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Assay for Sjogren--Larsson syndrome based on a deficiency of phytol degradation.

Sjogren--Larsson syndrome (SLS) is a metabolic disorder characterized by an accumulation of long-chain fatty alcohols in plasma (1, 2). Studies by Rizzo and coworkers (3-5) have identified the enzyme that is deficient in SLS as fatty aldehyde dehydrogenase (FALDH; EC, which is encoded by the ALDH10 gene. FALDH is part of the microsomal alcohol [NAD.sup.+]-oxidoreductase complex, which functions in the conversion of long-chain fatty alcohols into fatty acids (3). A deficiency of FALDH leads to the accumulation of fatty alcohols in plasma (4,5). Interestingly, FALDH also plays a role in the degradation of leukotriene [B.sub.4] (6).

In our laboratory, we recently established that SLS patients are also deficient in the degradation of phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), a branched-chain fatty alcohol commonly found in nature as part of the chlorophyll molecule (7). Interest in phytol has focused mainly on its metabolism to phytanic acid, a fatty acid that plays an important role in Refsum disease and several other peroxisomal diseases (8,9). However, very little was known about the precise mechanism of the conversion of phytol to phytanic acid until our discovery of the involvement of FALDH in this pathway. Indeed, when we incubated fibroblasts derived from SLS patients in the presence of phytol in the culture medium, we observed a marked deficiency in the conversion of phytol to phytenic acid (Fig. 1A).

In an effort to confirm this finding, we set up an assay in which fibroblast homogenates were incubated with phytol in the presence of [NAD.sup.+] and assessed activity by quantifying the amount of phytenic acid formed in this reaction by gas chromatography-mass spectrometry (GC-MS) (7). This assay was both sensitive and reliable for the diagnosis of SLS patients, which is of interest because the assays described previously in the literature have several drawbacks, including the use of noncommercially available or radioactive substrates and high residual activities in patients who were expected to be completely deficient because of nonsense mutations in the ALDH10 gene (3,10). For this reason, we performed a detailed analysis of the enzymatic assay for the reliable diagnosis of SLS patients. The assay makes use of cultured skin fibroblasts grown in Ham's F-10 nutrient mixture containing L-glutamine and 25 mmol/L HEPES (Gibco, Invitrogen) and supplemented with 100 mL/L fetal calf serum, 100 kIU/L penicillin, and 100 mg/L streptomycin (all purchased from Gibco) at 37[degrees]C in 5% C[O.sub.2]. Fibroblasts were harvested by trypsinization (Gibco) and stored as cell pellets at -80[degrees]C. The FALDH-deficient fibroblasts were from established SLS patients as concluded from the clinical history, deficient FALDH activity, and distinct mutations in the ALDH10 gene (11).

For the measurements, fibroblast pellets were suspended in phosphate-buffered saline and homogenized by sonication (two cycles of 10 s at 9 W) on ice. The incubation mixture consisted of 40 mg/L protein, 50 mmol/L glycine buffer (pH 9.2), 1 mmol/L [NAD.sup.+] (Roche), 1 g/L sodium cholate, and 1 g/L methyl-[beta]-cyclodextrin (Fluka) in a total volume of 500 [micro]L. Reactions were performed at 37[degrees]C and initiated by the addition of 200 [micro]mol/L phytol (mixture of Z and E isomers; Merck) dissolved in dimethyl sulfoxide. After 60 min, the incubation was terminated by the addition of 100 [micro]L of 1 mol/L HCl. As internal standard, 49.3 pmol of [sup.2][H.sub.3]- phytanic acid dissolved in toluene (Dr. H.J. ten Brink, Free University Hospital, Amsterdam, The Netherlands) was added. Subsequently, 2 mL of hexane was added, after which the organic layer was evaporated to dryness under nitrogen at 40[degrees]C. The sample was then derivatized with N-tert-butyldimethylsilyl-N-methyl-trifluoroacetamide (Aldrich) and pyridine (50 [micro]L each) at 80[degrees]C for 30 min, dried under nitrogen, and redissolved in hexane. The sample was analyzed on an Agilent Technologies Model 5890/5973 GC-MS system equipped with a CPsil 19CB capillary column [25 m x 0.25 mm (i.d.); film thickness, 0.2 [micro]m; Varian]. The GC was operated in the splitless mode, with a helium flow rate of 1.5 mL/min and an oven temperature program starting at 60[degrees]C for 1.5 min followed by an increase of 30[degrees]C/min up to 240[degrees]C, an increase of 10[degrees]C /min up to 270[degrees]C, and finally an increase of 30[degrees]C /min up to 300[degrees]C, which was held for 5 min. The injection and detector temperatures were 300 and 290[degrees]C, respectively. MS detection was performed with electron impact ionization applied at 70 eV. MS acquisition was performed in the single-ion monitoring mode, specifically the [[M - 57].sup.+] ions of 353.3, 367.3, 369.3, and 372.3 for phytol, phytenic acid, phytanic acid, and the internal standard, respectively. The metabolites were quantified by use of calibration curves for phytanic acid because phytenic acid is not available commercially. Calibration curves were generated in a concentration range of 0-100 [micro]mol/L, and phytenic acid concentrations were calculated by a linear fit.


We first optimized the assay for optimal pH by incubating control fibroblast homogenates in the standard reaction medium with a pH ranging between 8 and 10. Maximum activity was at pH 9.2 (Fig. 1A in the Data Supplement that accompanies the online version of this Technical Brief at Addition of methyl-[beta]-cyclodextrin, a compound that is often used to solubilize hydrophobic compounds (12), was found to have a positive effect on enzyme activity. A maximum increase in activity of approximately twofold was observed at 1 g/L methyl-[beta]-cyclodextrin (data not shown).

On analysis, we found a small quantity of phytenic acid in the phytol that was used as substrate (Fig. 1B). This contamination amounted to <5% of the phytenic acid produced under standard assay conditions. Therefore, for each assay, we prepared a sample that was stopped immediately. The amount of phytenic acid measured in this sample was subtracted from the samples that were incubated for the usual time period to correct for this contamination.

With the assay conditions described above, we found a dependence on [NAD.sup.+] with a [K.sub.m] of ~0.1 mmol/L, calculated from the Michaelis--Menten plot (Fig. 1B in the online Data Supplement). Incubations of fibroblast homogenates with 1 mmol/L [NAD.sup.+] and various concentrations of phytol showed first-order kinetics (Fig. 1C in the online Data Supplement), and a [K.sub.m] of ~0.1 mmol/L for phytol was calculated. Under these conditions, the assay was linear with time up to 30 min at a fixed protein concentration of 50 mg/L (data not shown). As standard assay conditions, 50 mg/L of fibroblast homogenate with an incubation time of 30 min was chosen because this allowed for reliable GC-MS quantification of the reaction product phytenic acid.

The intraassay imprecision (CV), obtained by measuring the phytol-degradation activity of 10 fibroblast homogenates derived from separate pellets of a single control cell line in a single experiment, was 11%. The interassay CV, obtained by measuring the activity of fibroblast homogenates derived from separate pellets of the same control cell line in 10 separate experiments, was 13%.

We measured phytol-degradation activity in homogenates of 10 different control fibroblasts cell lines and found a mean (SD) specific activity of 11.3 (5.1) nmol x [min.sup.-1] x [(mg protein).sup.-1] [range, 4.0-22.8 nmol x [min.sup.-1] x [(mg protein).sup.-1]], whereas in fibroblasts derived from 15 different previously diagnosed SLS patients, the activity was 0.6 (0.5) nmol x [min.sup.-1] x [(mg protein).sup.-1] [range, 0.0-1.9 nmol x [min.sup.-1] x [(mg protein).sup.-1]; Fig. 1C]. Additionally, we performed measurements on fibroblast homogenates that were derived from three obligate heterozygotes, and the specific activities were in an intermediate range between the controls and SLS patients [4.2 (1.3) nmol x [min.sup.-1] x [(mg protein).sup.-1]; range, 2.7-5.1 nmol x [min.sup.-1] x [(mg protein).sup.-1]].

When we assessed FALDH activity by measuring production of NADH fluorometrically (3), we observed high residual background activity in fibroblasts derived from SLS patients with nonsense mutations in ALDH10. This can be explained by the fact that as many as 12 different aldehyde dehydrogenases have been described in mammals (11), which makes the existence of overlapping substrate specificities quite likely. In contrast, the assay we describe here, which is based on phytol degradation, shows only a very marginal residual activity, which makes diagnosis of patients more straightforward.


(1.) Rizzo WB, Craft DA. Sjogren-Larsson syndrome: accumulation of free fatty alcohols in cultured fibroblasts and plasma. J Lipid Res 2000;41:1077-81.

(2.) Willemsen MA, IJlst L, Steijlen PM, Rotteveel JJ, de Jong JG, van Domburg PH, et al. Clinical, biochemical and molecular genetic characteristics of 19 patients with the Sjogren-Larsson syndrome. Brain 2001;124:1426-37.

(3.) Rizzo WB, Craft DA. Sjogren-Larsson syndrome. Deficient activity of the fatty aldehyde dehydrogenase component of fatty alcohol:[NAD.sup.+] oxidoreductase in cultured fibroblasts. J Clin Invest 1991;88:1643-8.

(4.) Rizzo WB, Dammann AL, Craft DA, Black SH, Tilton AH, Africk D, et al. Sjogren-Larsson syndrome: inherited defect in the fatty alcohol cycle. J Pediatr 1989;115:228-34.

(5.) Rizzo WB, Carney G, Lin Z. The molecular basis of Sjogren-Larsson syndrome: mutation analysis of the fatty aldehyde dehydrogenase gene. Am J Hum Genet 1999;65:1547-60.

(6.) Willemsen MA, Rotteveel JJ, de Jong JG, Wanders RJ, IJlst L, Hoffmann GF, et al. Defective metabolism of leukotriene B4 in the Sjogren-Larsson syndrome. J Neurol Sci 2001;183:61-7.

(7.) van den Brink DM, van Miert JN, Dacremont G, Rontani JF, Jansen GA, Wanders RJ. Identification of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid. Mol Genet Metab 2004;82:33-7.

(8.) Wanders RJ, Jansen GA, Lloyd MD. Phytanic acid a-oxidation, new insights into an old problem: a review. Biochim Biophys Acta 2003;1631:119-35.

(9.) Wierzbicki AS, Lloyd MD, Schofield CJ, Feher MD, Gibberd FB. Refsum's disease: a peroxisomal disorder affecting phytanic acid [alpha]-oxidation. J Neurochem 2002;80:727-35.

(10.) Rizzo WB, Dammann AL, Craft DA. Sjogren-Larsson syndrome. Impaired fatty alcohol oxidation in cultured fibroblasts due to deficient fatty alcohol: nicotinamide adenine dinuclectide oxidoreductase activity. J Clin Invest 1988;81:738-44.

(11.) Yoshida A, Rzhetsky A, Hsu LC, Chang C. Human aldehyde dehydrogenase gene family. Eur J Biochem 1998;251:549-57.

(12.) Singh I, Kishimoto Y. Effect of cyclodextrins on the solubilization of lignoceric acid, ceramide, and cerebroside, and on the enzymatic reactions involving these compounds. J Lipid Res 1983;24:662-5.

DOI: 10.1373/cl i nchem.2004.037879

Daan M. van den Brink, Joram M. van Miert, and Ronald J.A. Wanders *

Academic Medical Center, Laboratory of Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children's Hospital, University of Amsterdam, Amsterdam, The Netherlands; * address correspondence to this author at: Laboratory for Genetic Metabolic Diseases (F0-224), Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands; fax 31-20-696-2596, e-mail
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Title Annotation:Technical Briefs
Author:van den Brink, Daan M.; van Miert, Joram M.; Wanders, Ronald J.A.
Publication:Clinical Chemistry
Date:Jan 1, 2005
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