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The enzyme that catalyzes the oxidation of fatty aldehyde derived from ether glycerolipid catabolism has not been identified. To determine whether microsomal fatty aldehyde dehydrogenase (FALDH) is responsible, we investigated the metabolism of 1-O-[9, 10-(3)H-octadecyl]-glycerol ([(3)H]OG) in FALDH-deficient cultured cells from patients with Sjögren-Larsson syndrome (SLS) and in mutant Chinese hamster ovary (CHO) cells. Intact fibroblasts from SLS patients incubated with [(3)H]OG showed a selective deficiency (38+/-7% of normal) in the incorporation of radioactivity into fatty acid, but no decrease in incorporation of radioactivity into fatty alcohol, total lipids and phosphatidylethanolamine (PE). Consistent with fatty aldehyde accumulation, incorporation of radioactivity into N-alkyl-phosphatidylethanolamine, which is derived from Schiff base formation of free aldehyde with PE, was 4-fold higher in SLS fibroblasts compared to normal controls. Similar results were seen with SLS keratinocytes, whereas FALDH-deficient CHO cells showed a more profound reduction in radioactive fatty acid to 12+/-2% of normal. These results implicate FALDH in the oxidation of ether-derived fatty aldehyde in human and rodent cells. Metabolism of ether glycerolipids is a previously unrecognized source of fatty aldehyde that may contribute to the pathogenesis of SLS.

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Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with deficient oxidation of long-chain aliphatic alcohols. Previous studies have reported modest elevations in total (free + esterified) fatty alcohols in SLS, but free fatty alcohols have not been selectively measured, in part because of their low concentrations in most tissues and the presence of trace fatty alcohol contaminants in some solvents used for their analysis. We adapted methods to measure free fatty alcohols in cultured cells and plasma that minimize exogenous alcohol contamination. Fatty alcohols were analyzed as acetate derivatives, using capillary column gas chromatography. By this method, cultured skin fibroblasts from SLS patients were found to have 7- and 8-fold elevations in the mean content of hexadecanol (16:0-OH) and octadecanol (18:0-OH), respectively. The mean plasma 16:0-OH and 18:0-OH concentrations in SLS patients (n = 11) were 9- and 22-fold higher than in normal controls, respectively. In SLS fibroblasts, most of the fatty alcohol (59%) that accumulated was free rather than esterified alcohol, whereas free alcohol accounted for 23% of the total alcohol in normal cells. These results indicate that elevations in free fatty alcohols provide a sensitive marker for the enzymatic defect in SLS. The ability to measure free fatty alcohols in cultured cells and plasma should prove useful for investigations of normal fatty alcohol metabolism and the deranged metabolism in SLS.

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Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by the presence of congenital ichthyosis, mental retardation, and spasticity. The primary biochemical defect in SLS has recently been identified to be a deficiency of fatty aldehyde dehydrogenase (FALDH), which is a component of fatty alcohol:NAD+ oxidoreductase (FAO). We monitored four pregnancies at risk for SLS by measuring FAO and FALDH in cultured amniocytes or cultured chorionic villus cells. The enzymatic results in one case using amniocytes obtained during the second trimester predicted an affected SLS fetus, which was confirmed at termination of the pregnancy. Another at-risk fetus was predicted to be affected with SLS using cultured chorionic villus cells obtained in the first trimester, and fetal skin fibroblasts confirmed a profound deficiency of FAO and FALDH. Two other fetuses were correctly predicted to be unaffected. These results demonstrate that SLS can be diagnosed prenatally using enzymatic methods.

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Patients with the autosomal recessive form of rhizomelic chondrodysplasia punctata (AR-RCDP) and other generalized peroxisomal disorders are deficient in the incorporation of fatty alcohol into plasmalogen lipids. To determine whether these patients accumulated fatty alcohol, we measured their plasma fatty alcohol concentrations. Plasma octadecanol levels were elevated in six patients with AR-RCDP but tended to be normal in other generalized peroxisomal disorders such as neonatal adrenoleukodystrophy and Zellweger syndrome. Cultured skin fibroblasts from AR-RCDP patients accumulated six-fold more hexadecanol than normal when cells were incubated in the presence of palmitate but had normal hexadecanol content when palmitate was not present in the culture medium. These cells were profoundly deficient in the incorporation of hexadecanol into ether lipids but oxidized hexadecanol to fatty acid normally. AR-RCDP fibroblasts also showed a two- to seven-fold increase in the rate of hexadecanol synthesis, which was associated with an increase in the activity of acyl-CoA reductase. We conclude that patients with AR-RCDP accumulate fatty alcohol due to its impaired incorporation into ether lipids and a greatly increased rate of fatty alcohol synthesis.

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Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder associated with reduced activity of the fatty alcohol: NAD+ oxidoreductase complex (FAO). Recent studies indicate that SLS patients are specifically deficient in the fatty aldehyde dehydrogenase (FALDH) component of FAO. To investigate the possibility of carrier detection for SLS, FAO and FALDH activities were measured in cultured skin fibroblasts from normal controls, obligate SLS heterozygotes, and SLS homozygotes using the 18-carbon substrates octadecanol and octadecanal. Three of 11 heterozygotes for SLS had FAO activities that were within the normal range; the other 8 SLS heterozygotes had FAO activities below normal. In contrast, fibroblast FALDH activity was more effective than FAO in discriminating SLS heterozygotes from normal controls. FALDH activity (nmol min-1 (mg protein)-1) in normal controls was 8.54 +/- 1.16 (mean +/- SD; range 6.95-10.77; n = 12) and in SLS heterozygotes was 5.12 +/- 1.31 (range 3.28-6.96; n = 11), or 60 +/- 15% of mean normal activity. One SLS heterozygote had an FALDH activity within the lower range of normal; this heterozygote had an FAO activity below normal. None of the SLS heterozygotes had an FAO or FALDH activity that was in the range of that measured in SLS homozygotes. These results indicate that measurement of FAO and FALDH activities in cultured skin fibroblasts using 18-carbon substrates is useful for SLS carrier detection.

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Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with impaired fatty alcohol oxidation due to deficient activity of fatty alcohol:NAD+ oxidoreductase (FAO). FAO is a complex enzyme which consists of two separate proteins that sequentially catalyze the oxidation of fatty alcohol to fatty aldehyde and fatty acid. To determine which enzymatic component of FAO was deficient in SLS, we assayed fatty aldehyde dehydrogenase (FALDH) and fatty alcohol dehydrogenase in cultured fibroblasts from seven unrelated SLS patients. All SLS cells were selectively deficient in the FALDH component of FAO, and had normal activity of fatty alcohol dehydrogenase. The extent of FALDH deficiency in SLS cells depended on the aliphatic aldehyde used as substrate, ranging from 62% of mean normal activity using propionaldehyde as substrate to 8% of mean normal activity with octadecanal. FALDH activity in obligate SLS heterozygotes was partially decreased to 49 +/- 7% of mean normal activity using octadecanal as substrate. Differential centrifugation studies in fibroblasts indicated that this FALDH enzyme was largely particulate; soluble FALDH activity was normal in SLS cells. Intact SLS fibroblasts oxidized octadecanol to fatty acid at less than 10% of the normal rate, but oxidized free octadecanal normally, suggesting that the FALDH affected in SLS is chiefly involved in the oxidation of fatty alcohol to fatty acid. These results show that the primary enzymatic defect in SLS is the FALDH component of the FAO complex, which leads to deficient oxidation of fatty aldehyde derived from fatty alcohol.

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To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.

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We investigated the biochemical and clinical efficacy of dietary erucic acid (C22:1) therapy for X-linked adrenoleukodystrophy (ALD). In a double-blind crossover study of patients who were on chronic oleic acid (C18:1) therapy, addition of erucic acid to the diet led to a further reduction in plasma hexacosanoic acid (C26:0) concentration. We treated 12 newly diagnosed ALD patients with a diet enriched with erucic acid and oleic acid for 2 to 19 months. Mean plasma C26:0 concentration decreased to normal by 4 weeks, and the C26:0 composition of plasma sphingomyelin and phosphatidylcholine became normal by 4 months on therapy. Fatty acid analysis of postmortem tissues from 1 boy treated for 10 months suggested that dietary erucic acid entered the heart, liver, adrenal gland, and brain. Eight patients remained on treatment long enough (mean, 12 +/- 3 months) to evaluate their clinical response; 6 of these patients with moderate to advanced disease deteriorated neurologically or showed progression of white matter disease on brain magnetic resonance imaging whereas 2 mildly affected patients remained clinically stable after 10 and 19 months. No adverse effects of the diet occurred. We conclude that dietary erucic acid therapy is effective in lowering plasma C26:0 to normal in ALD patients, and may prevent further demyelination in some mildly affected boys.

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We investigated fatty alcohol metabolism in eight patients with Sjögren-Larsson syndrome, and in nine obligate heterozygotes. Fatty alcohol: nicotinamide-adenine dinucleotide oxidoreductase (FAO) activity was deficient in cultured skin fibroblasts (mean 18% of normal, n = 8) and peripheral blood leukocytes (mean 22% of normal, n = 3) from patients with Sjögren-Larsson syndrome. The palmitoyl coenzyme A-inhibitable component of FAO activity was decreased to 10% and 15% of normal in fibroblasts and leukocytes, respectively, of patients with Sjögren-Larsson syndrome. Most affected patients accumulated long-chain fatty alcohol in plasma, with a greater relative accumulation of octadecanol (mean threefold greater than normal) than hexadecanol (mean twofold greater than normal). Erythrocyte lipid alkyl ether linkages derived from hexadecanol were slightly increased in three of four patients. Fibroblasts and leukocytes from heterozygotes with Sjögren-Larsson syndrome showed mean FAO activities that were intermediate between those seen in homozygotes and in normal control subjects. The heterozygotes had normal fatty alcohol concentrations in plasma. These studies demonstrate FAO deficiency in patients with Sjögren-Larsson syndrome, and suggest that accumulation of fatty alcohol or its metabolic products may be important in the pathogenesis of this disorder.

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Lipid metabolism was studied in cultured skin fibroblasts from patients with the inherited disorder, Sjögren-Larsson syndrome (SLS). Intact SLS fibroblasts incubated in the presence of [1-14C]palmitate accumulated more radioactive hexadecanol than did normal cells, whereas incorporation of radioactivity into other cellular lipids was unaltered. The hexadecanol content of SLS fibroblasts was abnormally elevated. Hexadecanol accumulation was not due to increased fatty alcohol synthesis nor its deficient utilization for glycerol ether synthesis. The half-life of intracellular hexadecanol loaded into SLS fibroblasts was increased (70 min) compared with normal (15 min), and intact SLS fibroblasts showed impaired oxidation of [14C]-hexadecanol to fatty acid. Fatty alcohol:NAD+ oxidoreductase, the enzyme catalyzing this reaction, was deficient in SLS fibroblasts. Mean total activity in SLS fibroblasts (n = 5) was 13% of that in normal fibroblasts, and palmitoyl CoA-inhibitable activity was 1% of normal. Fibroblasts from two obligate SLS heterozygotes had enzyme activities intermediate between that in normal fibroblasts and individuals with SLS. These results suggest that the primary defect in SLS is deficiency of fatty alcohol:NAD+ oxidoreductase. SLS represents the first inherited disorder in man associated with an isolated abnormality in fatty alcohol metabolism.

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Intact cultured human fibroblasts reduced [1-14C]palmitate to radioactive hexadecanol in a concentration-dependent manner. In the presence of 30 microM radioactive palmitate, cellular levels of labeled hexadecanol increased over time and reached a steady state corresponding to at least 0.1% of cell-associated radioactive palmitate. These levels of [14C]hexadecanol were increased up to 10-fold when exogenous nonradioactive hexadecanol was present, suggesting that radioactive hexadecanol was actively metabolized. Cells incubated in fatty acid-free medium with [1-14C]hexadecanol rapidly oxidized it to palmitic acid; less than 2% of the hexadecanol taken up by the cells was incorporated into the ether linkage of phosphatidylethanolamine, and no incorporation into wax esters was detected. Double-label experiments involving incubation of intact fibroblast with [3H]palmitate and [14C]hexadecanol demonstrated simultaneous synthesis of hexadecanol from palmitate and oxidation of hexadecanol to palmitate. Addition of exogenous palmitate to the medium of intact cells inhibited the oxidation of hexadecanol to fatty acid in a concentration-dependent fashion. This was associated with an increase in the fibroblast content of hexadecanol and loss of hexadecanol into the medium. Activity of fatty alcohol:NAD+ oxidoreductase, which catalyzes the oxidation of hexadecanol to palmitic acid, was inhibited by palmitoyl-CoA and NADH, but not by palmitic acid. These results are consistent with the presence of a "fatty alcohol cycle" in which hexadecanol is synthesized from palmitate via acyl-CoA and simultaneously oxidized back to free fatty acid. Fatty acyl-CoA, which is the primary substrate for fatty alcohol synthesis, may also regulate the intracellular level of fatty alcohol by inhibiting its oxidation.

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The stability of aminophylline and methylprednisolone sodium succinate in admixtures containing both drugs was studied. Admixtures containing aminophylline 1.0 mg/mL and methylprednisolone sodium succinate 2.0 and 0.5 mg/mL were prepared in both 5% dextrose injection and 0.9% sodium chloride injection. Each admixture was prepared in triplicate and samples were kept at room temperature in glass. Immediately after admixture and at one, two, and three hours, samples were visually inspected, tested for pH, filtered, and assayed in duplicate by high-performance liquid chromatography for theophylline concentration and for both methylprednisolone sodium succinate and methylprednisolone alcohol. Control solutions containing only one of the two drugs were also tested. No visual changes were observed. The admixtures had higher pH values after aminophylline was added, but pH of the samples did not change significantly. Aminophylline concentrations did not change significantly throughout the study period. In 0.9% sodium chloride admixtures with methylprednisolone sodium succinate 0.5 mg/mL, less than 90% of the initial methylprednisolone concentration remained at two hours at the 2.0 mg/mL initial concentration, less than 90% remained at three hours. However, methylprednisolone alcohol (a pharmacologically active form of methylprednisolone sodium succinate) was detected in increasing concentrations after the first hour. Aminophylline in a final concentration of 1.0 mg/mL or less can be mixed with methylprednisolone sodium succinate in a final concentration of 2.0 mg/mL or less in 5% dextrose injection or 0.9% sodium chloride injection and administered intravenously within three hours after mixing.

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