ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 250, No. 1, October, pp. 1’7-179, 1986

A Comparative Study of Stearic and Lignoceric Acid Oxidation by Human Skin Fibroblasts

HARMEET SINGH’ AND ALF POULOS

Department of Chemical Pathology, The Adelaide Children k Hospital, North Adelaide, Seth Australia 5006

Received February 24,1986, and in revised form June 3, 1986

Sensitive assays were developed for long chain and very long chain fatty acid oxidation in human skin fibroblast homogenates. Stearic and lignoceric acids were degraded by the fibroblasts by the P-oxidation pathway. The cofactor requirements for stearic and lignoceric acid P-oxidation were very similar but not identical. For example, appreciable lignoceric acid oxidation could be demonstrated only in the presence of a-cyclodextrin and was inhibited by Triton X-100. In Zellweger’s syndrome, stearic acid P-oxidation was partially reduced whereas lignoceric acid P-oxidation was reduced dramatically (~12% activity compared to the controls). The results presented suggest that stearic acid P-oxidation occurs in mitochondria as well as in peroxisomes, but lignoceric acid oxidation occurs entirely in the peroxisomes. We suggest that the P-oxidation systems for stearic acid and lignoceric acid may be different. 0 1986 AcademicPress, Inc.

Very long chain fatty acids (VLCFA), especially those greater than Cz2 in length, are known to accumulate in several disease states including adrenoleukodystrophy (ALD), adrenomyeloneuropathy (AMN), infantile Refsum’s disease and cerebrohe- pato-renal (Zellweger’s) syndrome (l-6). The increase in VLCFA has been shown to occur in several tissues including skin fi- broblasts and leukocytes (1, 4-7). In addi- tion some monounsaturated fatty acids (C26:1, probably C27:1 and others) have been reported to accumulate in Zellweger’s syn- drome (5, 8, 9). However, the complete structural identification of these long chain fatty acids has not been carried out (5). Al- though the VLCFA are known to accu- mulate in several disease states, the exact molecular defect remains to be determined. Some evidence has been provided that VLCFA oxidation may be defective in ALD

1 To whom correspondence should be addressed. * Abbreviations used: VLCFA, very long chain fatty

acids; ALD, adrenoleukodystrophy; AMN, adreno- myeloneuropathy; HPTLC, high-performance TLC.

and Zellweger’s syndrome (5,10-12). Also, VLCFA synthesis in skin fibroblasts of ALD patients is reported to be increased (30, 31).

The exact mechanism of oxidation of VLCFA, that is whether it be an (Y-, p-, or an w-oxidation system, the relative contri- bution of any of these oxidation systems in ‘uiz)o, and the intracellular localization of the oxidation of VLCFA are conjectural. To understand the molecular defect(s) in the oxidation of VLCFA in several disease states, we optimized the assay conditions for P-oxidation of CtiIO (lignoceric acid) and C1s:O (stearic acid) in homogenates of skin fibroblasts. The results presented in this paper provide strong evidence that p-oxi- dation of lignoceric acid occurs in an or- ganelle other than mitochondria. The pos- sible involvement of peroxisomes (micro- bodies) in P-oxidation of lignoceric acid is discussed.

MATERIALS

Potassium [%]cyanide (51.1 mCi/mmol) and [l- %]stearic acid (56 mCi/mmol) were purchased from

171 0003-9861/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

172 SINGH AND POULOS

New England Nuclear, Boston, Massachusetts. Tri- cosanol was purchased from Nu-Chek Preparations, Inc., Elysian, Minnesota, and methanesulfonyl chlo- ride from Aldrich Chemical Company, Milwaukee, Wisconsin. pBromophenacy1 bromide was obtained from TCI, Japan. Preswollen DEAE-cellulose (DE-52) was purchased from Whatman Chemical Separation, Ltd., England. Reverse-phase KC-18 thin-layer plates were purchased from Whatman, Inc., Clifton, New Jersey, and thin-layer silica gel 60 plates were from E. Merck, Darmstadt, West Germany. Dulbecco’s modified Eagle’s medium was purchased from Flow Laboratories, McLean, Virginia, and fetal calf serum from GIBCO New Zealand Ltd., New Zealand. Dul- becco’s phosphate buffer (Caz+- and Mg’+-free) and trypsin-versene solution was obtained from Com- monwealth Serum Laboratories, Melbourne. All other chemicals and reagents were purchased from Sigma Chemical Company, St. Louis, Missouri. All the sol- vents used were either from May and Baker Australia Pty., Ltd., Victoria, or from Ajax Chemicals, Sydney, Australia.

Two of the Zellweger lines (lines GM 4340 and GM 0288) were obtained from the Human Mutant Cell Re- pository (Camden, N.J.). The other two Zellweger cell lines were kindly provided by Professor David Danks (Birth Defects Institute, University of Melbourne). The remaining fibroblast cell lines were obtained from patients with Zellweger’s syndrome where the diag- nosis was confirmed on the basis of family history and clinical and biochemical investigations including long chain fatty acid analysis (13).

METHODS

Synthesis of[l-“C]Zigrzoc& acid. [1-“C&ignoceric acid was synthesized as described (14) with some modification. Tricosanol(O.25 mmol) was dissolved in 20 ml of dry pyridine and 200 ~1 of methanesulfonyl chloride (2.6 mmol) was added slowly at 4°C. The mixture was brought to room temperature and stirred for 3 h. The excess reagent was decomposed with 5 ml of ice-cold water. The reaction product was ex- tracted with 50 ml of diethyl ether. The ether extract was washed four times with a saturated solution of copper sulfate (25 ml each time) to remove the pyridine and washed a further two times with water (25 ml each time). The solvent was removed and the product was redissolved in 3-4 ml of dry diethyl ether and crystallized at -20°C overnight. The purity of the product was checked by thin-layer chromatography (TLC) on silica gel 60 (solvent system, hexane:ether 21, v/v). Tricosanol was found to be quantitatively converted to its methanesulfonate derivative as de- termined by TLC (Rf value for tricosanol = 0.34; methanesulfonate derivative of tricosanol = 0.43).

The methanesulfonate derivative of tricosanol (35 rmol) was dissolved in 2.25 ml of ethanol and 1 mCi

of Ki4CN (20 rmol) dissolved in 250 ~1 of 50 mM KOH was added, mixed, and heated at 80°C for 26 h. Water (2 ml) was added and the radioactive product was ex- tracted four times with diethyl ether (5 ml each time). The ether extract was washed two times with water (5 ml each time). After evaporation of the solvent, radiolabeled product was dissolved in 2 ml of 95% ethanol, 0.2 g of KOH was added, and the reaction mixture was heated at 100°C for 48 h. The reaction mixture was cooled at 4°C and acidified with 400 ~1 of 10 M HCl, 2 ml water was added, and the radiola- beled product was extracted five times with diethyl ether (5 ml each time). The ether extract was washed twice with 5 ml water, the solvent was evaporated, and the radiolabeled product was dissolved in 2 ml of chloroform:methanol:water (30:60:4, by volume), and applied to a DE-52 column (1.2 X 10 cm). The column was washed with 100 ml of the above solvent and the radioactive product was eluted from the column with 40 ml of chloroform:methanol:0.8 M ammonium ace- tate (30:60:4, by volume). The solvent from the radio- active product was removed, and the product was re- dissolved in 5 ml of chloroform:methanol (2:1, v/v) and partitioned with 1 ml of water. The solvent from the chloroform extract was evaporated and the above procedure was repeated. The purity of the product was checked by TLC on silica gel 60 [solvent system, hexane:ether:acetic acid (60:4&l, by volume)]. A small fraction of the radiolabeled product was converted to the methyl ester by treatment with 1.5% (v/v) H&SO, in methanol at 80°C for 3 h. The methyl ester(s) was separated on reverse-phase KC-18 TLC plates [solvent system, acetonitrile:tetrahydrofuran; (85:15, v/v)] and the radiolabeled purity was checked by subjecting the plates to autoradiography. The purity of the methyl ester was further determined by capillary gas chro- matography. The radiolabeled product was identified as lignoceric acid (100% purity) with high specific ac- tivity (51 mCi/mmol).

Preparation of jibrobhst homogenates. Fibroblasts were grown under sterile conditions for l-4 weeks in tissue culture flasks (75 cm2) in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal calf serum and benzyl penicillin (80 units/ml). Before the cells were harvested, the culture medium was removed and the cells were rinsed with Dulbecco’s phosphate buffer (Caz+ and Mg2+ free). The cells were removed from tissue culture flasks by incubating at 37°C for 2-5 min with trypsin-versene solution. The cells were collected by low speed centrifugation (400g X 5 min). The cell pellet was washed three times with 4-5 ml of Dul- becco’s phosphate buffer to remove residual trypsin. The cell pellet was dispersed in 0.3-0.4 ml of 0.25 M sucrose and the cell suspension was briefly sonicated (5-10 s) using a microtip (Ystrom) to disrupt the cells. The homogenate was centrifuged at 400g X 10 min at 4°C to remove unbroken cells and the supernatant was used for enzyme assays.

FATTY ACID /J-OXIDATION BY HUMAN SKIN FIBROBLASTS 173

Preparation of substrates for enzyme assays. [l- %]Stearic acid (30-40 nmol) and 50 pg of phospha- tidylcholine were pipetted into a test tube. The solvent was evaporated under nitrogen and 0.6-0.7 ml of water was added. The mixture was sonicated for 20-30 min in an ultrasonic water bath (Branson Ultrasonic Cleaner, Shelton, Conn.) and 25 ~1 of this substrate was used for each assay. [1-‘%&ignoceric acid (15-20 nmol) was pipetted, the solvent was evaporated under nitrogen, and 0.6-0.7 ml of a-cyclodextrin (20 mg/ml) was added. The mixture was sonicated for 20-30 min in an ultrasonic bath, and 25 ~1 of this substrate was used for each assay.

Enzyme Assay. For stearic acid oxidation, the re- action mixture consisted of Tris-HCl buffer, pH 8.0 (50 mM), MgClz (1 mM), ATP (2 mM), dithiothreitol(1 mM), NAD (0.4 mM), L-carnitine (0.4 mM), FAD (60 fiM), coenzyme A (60 PM), [l-%]stearic acid (3-4 PM),

and antibiotics (0.3 mg/ml each of streptomycin and benzyl penicillin) in a total volume of 0.5 ml. The en- zyme reaction was started by the addition of lo-20 fig of fibroblast protein, and the reaction was carried out at 3’7°C for 60 min. The reaction was stopped by the addition of 4 ml of chloroform:methanol (2:1, v/ v) and partitioned by the addition of 0.5 ml of water (15). The lower phase (containing unreacted [l- %]stearate) was removed and the upper phase was washed twice with hexane (2 ml each time). An aliquot of the upper phase was taken for counting the radio- activity to assess the total amount of water soluble radioactivity, which consisted of both stearoyl CoA and the product acetyl CoA. To the remaining upper phase, 10 M KOH (final concentration 0.1 M) was added and the reaction mixture was heated at 55°C for 16- 18 h to hydrolyze the stearoyl CoA. The reaction mix- ture was acidified (pH 1-1.5) with 10 M HCl, and the product (unreacted [1-14C]stearate) was extracted twice with 2 ml of hexane. The hexane unextractable and water soluble radioactivity ([1-i4C]acetate) was counted to determine the actual amount of oxidation of radiolabeled stearic acid to radiolabeled acetate.

For lignoceric acid oxidation, the reaction mixture consisted of Tris-HCl buffer, pH 8.0 (50 mM), MgCl, (2.5 mM), ATP (5 mM), dithiothreitol (1 mM), NAD (1 mM), L-carnitine (1 mM), FAD (150 PM), coenzyme A (150 HIM), [1-i4C]lignoceric acid (4-5 PM), cu-cyclodextrin (2.5 mg/ml), and antibiotics (0.3 mg/ml each of strep- tomycin and benzyl penicillin) in a total volume of 0.2 ml. The reaction was started by the addition of 20- 50 pg of fibroblast protein and carried out at 37°C for 120 min. The reaction was stopped by the addition of 4 ml chloroform:methanol(2:1, v/v) and the reaction mixture was partitioned by the addition of 0.8 ml wa- ter (15). The upper phase was washed twice with 2 ml hexane and processed as for stearic acid oxidation.

Protein estimation in the fibroblast homogenates was performed by a dye binding assay using human albumin as standard (16).

Icknti&xztion of the radiolabeled product. Incubation of radiolabeled fatty acids, namely stearic acid and lignoceric acid, with fibroblast homogenates and sub- sequent partitioning (15) of the incubation mixtures resulted in the appearance of the radioactivity in the upper aqueous phase. After alkaline hydrolysis of the upper phase extract the radioactivity associated with lipids was extracted twice with either hexane or chlo- roform. The radioactivity remaining in the upper phase was designated as water soluble product. Under acidic conditions, the water soluble radioactive prod- uct was extractable (>95%) in ethyl acetate. Evapo- ration of the ethyl acetate under nitrogen resulted in greater than 90% loss of the radioactivity suggesting that the radiolabeled product was highly volatile.

The solvent from upper phase extracts (15) before alkaline hydrolysis (described above) was evaporated under nitrogen. The radiolabeled product was dis- solved in chloroform:methanol:water (66:33:5, by vol- ume) and applied to a high-performance thin-layer chromatographic (HPTLC) silica gel 60 plate. The plate was developed twice in butanol:acetic acid:water (5:2:3, by volume). In the above solvent system, coen- zyme A derivatives of stearic or lignoceric acid were well separated from acetyl coenzyme A, coenzyme A, acetoacetyl coenzyme A, 3-hydroxy-3-methylglutaryl coenzyme A, and other coenzyme A derivatives of short chain acids.

The radiolabeled product in the upper phase (de- scribed above) was dissolved in 200 ~1 of water and 20 ~1 of 1 M KOH was added. The mixture was heated at 80°C to hydrolyze coenzyme A derivatives of both long and short chain acids. After 3 h, the reaction mixture was cooled, 1 ml of acetonitrile was added, and the pH was adjusted to 9.0 with 1 M HCI. One milliliter of acetonitrile containing p-bromophenacyl bromide (2 mg) and l&crown-6 ether (0.5 mg) was added, and the heating was continued at 80°C. After 1 h, the solvent from the reaction mixture was evap- orated under nitrogen, and the bromophenacyl deriv- ative was extracted with 3 ml of diethyl ether. The ether extract was washed twice with 1 ml of water, and the bromophenacyl derivative was applied to a thin-layer silica gel 60 plate. The plate was developed using chloroform. In the above solvent system, bro- mophenacyl derivatives of acetate, propionate, bu- tyrate, 3-hydroxy-3-methylglutarate, and others were well separated from each other. Bromophenacyl de- rivatives of stearic or lignoceric acid were well sep- arated from those of short chain acids.

RESULTS

Under the assay conditions described, 20-30% of the radioactivity from stearic acid and lo-20% of that from lignoceric acid incubated with fibroblast homogenates

174 SINGH AND POULOS

appeared in the upper aqueous phase (15). Most of the radioactivity in the upper phase could not be extracted by hexane or chlo- roform, suggesting that stearic acid and lignoceric acid were converted to water soluble products. Thin-layer chromato- graphic analysis of the aqueous upper phase material indicated that both stearic acid and lignoceric acid were converted to two major radiolabeled products (Fig. la). Band A migrated on TLC with radiolabeled stearoyl CoA, and band B migrated with unlabeled acetyl CoA (acetyl CoA standard not shown). It is known that coenzyme A derivatives of short chain acids (two to four carbons in length) are clearly separated by TLC from the coenzyme A derivatives of long chain acids (carbon 12 and longer) (1’7). After further treatment, namely al- kaline hydrolysis of the upper phase ma- terial, a large fraction (SO-SO%) of the ra- dioactivity was extractable in hexane or chloroform. Thin-layer chromatographic separation of the radiolabeled lipid pro- duced after the treatment (in the hexane

extract) indicated that the radiolabeled material was stearic acid or lignoceric acid (data not shown). The remaining water soluble radioactivity was extractable in ethyl acetate only under acidic conditions and was highly volatile, suggesting that the product may be a short chain acid (two to four carbons in length).

Bromophenacyl derivatization of the upper phase extract (see Methods) and the separation of the bromophenacyl deriva- tives by TLC showed a number of radio- labeled products (Fig. lb). The fast moving band (band A) was identified as the bro- mophenacyl derivative of the long chain fatty acid (stearic acid or lignoceric acid). The second band (band B) probably was the bromophenacyl derivative of the oxidation product of the long chain acid. This band migrated ahead of the bromophenacyl de- rivative of butyric acid. The third band (band C) migrated with the bromophenacyl derivative of acetic acid. The low intensity of the radioactive product comigrating with the bromophenacyl derivative of

12 3 4 5 6 " _.

O* (b)

FIG. 1. Autoradiographs of the thin-layer separation of the product (a) and bromophenacyl de- rivatives of the product (b) generated by the incubation of the radiolabeled fatty acids with fibroblast homogenates as described under Methods. 0 represents site of application of sample and SF represents solvent front on chromatogram. (a) Standard stearic acid (lane l), synthetic stearoyl CoA (lane 2), upper phase extracts (duplicates) after incubation with [1-‘%]stearic acid (lanes 3 and 4), and [1-‘*C]lignoceric acid (lanes 5 and 6). Minor radioactive bands migrating ahead of the major stearoyl CoA band (lane 2) represent the partial esterification of the coenzyme A moiety of stearoyl CoA during chromatography in the acidic solvent system. The minor band near the solvent front migrates with free fatty acid. (b) Standard acetic and butyric acids (lower band and top band, respectively, in lane 5), upper phase extracts (duplicates) after incubation with [I-“CJstearic acid (lanes 1 and 2), and [1-‘%]lignoceric acid (lanes 3 and 4). The round spots between lanes 2 and 3 are X-ray film artifacts.

FATTY ACID @-OXIDATION BY HUMAN SKIN FIBROBLASTS 175

acetic acid is due to loss of this volatile The cofactor requirements for stearic radioactive product during experimental acid and lignoceric acid P-oxidation were manipulation. The fourth band (band D) similar, but not identical (Figs. 2, 3). was identified as underivatized fatty acid. Stearic acid and lignoceric acid @-oxidation The radioactive band at the origin (band depended upon the presence of ATP, Mg2+, E) could not be identified. and coenzyme A. Omission of any one of

0 2

I a b

0 100 200 300 400

FINAL CONCENTRATION (PM)

ow FINAL CONCENTRATION (DM)

0 1 2 3 4 5

FINAL CONCENTRATION (mM)

FINAL CONCENTRATION (mM)

FIGS. 2,3. Addition of different concentrations of cofactors on the o-oxidation of stearic acid (Fig. 2) or lignoceric acid (Fig. 3) by fibroblast homogenates. The enzyme assays were performed in duplicate as described under Methods except that the concentration of one of the cofactors was varied. The amount of water-soluble radiolabeled acetate produced was measured. (a) 0, NAD; 0, L-carnitine; W, coenzyme A; El, FAD. (b) 0, Mg’+; 0, ATP, 0, DTT.

176 SINGH AND POULOS

these cofactors resulted in a drastic reduc- tion in P-oxidation of these acids. Higher concentrations of coenzyme A (Figs. 2a, 3a) and Mg2+ (Figs. 2b, 3b) resulted in inhi- bition of P-oxidation. Higher concentra- tions of coenzyme A were much more in- hibitory for lignoceric acid oxidation (Fig. 3a) than for stearic acid oxidation (Fig. 2a). Higher concentrations of Mg2+ were much more inhibitory for stearic acid oxidation (Fig. 2b) than for lignoceric acid oxidation (Fig. 3b). It is evident that both stearic acid oxidation and lignoceric acid oxidation have an absolute requirement for ATP (Figs. 2b, 3b). Even at the highest concen- tration of ATP tested (5 mM), lignoceric acid oxidation was found to be increasing with ATP concentration. The effect of ATP on lignoceric acid oxidation was tested us- ing l-h incubation time (same as for stearic acid oxidation), and the oxidation increased with ATP concentrations up to 5 mM.

Addition of NAD resulted in a sixfold increase in stearic acid oxidation (Fig. 2a) and a threefold increase in lignoceric acid (Fig. 3a). A similar stimulation of peroxi- somal palmitic or oleic acid oxidation by NAD in rat liver has been described (18). The addition of FAD resulted in some stimulation (less than twofold) of stearic

acid oxidation but had no effect on ligno- ceric acid oxidation (Figs. 2a, 3a). Under our assay conditions L-carnitine and di- thiothreitol (DTT) had no effect on stearic acid oxidation but slightly stimulated lig- noceric acid oxidation (Figs. 2, 3). The co- factor requirements for stearic acid oxi- dation were the same whether the oxida- tion was measured in the presence of phosphatidylcholine or a-cyclodextrin.

P-Oxidation of stearic acid by fibroblast homogenates was linear with time up to 2 h. Increasing concentration of protein up to 24 pg/assay resulted in a linear increase in the oxidation of stearic acid. Under sim- ilar but not identical assay conditions (see Methods), lignoceric acid P-oxidation was linear with time up to 2 h, and was linear with protein concentration up to 96 pg/assay.

The method of presentation of the lipid substrate (stearic or lignoceric acid) on the rate of P-oxidation by fibroblast homoge- nates was investigated (Table I). Under all three procedures employed for the solubi- lization of the substrate, stearic acid p-ox- idation was linear with protein (at least up to 20 pg protein/assay). A higher rate of P-oxidation of stearic acid was seen in the presence of phosphatidylcholine or Triton

TABLE I

THE METHOD OF PRESENTATION OF THE SUBSTRATE vs THE /~-OXIDATION OF LONG CHAIN FAITY ACIDS BY FIBROBLAST HOMOGENATES

Treatment

Stearic acid oxidation (pmol acetate/h)

Protein content/assay: 10 pg 20 pg

Lignoceric acid

oxidation (pmol acetate/h)

20 pg 40 Irg

1. Phosphatidylcholine 55.6 108.9 1.0 1.6

(10 pg/ml) (4.55 PM) (7.69 PM)

2. Triton X-100 48.9 103.2 0.5 0.6

(0.01%) (5.98 /.&M) (8.80 /.LM)

3. a-Cyclodextrin 28.1 58.8 4.4 8.8

(2.5 mg/ml) (6.25 PM) (3.10 PM)

Note. Figures in parentheses indicate the final concentration of the substrate or the compound in the assay mixture. The substrate (fatty acid) was dispersed in phosphatidylcholine or Triton X-100 or a-cyclodextrin by sonication (see Methods) and added to the incubation mixture. The enzyme assays were performed as described under Methods.

FATTY ACID &OXIDATION BY HUMAN SKIN FIBROBLASTS 177

X-100 than with a-cyclodextrin. On the other hand, maximum lignoceric acid p- oxidation was observed in the presence of cu-cyclodextrin. However, lignoceric acid oxidation was negligible in the presence of phosphatidylcholine or Triton X-100 (Table I).

In control fibroblasts, stearic acid oxi- dation was approximately 19-fold higher than lignoceric acid oxidation (Table II). In patients with Zellweger’s syndrome, stearic acid P-oxidation was partially de- creased (60% of controls) but lignoceric acid P-oxidation was nearly absent. Thus, the ratio of stearic acid to lignoceric acid oxidation was very high in Zellweger’s syndrome.

DISCUSSION

The method described here was devel- oped in view of the difficulties encountered in the measurement of very low activities of P-oxidation enzymes in fibroblast ho- mogenates. Precipitation of unreacted substrates (fatty acids) by perchloric acid in the presence of serum albumin and measurement of acid soluble radioactivity as an index of P-oxidation could not be em- ployed for two reasons. First, stearic acid oxidation in fibroblast homogenates is very low (at least 100 times lower compared with liver homogenates (unpublished)), and lignoceric acid oxidation in fibroblast homogenates is even lower (Table II). Sec- ond, lignoceric acid binds poorly to serum

albumin (unpublished) and therefore easily escapes precipitation, as does some of the stearic acid. The present method can be used reliably for the measurement of p-ox- idation of stearic or lignoceric acid by fi- broblast homogenates, and the method re- quires only microgram quantities of ho- mogenate protein. Our method is at least 10 times more sensitive than the one re- ported for the measurement of water sol- uble radioactivity from fatty acids where the reaction product was not identified (12). Identification of the product is important in view of the observations in rat brain where lignoceric acid was shown to be con- verted by a coenzyme A independent path- way to glutamic acid and not acetyl CoA (25). Also, the previously reported assays, which measured COB production from pal- mitic, stearic, or lignoceric acid by fibro- blast homogenates, are much less sensitive, requiring at least 20 times more protein per assay (5, 10, 11). Moreover, the mea- surement of CO, production from radio- labeled fatty acids has the disadvantage that it does not differentiate between (Y- and ,&oxidation. Thus, although COz may arise from decarboxylation (a-oxidation) of the fatty acids, it may also be produced from acetyl CoA which is formed by p-ox- idation of fatty acids in mitochondria. Also COz measurements are not suitable when the P-oxidation pathway is confined to or- ganelles other than mitochondria.

Using our assay procedure, we observed a number of differences in the cofactor re-

TABLE II

LONG CHAIN FATTY ACID ~-OXIDATION BY HOMOGENATES OF SKIN FIBROBLASTS

Long chain fatty acid oxidation (pmol * mg protein-‘. h-l)

Clinical diagnosis Stearic acid Lignoceric acid

(Cl&O) (C24:O) Ratio

(C18:O/C24:0)

Control 4397 f 763 237 f 18 18.6 (n=6) (n = 6)

Zellweger’s syndrome 2666 + 304 26 f 10 102.3 (n = 5) (n-=5)

Note. The enzyme assays were performed as described under Methods. The values represent means f SD and n is the number of different cell lines tested.

1’78 SINGH AND POULOS

quirements for stearic and lignoceric acid oxidation. For example, lignoceric acid ox- idation was stimulated by the addition of a-cyclodextrin (Table I). It is unlikely that this relates entirely to the solubilization effect of a-cyclodextrin because Triton X- 100 appears to inhibit lignoceric acid but not stearic acid oxidation. These data, taken in conjunction with differences in response to ATP, Mg’+, and FAD, indicate that the two oxidation systems are differ- ent. The observations that rat and chicken liver fatty acyl CoA oxidase had high spec- ificity toward C12:O or C14:O fatty acyl CoA (28, 29) but not longer chain acyl CoAs support the idea that at least one or more of the ,&oxidation enzymes required for lignoceric acid oxidation are different. Further support of this hypothesis is pro- vided by the marked reduction in lignoceric acid ,&oxidation, but only a minor reduc- tion in stearic acid P-oxidation, in Zell- weger fibroblasts. Immunocytochemical evidence indicates that Zellweger fibro- blasts have a greatly reduced number of peroxisomes (24). It is, therefore, probable that the reduced lignoceric acid oxidation activity results from diminished activity of the peroxisomal P-oxidation pathway. The decreased stearic acid oxidation in Zellweger fibroblasts suggests that a sig- nificant amount (about 40%) of the oxida- tion observed is carried out by peroxisomes but the bulk of the activity is probably mi- tochondrial. However, it is possible that the reduced stearic acid ,&oxidation may reflect the mitochondrial abnormalities which have also been reported in Zellweger’s syn- drome (22,23). The deficiency in lignoceric acid P-oxidation in Zellweger fibroblasts suggests that either the peroxisomal p-ox- idation complex or one or more of the in- dividual peroxisomal P-oxidation enzymes is defective. Recent studies by Santos et al. (26) indicate that the activity of at least one of the peroxisomal P-oxidation en- zymes, fatty acyl CoA oxidase, is present in skin fibroblasts of Zellweger syndrome patients, although Tager et al. (27) have reported that some peroxisomal P-oxida- tion enzymes, notably the bifunctional protein and 3-ketothiolase, are absent in Zellweger liver. Interestingly, Tager et al.

(27) demonstrated immunochemically that the peroxisomal fatty acyl CoA oxidase was absent in liver of a Zellweger syndrome patient. The observations that fatty acyl CoA oxidase was absent in Zellweger liver (27) but was present in Zellweger fibro- blasts (26) may reflect either tissue-specific differences or methodological difficulties. Our findings concerning lignoceric acid ox- idation in Zellweger fibroblasts support an earlier report (12) but we have extended these studies to show that the major water soluble product formed from stearic and lignoceric acids is acetate, thereby con- firming that both fatty acids are degraded via a P-oxidation mechanism. Further- more, we present evidence that in human skin fibroblasts, the lignoceric acid oxida- tion pathway is different from the stearic acid oxidation pathway. Further studies are in progress to delineate the specific dif- ferences in the P-oxidation enzymes in- volved in stearic and lignoceric acid oxi- dation.

ACKNOWLEDGMENTS

The authors are thankful to Ms. Greta Richardson

for growth and maintenance of cell cultures, Dr. Bar-

bara Paton for critical review of the manuscript, and Mrs. Vivian Davies for typing. This work was sup-

ported by a grant from the National Health and Med- ical Research Council of Australia.

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