmonophosphate, oleic acid, galactosylceramide, phosphatidylethanolamine and phosphatidylserine, phosphatidylcholine and phosphatidylinositol) were obtained from Sigma Chemical Co. (St. Louis, MO). All reagents and solvents were of analytical grade, and the HPTLC plates were routinely prewashed by developing with chloroform : methanol : water (60:40:10, vlv) and activated for 1 h at 150°C before use. Gas chromatography-mass spectrometry (GCMS) analysis was performed on a Hewlett-Packard model 5890 A gas chromatograph coupled to a model 5970 mass-selective detector (Hewlett-Packard Corp., Palo Alto, CAI. The capillary column (0.20 mm x 25 m) in the gas chromatograph contained fused silica crosslinked with 5% phenyl methyl silicone with a 0.33 nm film thickness. Dog and human autopsy samples were obtained from Dr. A. Siakotos, Indiana University Medical Center (Indianapolis, IN 46202), and mannosidosis goat tissue was obtained from Dr. M. Z. Jones (Michigan State University, East Lansing, MI 48824).
Storage of Lipids Each standard lipid or extracted lipid mixture was weighed and dissolved in a specific volume of chloro-
form :methanol (2 : 1, v/v) (C/M) and stored under nitrogen in a Teflon seal-screw cap vial a t - 70°C until use.
Extraction of Lipids and High Performance Thin Layer Chromatography Normal or affected tissue (1g) was homogenized in ice in a mixture of water (2 ml) and chloroform :methanol (1: 1) (12 ml) using first a Polytron and then a PotterElvehjem homogenizer. The fine suspension thus obtained was diluted with chloroform: methanol (1:1) (12 ml), and the total mixture was transferred to a Teflon seal-screw cap vial; the air above the mixture displaced by blowing in dry, oxygen-free nitrogen; the tube sealed using the cap, covered from light using aluminum foil, and then gently rocked using a nutator a t room temperature (23°C) for 4 h. Following extraction, the undissolved matter (mostly protein) was separated by centrifugation at 2,OOOg for 15 min and the clear organic extract was quickly flash evaporated in dimly lit room (-10-15 min per sample) to a syrup or semi-solid. The residue thus obtained was dissolved in chloroform : methanol (2: 1) (5 ml) by bath sonication and centrifuged again to remove a small amount of undissolved matter left in the extract. The supernatant thus obtained was evaporated to 1 ml by blowing in nitrogen, quantitatively transferred to a pre-weighed Teflon sealscrew cap vial, and evaporated by blowing in nitrogen (dried in the end by evaporation in the presence of a few drops of acetone). The lipid mixture thus obtained was weighed and dissolved in chloroform :methanol (2 : 1)to a concentration of 30 mg/ml. HPTLC was carried out using solvent combinations described in the figure legends, the plate dried and partially exposed (only the standard or reference lane) to iodine vapor (2 min) by covering the sample lanes by a glass plate. The bands of interest were thus marked, scraped, and used for GUMS as described in the following section. Identical plates were dried, sprayed with 10%CuSOp in 8%H3P04,and charred at 190°C for 10 min [Banerjee et al., 19901. Gas Chromatography-MassSpectrometry Silica gel scrapings obtained from a band of interest was transferred to a 5 ml screw-cap vial and mixed with 14% (w/w) boron trifluoride in methanol (3.5 ml). After capping under nitrogen, the vial was heated in a boiling water bath for 15 min, cooled to room temperature and then water (4 ml) was added to the product. The fatty acid methyl esters thus obtained by esterification (for free fatty acid) or trans-esterification (for triacylglycerol (TG)were extracted twice with 15ml hexane, the extract neutralized with Na2C0, and dried on anhydrous Na2S0,. Finally the esters were purified by silicic acid chromatography using hexane :ether (80 :20, v/v) as the eluting agent. The solution obtained was injected (2 ~ 1 ) into the GUMS system. Densitometry Densitometric analysis of the HPTLC-lipid-bands was carried out using a Hoefer GS 300 densitometer as detailed by Banerjee et al. [19901.
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551
RESULTS Dog Pancreas Comparison of the lipids from NCL pancreas (dog 28) with normal (dog 107),by HPTLC and charring, showed a decrease in cholesterol and phospholipids (the phospholipids are not shown here) and a large increase (sixfold peak area by densitometry) in a band just above the FFA band (Fig. 1, lanes 1, 2; Fig. 3a). This band was later identified as mixed TG by comparisonwith triolein (lanes 7 and 8). A prominent increase in the intensity of the free fatty acid band was also observed for the NCL sample (lanes 1and 2). GUMS analysis of these bands revealed a twofold increase in the proportion of oleic acid (18: 1)in the TG and FFA bands and a 50%decrease in palmitic acid (16 :0) in the free fatty acid band in NCL pancreas (Table I). The previously observed elevation of dolicholmonophosphate can be clearly seen (Fig. 1, lane 2). Dog Spleen HPTLC-charring showed a prominent increase in the intensity of the TG band (twofold peak area by densitometry) for the NCL dog (No. 28) as compared to normal 2. a: Total lipid obtained from normal dog (No. 107) spleen (age (No. 107) even though there was no appreciable change at Fig. autopsy, 18months) (lane1,150 pg), NCL dog (No. 28)spleen (age at in the free acid band (Fig. 2a, lanes 1,2). GC/MS anal- autopsy, 18 months) (lanes 2,7, 150 pg each), 0-mannosidosis goat ysis revealed similar results with a 70% increase in spleen (lane 3, 150 Fg), normal goat spleen (lane 41, human infantile spleen (H.R., age at autopsy, 9 yrs) (lane 5, 150 pg), human linoleic (18: 2) acid and a 38% decrease in stearic acid NCL splenomegaly spleen (age at autopsy, 8 yrs) (lane 6, 150 pg), normal (Table I) in the TG band. A repeat experiment (Fig. 2b) human brain (age a t autopsy, 20 yrs) (lane 8,300 pg), and human juvenile NCL brain (B.P., age at autopsy, 18 yr) (lane 9, 300 pg) were showed that the HPTLC profiles were reproducible. compared with triolein (TrOle, lane lo), methyloleate (MeOle, lane l l ) , and dolichol (Dol, lane 11). The fastest moving band in all the Human Brain plates was due to cholesteryl esters (note the increase in mannosidosis An increase in the TG band (twofold peak area by goat spleen, lane 3,as compared to normal, lane 4). b A second experidensitometry) was observed for the juuenile NCL brain ment to compare lipids (300pg/lane) obtained from Batten and normal normal dog (No. 107) pancreas (lane 1) and NCL dog (No. 28) sample (B.P.) as compared to normal (Fig. 2a, lanes 8,9; tissues: pancreas (lane 2); normal dog (No. 107) spleen (lane 3)and NCL dog Fig. 3b) after HPTLC-charring. GUMS results showed a (No. 28) spleen (lane 4); normal human spleen (lane 5) and human 2.5-fold increase in linoleic acid (18:2) in the TG band infantile NCL (H.R.) spleen (lane6);normal human brain (lane7)and juuenile NCL (B.P.) brain (lane 8); standard lipids (lane 9). and appreciable increase in both linoleic (95%)and oleic human Both plates developed in hexane: ether: acetic acid, 75 : 21 :4 (vlv). acid (73%)as well as a decrease in palmitic (16 :0 ) (42%) and stearic (18: 0) (44%) acids in the free fatty acid band Human Spleen (Table I). Other lipids were essentially as normal apart from an elevation of dolichol (Fig. 2, lanes 8, 91, which In this case an infantile NCL (INCL)spleen (H.R.)was has been reported before [Ivy et al., 19841. compared with one normal and 2 pathological controls which were a patient with splenomegaly associated with lymphoma and one with Gaucher disease (M.H.).While the TG band for the normal was essentially undetectable even by GUMS (not shown here), a strong triacylglycerol band (15-fold increase in peak area by densitometry as compared to normal, Fig. 3c)was observed in the NCL, splenomegaly, and Gaucher spleen samples (Fig. 2a, lanes 5,6). GUMS analysis revealed a 10-fold increase in linoleic acid, a 60% increase in palmitoleic acid (16: 1)and a 50% decrease in both palmitic and stearic acids in the NCL sample as compared to splenomegaly spleen. A 25% decrease in oleic acid was also observed in the TG band of the INCL sample, and GCI Fig. 1. Total lipid extractsfrom normal dog (No. 107) pancreas (age a t autopsy, 18 months) (lanes 1,6,150 pg each) and NCL dog (No. 28) MS of the FFA bands revealed a 3.2-fold increase in pancreas (age at autopsy, 18 months) (lane 2, 150 pg; lane 7,30 pg) were compared with standard lipids: dolichol (Dol, lane 3), dol- linoleic acid and a 24% loss of palmitic acid accompanied icholmonophosphate (DolP, lane 4), methyloleate (MeOle,lanes 5, lo), by a striking appearance of 6.5% of arachidonic acid free fatty acid (oleicacid was used) (FFA, lane 5)triolein (TrOle,lane 8), (Table I). Comparison with a normal spleen sample, and diolein (DiOle, lane 9). Note the large increase in the band correwhich contained saturated and unsaturated fatty acids sponding to triolein in the NCL samples in lanes 2 and 7. Developed in in the ratio saturated: unsaturated :-6 :4, showed an hexane :ether: acetic acid 70 : 15 : 2 (v/v).
Banerjee et al.
552 255,
portions of free oleic acid (22.6%) and linoleic acid (10.9%) were lower than or similar to splenomegaly spleen oleic acid (35.6%) and linoleic acid (8.2%). Mannosidosis Goat Spleen as a Pathological Control As can be seen from Figure 2a (lanes 3,4) there was essentially no triacylglycerol band at all in either the normal (lane 4) or the mannosidosis goat spleen (lane 3) samples confirming that TG elevation is not just a nonspecific accompaniment to lysosomal storage. The only prominent change observed in the affected goat spleen sample was the appearance of the prominent cholesteryl ester band. The free fatty acid bands were of the same intensity, and it was difficult to detect any TG in either spleen, even by GUMS.
1
TO HCL Weon (Human H R )
C
CE
Fig. 3. Densitometric analysis of HFTLC bands of lipids: a) NCL dog (No. 28) pancreas lipids compared with normal dog (No. 107) pancreas; b)juvenile NCL brain (human, B.P) lipids compared with normal human brain lipids; c) infanfile NCL spleen (human, H.R) lipids compared with normal human spleen lipids.
even greater increase in unsaturated fatty acids in INCL spleen. The triacylglycerol-fatty acids were practically undetectable by GUMS for normal spleen, while in Gaucher spleen 19.9% of triacylglycerol-fatty acids and 15.3% of FFA was arachidonic acid (Table I). The presence of less commonly observed FFA like 20:3 (2.3%), 20 :2 (3.3%),and 20: 0 (0.6%) (not shown in Table I) was also observed in Gaucher triacylglycerol. However, unlike the human NCL spleen sample, the levels of other triacylglycerol-unsaturated fatty acids in the Gaucher spleen sample such as oleic acid (18: 1) (12.6%) and linoleic acid (18:2) (not detected) were lower than those in splenomegaly spleen (Table I). Pro-
DISCUSSION A general increase in free and TG forms of oleic and/or linoleic acids and in one case arachidonic acid at the expense of saturated fatty acids was observed in all the NCL samples as compared to normal. Sometimes the accumulation was large (up to 15-foldpeak area by densitometry following HPTLC) and such increase in oleic and linoleic acids was not observed in the pathological controls like mannosidosis goat spleen-, myeloma associated splenomegaly spleen-, and Gaucher spleenderived triacylglycerol and free fatty acids. An added feature distinguishing the NCL samples from the other storage diseases is that Batten disease is almost never known to be accompanied by splenomegaly. The lipid-pattern in the NCL samples were clearly distinguishable from that in Wolman disease (which involves splenomegaly) in which mainly cholesteryl esters and, sometimes, TGs are stored [Fredrickson and Ferrans, 19781and the fatty acid composition is normal. Wolman disease is an acid lipase deficiency, suggesting that NCL could be a more specificunsaturated fatty acid lipase deficiency. In current literature on Batten disease, the major focus has been on characterizing the constituents of the isolated ceroid pigment which is characteristic of NCL tissue. This is a lipid-protein complex of which over half the protein is claimed to be the strongly hydrophobic c-subunit of mitochondria1 ATP synthase [Hall et al., 1990;Palmer et al., 19891,but this could be an artifact of the isolation procedure. In addition the presence of P-amyloid precursor protein [Wisniewski and Maslinska, 19891,S-methylcysteine- and methylated lysinecontaining proteins [Katz and Gerhardt, 19901and abnormal lipids such as conjugated dolichols have been reported. However, the structure of the autofluorescent material remains controversial and seems most likely to be lipid derived. It has been reported by Esterbauer et al. [19841, van Kujik et al. [19861, and Siakotos et al. L19881 that linoleic acid can undergo hydroperoxidation followed by breakdown to produce 4-hydroxynonenal (HNE). This could lead to the formation of autofluorescent ceroid pigment via the formation of Schiff bases. The failure to normally metabolize glycerides containing such fatty acids could be the source of this HNE [Ivy et al., 19841.
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TABLE I. Relative Abundance (Weight %) of Fatty Acids in the Free Fatty Acid and Triacylglycerol Bands* ~
Source of tissue Triacylglycerols Normal dog pancreas NCL dog pancreas Normal dog spleen NCL dog spleen Normal human brain NCL human Guvenile) brain Human splenomegaly spleen NCL human (infantile) spleen Gaucher human spleen Free fatty acids Normal dog pancreas NCL dog pancreas Normal dog spleen NCL dog spleen Normal human brain NCL human Guvenile) brain Human splenomegaly spleen NCL human (infantile) spleen Gaucher human spleen
~
~~~~~
14:O
16:l
16:O
18:2
18: 1
18:O
20:4
4.8 3.8 4.4 3.4 6.8 5.0 2.3 1.5 5.5
11.9 7.8 7.0 6.2 13.7 10.3 5.7 8.9
32.6 21.0 27.2 25.6 35.1 28.3 34.0 19.8 31.3
9.1 2.3 8.2 13.9 4.8 11.6 3.0 32.1
N.D.
22.3 51.6 31.8 37.6 23.0 26.2 43.2 32.8 12.6
16.1 13.4 21.4 13.4 12.5 16.1 11.0 4.9 30.7
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
34.2 16.1 29.5 35.3 42.4 24.9 30.6 20.3 20.4
17.4 12.9 7.7 6.9 4.4 8.6 8.2 26.2 10.9
25.4 47.7 23.9 24.5 22.0 38.1 35.6 22.6 22.6
22.9 14.8 25.6 26.2 24.1 13.4 16.1 16.5 16.9
N.D.
N.D. N.D. 4.2
N.D. N.D. 6.5 2.9 2.0 2.0
N.D. N.D. 2.6 6.8 7.0 7.1 8.5 5.1 5.8 4.4
19.9 2.3
N.D. N.D. N.D. N.D. N.D.
6.5 15.3 * N.D. = not detected. When the same tissue homogenate was extracted, variability of results among several extractions in our hands was
