Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Abstract

Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.

Figures

Fig. 1

Enzyme activities in cultured keratinocytes from normal controls and Sjögren-Larsson syndrome (SLS) patients. Data are expressed as means ± SD for four patients and four normal controls. A: Fatty aldehyde dehydrogenase (FALDH) activity. B: Fatty alcohol:NAD oxidoreductase (FAO) activity.

Fig. 2

High-performance thin-layer chromatography analysis of keratinocyte neutral lipids from three normal controls and three SLS patients. Total cellular lipids (corresponding to 1.2–1.9 mg of cell protein) were spotted on each lane. Lipids were separated using a two-step solvent system consisting of hexane-diethyl ether-acetic acid (32:17:1) developed to 40% up the plate, followed by development in toluene to the top. Lipids were visualized by charring. Alk, alkane; CE, cholesteryl ester; WE, wax ester; ?, unidentified lipid; Alk-DG, alkyl-diacylglycerol; TG, triglyceride; Chol, cholesterol; PL, phospholipid.

Fig. 3

Identification of 1-O-alkyl-2,3-diacylglycerol derived from SLS keratinocytes. The alkyl-diacylglycerols were purified by TLC and subjected to alkaline hydrolysis to produce deacylated 1-O-alkylglycerols before the formation of isopropylidine derivatives and analysis by chemical ionization-GC-MS. 1-O-Heptadecylglycerol (peak 4) served as an internal standard. A, B: Total ion chromatograms of SLS keratinocyte lipids (A) and normal keratinocyte lipids (B). Peak 1, 1-O-pentadecylglycerol; peak 2, 1-O-hexadecenylglycerol; peak 3, 1-O-hexadecylglycerol; peak 4, 1-O-heptadecylglycerol internal standard; peaks 5, 6, 1-O-octadecenylglycerol; peak 7, 1-O-octadecylglycerol. C: Mass spectrum of peak 3 confirms its structure as 1-O-hexadecyl-2,3-isopropylidine-glycerol.

Fig. 4

Incorporation of [3H]octadecanol (18:0-OH) into keratinocyte lipids. Cells were incubated with [3H]18:0-OH for 24 h, after which cellular lipids were extracted and separated by TLC. Black bars, normal control cells; gray bars, SLS cells. Data are expressed as cpm/mg protein (means ± SD) for four determinations from two experiments. * P ≤ 0.01 using Student's t-test. Alkyl-DG, alkyl-diacylglycerol.

Fig. 5

Fatty alcohol cycle and metabolic pathways altered in SLS keratinocytes. FAO consists of two components, fatty alcohol dehydrogenase (reaction 1) and FALDH (reaction 2), that catalyze the sequential oxidation of fatty alcohol to fatty acid. The line across reaction 2 indicates the metabolic block in SLS. Increased fatty alcohols in SLS are diverted into the biosynthesis of wax esters and ether glycerolipids. PE, phosphatidylethanolamine; N-Alkyl-PE, N-alkyl-phosphatidylethanolamine.