ANALYTICAL

196,95-98

BIOCHEMISTRY

(1991)

Separation of Phytanic and Pristanic Acid by High-Pressure Liquid Chromatography: Application of the Method Bengt Frode Kase, *,l Jonas Glund,ty2 and Leslie Sisfontest *Department of Pediatric Research, Rikshospitalet, The National Hospital, N-0027 Oslo 1; and tDepartment of Clinical Chemistry I, Huddinge University Hospital, S-141 86 Sweden

Received

November

28, 1990

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation and purification of these acids, is described. A base-line separation of [U-‘Hlphytanic and [U-‘Hlpristanic acid is achieved with a graphitized carbon column. The isoprenoid metabolites formed after incubation of cultured fibroblasts with phytanic or pristanic acids are extracted with a Sep-Pak C,, cartridge and separated from the substrates by the same reversed-phase HPLC used for substrate purification. The methods are suitable for studies on the mechanisms for degradation of phytanic acid. Recently, different inborn errors with accumulation of phytanic acid have been defined. The present method will be a useful tool in our efforts to define these metabolic defects and their subcellular localization. 0 1991 Academic Press. Inc.

Phytol is the alcohol moiety of chlorophyll and the precursor to the polyisoprenoid acid, phytanic acid, that humans get mainly from animal fat (1). Both animals and humans have been shown to degrade phytanic acid very efficiently. Normal P-oxidation of fatty acids is blocked or hindered by the presence of a 3-methyl substitute. The sequence for degradation of phytanic acid in experimental animals has been shown to include (Yoxidation followed by a @-oxidative degradation with losses of 3- and Z-carbon fragments (1). The mechanism of a-oxidation and the formation of the (n-l) lower homolog, pristanic acid, is still not un1 To whom correspondence should be addressed at Department of Pediatric Research, Rikshospitalet, The National Hospital of Norway, N-0027 Oslo 1, Norway. * Dr. Jonas ijlund died on the 7th of October 1990. 0003-2697/91$3.00 Copyright

0 1991

derstood. Also, the accumulation of phytanic acid and the defective phytanic acid oxidase in diseases with lack of peroxisomes is difficult to explain. The main chromatographic systems available for purification of phytanic and pristanic acid until now have been thin layer chromatography (TLC) using silicic acid as adsorbent (2) and preparative gas liquid chromatography (GLC) (3,4). The chromatographic performance of TLC fails to separate pristanic from phytanic acid. Preparative GLC with packed columns has a better chromatographic performance but a restricted capacity to separate and purify from microgram up to milligram amounts of phytanic and pristanic acid. Standard straight-phased or reversed-phase high-pressure liquid chromatography (HPLC) has the same limitation as TLC. This paper describes baseline separation of uniformly tritium-labeled phytanic acid and pristanic acid by a simple HPLC system using a new reversed-phase column with retention of solutes by an adsorption mechanism. The method, improvements compared to existing chromatographic methods, and possible applications of it for further research on phytanic acid degradation will be discussed. MATERIALS

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METHODS

Synthesis of unlabeled and tritium-labeled methylpristanate and methyl phytanate. Phytanic acid was synthesized as described previously (5). To 24 mmol of phenylmagnesium bromide in 5 ml of diethyl ether, 1.4 g of 80% methyl phytanate in 3 ml diethyl ether was added dropwise. The mixture was stirred for 30 min and then hydrolyzed by careful addition of 10 ml 6 M HCl dissolved in diethyl ether. After workup under aqueous acidic conditions the organic phase was dried, filtered, and evaporated. 95

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KASE,

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FIG. 1. Reversed-phase high-pressure liquid chromatogram. [UsH]Phytanic acid and [U-3H]pristanic acid were eluted with 0.2% cont. acetic acid in methanol and separated by a Hypercarb column.

The diphenylcarbinol was dissolved in 30 ml acetic anhydride and refluxed for 8 h. After cooling and addition of 50 ml of water the mixture was extracted with 50 ml of chloroform three times. The organic phase was dried, filtered, and evaporated. This product was dissolved in 10 ml of cont. acetic acid. A solution of 4.0 g CrO, in 3.2 ml of water and 8.0

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ml of cont. acetic acid was added dropwise. The mixture was stirred at a temperature of 10°C during the addition and then stirred at room temperature for 45 min. Workup was performed by addition of 100 ml of water and extraction with three portions of 50 ml of chloroform each. The organic phase was washed with 50 ml diluted HCl and then with 50 ml of saturated brine before drying (MgSO,), filtration, and evaporation. The residue was dissolved in 10 ml of methanol and this solution was added dropwise to a refluxing solution of 2% HCl in 50 ml methanol. Reflux was maintained for 40 min. Cooling by addition of 50 ml of water and extraction with 50 ml of chloroform three times yielded, after drying, filtration, and evaporation, a crude residue containing benzophenone. This by-product was removed by refluxing the residue dissolved in 100 ml nhexane with 17 g semicarbazide on SiO, for 24 h. After cooling and filtration the filtrate was evaporated. This yielded 0.5 g of 80% methyl pristanate. A mixture of 0.2 g methyl pristanate and 0.4 g methyl phytanate was sent to Amersham Inc., Buckinghamshire, England, for tritiation according to Wilzbach. The total activity of the tritiated mixture was 5 Ci, giving a specific activity of 9.2 Ci/mmol of [U-3H]phytanic acid and [U-3H]pristanic acid. Hydrolysis and extraction. Phytanyl- and pristanylmethyl esters were hydrolyzed for 30 min at 70°C in ethanol with 2 M KOH. The mixtures were acidified with 5 M HCl and extracted on Sep-Pak C,, cartridges (Millipore Corp., Millford, MA) (6). The isoprenoid

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FIG. 2. Reversed-phase high-pressure liquid chromatograms. (A) Extract of cultured fibroblasts incubated with [U-3H]phytanic acid (5 x 10-s M) for 6 days and eluted with 0.2% glacial acetic acid in methanol and separated from more polar products by a Hypercarb column. (B) Extract of cultured fibroblasts incubated with [U-3H]pristanic acid (lo-’ M) for 3 days and eluted with 0.2% cont. acetic acid and 10% H,O in methanol and separated from more polar products by a Hypercarb column.

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acids were eluted with ether after two washings with water. The ether was evaporated under N, and the residue was redissolved in ethanol. The free acids were purified immediately before use by HPLC. Chemicals for solvent preparation were of HPLC or analytical grade. Chromatography. Aliquots of the extracts of the phytanic acid and pristanic acid mixture were injected into a LDC Consta Metric III HPLC instrument fitted with a Hypercarb graphitized carbon column, 100 X 4.7 mm i.d., 7 pm (Shandon Scientific Ltd, Cheshire, England). Phytanic and pristanic acid were separated with 0.2% cont. acetic acid in methanol at a flow rate of 1 ml/min. To elute more polar metabolites of pristanic acid, 0.2% cont. acetic acid and 10% H,O in methanol were used. The performance of the column was only slowly reduced and easily restored after several runs by using 1,4 dioxane (HPLC-grade) as solvent. Tissue culture. Skin biopsies were taken from the forearm. Cultures of skin fibroblasts were initiated and maintained in basal Eagle’s medium with 16% fetal calf serum. Penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin (2 U/ml), and L-glutamine (1.5 pmol/ml) were added to the medium. Monolayer cultures were established in 25-cm2 flasks containing 7 ml of the medium at 37°C and gassed with 5% CO, in air. Incubation, hydrolysis, and extraction. Phytanic acid and pristanic acid, used as substrates, were bound to albumin and sterilized by filtration with a Costar /*Star filter (0.22 pm). The incubations were initiated by adding the substrates directly to the medium in the flasks with cells growing in monolayer. The medium used in these incubations consisted of basal Eagle’s medium and was enriched with only 1.6% fetal calf serum. As blank incubations were used cell cultures in the same medium boiled for 5 min in water. After removal of the medium the cells were scraped off with a Costar disposable cell scraper (1.8 cm blade length), washed in 0.9% saline and ethanol. The cell sap and the medium was mixed and hydrolysed in 1 M KOH and 30% ethanol at 100°C for 3 h. After acidification, the hydrolysate was extracted with Sep-Pak C,, as described. In addition an elution step with 6 ml absolute ethanol was used before the final elution with 6 ml diethyl ether. Aliquots of the ethanol extracts were chromatographed on HPLC. RESULTS

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DISCUSSION

The mixture of methyl pristanate and methyl phytanate tritiated by Amersham Inc. was chromatographed by HPLC after hydrolytic cleavage of the methyl esters. Several peaks eluted and each of them was isolated for combined gas chromatography-mass spectrometry (GC-MS). The content of two of them was identified as phytanic acid and pristanic acid. These two peaks were mixed and rechromatographed as shown in Fig. 1.

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Extraction with diethyl ether on Sep-Pak C,, was almost complete for both phytanic and pristanic acid (>95%). The same degree of extraction was achieved with absolute ethanol prior to ether elution of the cell culture hydrolysate. Cultured human fibroblasts were incubated with newly purified pristanic and phytanic acid bound to bovine serum albumin and the incubates were extracted after alkaline hydrolysis. In the blank incubations the 3H activities in the water phase ranged from 0.5 to 1.8% of the total activity. The rest of the activities were obtained in the ethanol/ether phase with a recovery for the blank incubations above 92%. It is evident that the 3H-label in the substrates is stable and not exchanged with hydrogen during the steps of incubation, hydrolysis, and extraction. The end products of [U-3H]phytanic and [U-3H]pristanic acids are CO, and 3H20. The amount of tritium activity in the water phase should reflect the degree of complete oxidation. In addition, [3H]acetic and [3H]propionic acid are supposed to elute into the water phase. After 3 days of incubation with phytanic acid, 1.4% of the 3H activity was detected in the water phase. After 6 days the amount was 5%. The corresponding values for the pristanic acid incubations were 13 and 21%, respectively. It is evident that under the conditions used, pristanic acid is more efficiently oxidized than phytanic acid and that only a minor part of pristanic acid remains unmetabolized after 6 days of incubation. This indicates that one of the steps in conversion of phytanic acid into pristanic acid is rate limiting. The ethanol/ether phases were evaporated and parts of the extracts were chromatographed on reversedphase HPLC using the graphitized carbon HPLC column (Hypercarb). In Fig. 2A a chromatogram of the extract from a 6-day incubation with phytanic acid is shown. The least polar peak that eluted from 20-26 ml is phytanic acid and the peak that eluted from 12-15 ml has an elution volume identical to that of pristanic acid. The identity of the material corresponding to these peaks was verified with combined GC-MS. The more polar compounds at elution volumes 6-8 ml and 3-4 ml have not yet been identified. The chromatogram demonstrates the baseline separation of phytanic acid and pristanic acid. With this system it should be possible to detect intermediates in the conversion of phytanic acid to pristanic acid on HPLC. In Fig. 2B a chromatogram of the extract from the 3-day incubation with pristanic acid is shown. Pristanic acid eluted at 50-60 ml. This peak is split with better resolution (not shown) probably because of isomeric forms. More polar products eluted from 17-22 ml, from 7-8 ml, and from 3-5 ml. These polar products remain unidentified. This paper describes the synthesis of pristanate from phytanate, a simple extraction procedure, and an iso-

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cratic reversed-phase HPLC that easily separates protonized phytanic acid from pristanic acid. A merit of the present method is also the combined preparative and analytical properties. Both phytanic and pristanic acid can be purified in milligram amounts with simple equipment, and the same system is most suitable to separate and isolate intermediates and degradation products of these acids in biological assays for further identification. Thus a simple procedure is presented for the synthesis and purification of two isoprenoids commercially not available today. In addition, the uniformly tritiated substrates and the new chromatographic method described represent suitable tools to characterize the CYoxidation of phytanic acid. With these tools it should also be possible to study the P-oxidation, w-oxidation, elongation, phytanyl-CoA ester formation, and conjugation of isoprenoid acids. There is defective phytanic acid decarboxylation in skin fibroblasts from patients lacking peroxisomes. Surprisingly, however, the activity has been localized to the mitochondrial fraction both from rat (7,8) and human liver homogenates (9), and not to the peroxisomal fraction. Thus it remains an enigma why phytanic acid decarboxylation is defective in patients with peroxisomopathies. The methodologies presented here should be helpful in further attempts to understand the reaction mechanisms for the conversion of phytanic acid into

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pristanic acid and to localize the subcellular ments for each of the reaction steps.

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ACKNOWLEDGMENTS The skillful technical assistance of Odd Thoresen is appreciated. This work was supported by the Anders Jahres Foundation, by the Medical Innovation, The National Hospital, and by the Swedish Medical Research Council.

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K., Bjorkhem, I., and Pedersen, J. I. (1986) Biochem. Biophys. Res. Commun. 138,167-173. 7. Tsai, S-C., Avigan, J., and Steinberg, D. (1969) J. Biol. Chem.

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Separation of phytanic and pristanic acid by high-pressure liquid chromatography: application of the method.

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation ...
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