Unusual clinical presentation in two cases of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an inborn error of metabolism that combines the clinical features of late infantile metachromatic leukodystrophy and mucopolysaccharidosis. The characteristic biochemical abnormality is a reduction in the activities of several sulfatases, with consequent tissue accumulation of sulfatides, sulfated glycosaminoglycans, sphingolipids, and steroid sulfates. In this study we present two unusual cases of MSD with variable enzymatic deficiency of arylsulfatases A, B, and C. Both patients had ichthyosis, broad thumbs and index fingers, an unusually slow progression of the neurologic symptoms, and lacked the hepatosplenomegaly that is typical of MSD. Olivopontocerebellar atrophy was present and one patient had a large retrocerebellar cyst. Mucopolysaccharides were not detected in the urine from either subject. Leukocyte arylsulfatase A activity in patient 1 was 0.46 nmol/mg protein/hr and in patient 2 was 0.0 nmol/mg protein/hr (normal 0.7-5.0 nmol/mg protein/hr). Leukocyte arylsulfatase B activity in patient 1 was 24 nmol/mg protein/hr and in patient 2 was 22 nmol/mg protein/hr (normal 115-226 nmol/mg protein/hr). Leukocyte arylsulfatase C in patient 1 was 0.30 pmol/mg protein/hr and in patient 2 was 0.28 pmol/mg protein/hr (normal 0.84 pmol/mg protein/hr). In conclusion, these two patients with MSD had mild clinical presentations not previously reported and variable enzymatic deficiency of arylsulfatases A, B, and C.

Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.

BACKGROUND AND AIMS: The pathogenesis of nonalcoholic steatohepatitis (NASH) is unknown. We tested the hypothesis that NASH is associated with 2 defects: (1) peripheral insulin resistance, which increases lipolysis, delivery of free fatty acids (FFA) to the liver, and hepatic fatty acid beta oxidation, thereby creating oxidative stress; and (2) an abnormality within the hepatocytes that might render them more susceptible to injury from oxidative stress. METHODS: The hypothesis was tested by evaluation of (1) insulin resistance by a 2-step hyperinsulinemic (10 and 40 mU. m(-2). min(-1)) euglycemic clamp; (2) insulin effects on lipolysis by enrichment of [U-(13)C]glycerol; (3) frequency and severity of structural defects in hepatocyte mitochondria in vivo; (4) fatty acid beta oxidation from serum [beta-OH butyrate], release of water-soluble radioactivity from (3)H-palmitate by cultured fibroblasts and urinary dicarboxylic acid excretion; and (5) hepatic lipid peroxidation by immunohistochemical staining for 3-nitrotyrosine (3-NT). Subjects with NASH (n = 6-10 for different studies) were compared with those with fatty liver (n = 6) or normal controls (n = 6). RESULTS: NASH and fatty liver were both associated with insulin resistance, with mean glucose infusion rates (normal/fatty liver/NASH) of step 1, 4.5/1.6/0.9; step 2, 9.5/7.7/4.5 (P < 0.03 for both steps). Although baseline rates of glycerol appearance were higher in those with NASH than in those with fatty liver (means, 14.6 vs. 21.6 micromol. kg(-1). min(-1); P < 0.05), neither group significantly suppressed glycerol appearance at insulin infusion rates of 10 mU. m(-2). min(-1). NASH was associated with loss of mitochondrial cristae and paracrystalline inclusions in 9 of 10 subjects, compared with 0 of 6 subjects with fatty liver. However, no evidence of a generalized defect in fatty acid beta oxidation was noted in any group. Also, mean [beta-OH butyrate] was highest in those with NASH (means, 90 vs. 110 vs. 160 micromol/L; P < 0.04). Increased staining for 3-NT was present in fatty liver, and even greater staining was seen in NASH. CONCLUSIONS: These data indicate that peripheral insulin resistance, increased fatty acid beta oxidation, and hepatic oxidative stress are present in both fatty liver and NASH, but NASH alone is associated with mitochondrial structural defects.

Microsomal fatty aldehyde dehydrogenase catalyzes the oxidation of aliphatic aldehyde derived from ether glycerolipid catabolism: implications for Sjögren-Larsson syndrome.

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.

Skeletal muscle phosphatidylcholine fatty acids and insulin sensitivity in normal humans.

The fatty acid composition of skeletal muscle membrane phospholipids (PL) is known to influence insulin responsiveness in humans. However, the contribution of the major PL of the outer (phosphatidylcholine, PC) and inner (phosphatidylethanolamine, PE) layers of the sarcolemma to insulin sensitivity is not known. Fatty acid composition of PC and PE from biopsies of vastus lateralis from 27 normal men and women were correlated with insulin sensitivity determined by the hyperinsulinemic euglycemic clamp technique at insulin infusion rates of 0.4, 1.0, and 10.0 mU . kg-1 . min-1. Significant variation in the half-maximal insulin concentration (ED50) was observed in the normal volunteers (range 24.0-146.0 microU/ml), which correlated directly with fasting plasma insulin (r = 0.75, P < 0.0001). ED50 was inversely correlated with the degree of membrane unsaturation (C20-C22 polyunsaturated fatty acids; r = 0. 58, P < 0.01) and directly correlated with fatty acid elongation (ratio of 16:0 to 18:0, r = 0.45, P < 0.05) in PC. However, no relationship between fatty acid composition and insulin sensitivity was observed in PE (NS). These studies suggest that the fatty acid composition of PC may be of particular importance in the relationship between fatty acids and insulin sensitivity in normal humans.

Prenatal diagnosis of Sjögren-Larsson syndrome using enzymatic methods.

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.

Sjögren-Larsson syndrome: technique and timing of prenatal diagnosis.

BACKGROUND: Sjögren-Larsson syndrome is an autosomal recessive disease with sequelae including ichthyosis, mental retardation, and spasticity. Although fetal skin biopsy has permitted prenatal diagnosis of Sjögren-Larsson syndrome in the late second trimester, it is accompanied by substantial risks, including fetal loss, premature labor, and detection at a gestational age close to the legal limit for pregnancy termination in most states. A new technique involving biochemical assay of cultured amniocytes for reduced levels of fatty alcohol:oxidized nicotinamide-adenine dinucleotide (NAD+)-oxidoreductase may allow earlier and less invasive detection of Sjögren-Larsson syndrome. CASE: A 38-year-old Lebanese woman, gravida 6, para 3, presented for prenatal diagnosis of Sjögren-Larsson syndrome following a history of two children born with the disease. At 19 weeks' gestation, multiple fetal skin biopsies were obtained by ultrasound-guided transabdominal percutaneous insertion of biopsy forceps. Histologic examination of the specimen revealed no evidence of Sjögren-Larsson syndrome. However, assay of fatty alcohol:NAD(+)-oxidoreductase in cultured amniocytes obtained at fetal skin biopsy showed a profound enzymatic deficiency. Additional fetal skin biopsies were obtained at 23.5 weeks' gestation, and histologic examination was positive for Sjögren-Larsson syndrome. The patient elected to terminate the pregnancy, and a subsequent autopsy on the fetus confirmed Sjögren-Larsson syndrome. CONCLUSION: This case demonstrates the limitations of histologic examination of fetal skin specimens for the diagnosis of Sjögren-Larsson syndrome and indicates the potential value of biochemical detection from fetal amniocytes. This new technique may allow earlier diagnosis of Sjögren-Larsson syndrome, is less invasive, and may be less psychologically traumatic for the patient if she elects to terminate the pregnancy.

Sjögren-Larsson syndrome.

Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by the presence of congenital ichthyosis, mental retardation, and spastic diplegia or tetraplegia. The ichthyosis is generalized in distribution, and histological features of the skin include hyperkeratosis, papillomatosis, acanthosis, and a mildly thickened granular layer. Electron microscopy shows lamellar membranous inclusions in the granular and cornified cells. SLS patients have impaired fatty alcohol oxidation caused by deficient activity of fatty aldehyde dehydrogenase, a component of the fatty alcohol:NAD+ oxidoreductase complex. This enzymatic defect provides a reliable means for diagnosing SLS patients and unaffected SLS carriers. Prenatal diagnosis of SLS can be accomplished by enzymatic studies of amniocytes and cultured chorionic villi cells, or by histological examination of a fetal skin biopsy. Although still unproven, fatty alcohol accumulation in patients is thought to be responsible for the cutaneous symptoms.

Fatty alcohol accumulation in the autosomal recessive form of rhizomelic chondrodysplasia punctata.

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.

Sjögren-Larsson-like syndrome with bone dysplasia and normal fatty alcohol NAD+ oxidoreductase activity.

We report a boy and girl with a "new" multiple congenital anomalies/mental retardation syndrome which resemblances Sjögren-Larsson syndrome. Both patients had a concordant pattern of anomalies consisting of congenital lamellar ichthyosis with spontaneous improvement, moderate mental retardation, mild pyramidal involvement, telecanthus, flat facies, stubby long bones, and coxa valga. Severe myopia, ventriculoseptal defect, and postaxial polydactyly were present in the girl who had more severe bone involvement with dense, enlarged metaphyses, vertebral dysplasia, and advanced skeletal maturation of the lower limbs. Long-chain fatty alcohol NAD+ oxidoreductase (FAO) and steroid sulfatase were normal.

Peroxisomal abnormality in fibroblasts from involved skin of CHILD syndrome. Case study and review of peroxisomal disorders in relation to skin disease.

BACKGROUND AND DESIGN: Peroxisomal deficiency has been described in a number of syndromes characterized by chondrodysplasia punctata, including the Conradi-Hünermann (C-H) syndrome. Because of overlapping clinical features of X-chromosome inheritance, ichthyosis, and limb-reduction defects in C-H and CHILD (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) syndromes, we examined peroxisomal content using diaminobenzidine cytochemistry and peroxisomal functions in fibroblasts from involved vs uninvolved skin of CHILD syndrome. RESULTS: Fibroblasts from involved skin of a patient with CHILD syndrome accumulated cytoplasmic lipid, visualized with the fluorescent probe, nile-red. Ultrastructurally, fibroblasts of involved skin of CHILD syndrome accumulated lamellated membrane and vacuolar structures. By diaminobenzidine ultracytochemistry, fewer peroxisomes were present. Moreover, the activities of two peroxisomal enzymes, catalase and dihydroxyacetone phosphate acyltransferase, were decreased (approximately 30% of normal). However, peroxisomal oxidation of very-long-chain and branched-chain fatty acids was preserved. Moreover, plasma very-long-chain fatty acids, plasma phytanic acid, and erythrocyte plasmalogen content were normal. CONCLUSIONS: The CHILD, C-H, and rhizomelic chondrodysplasia punctata syndromes are all characterized by ichthyosis, chondrodysplasia punctata, and limb defects, as well as peroxisomal deficiency. Thus, these syndromes may be related pathogenically. Because peroxisomes are involved in prostaglandin metabolism, peroxisomal deficiency may directly contribute to the previously reported alterations in prostaglandin metabolism in fibroblasts of involved skin of fibroblasts.

Mutants in a macrophage-like cell line are defective in plasmalogen biosynthesis, but contain functional peroxisomes.

We have used a fluorescence-activated cytotoxicity protocol, 9-(1'-pyrene)nonanol (P9OH)/UV selection (Morand, O. H., Allen, L.-A. H., Zoeller, R. A., and Raetz, C. R. H. (1990) Biochim. Biophys. Acta 1034, 132-141), to isolate a series of plasmalogen-deficient mutants in a murine, macrophage-like cell line, RAW 264.7. Three of these mutants, RAW.7, RAW.12, and RAW.108, displayed varying degrees of plasmalogen deficiency (48, 17, and 14% of wild-type levels, respectively), and all three mutants were deficient in peroxisomal dihydroxyacetone phosphate (DHAP) acyltransferase activity (5% of wild-type). Unlike previously described Chinese hamster ovary (CHO) cell mutants, the RAW mutants contained intact, functional, peroxisomes and normal levels of alkyl-DHAP synthase activity, a peroxisomal, membrane-bound enzyme. In RAW.7 and RAW.108 cells, the loss of peroxisomal DHAP acyltransferase is the primary lesion. RAW.12 displayed not only a deficiency in the DHAP acyltransferase activity, but also displayed a second lesion in the biosynthetic pathway, a deficiency in delta 1'-desaturase activity (plasmanylethanolamine desaturase, EC 1.14.99.19), the final step in plasmenylethanolamine biosynthesis. The deficiencies expressed in the mutants represent unique lesions in plasmalogen biosynthesis. Since the RAW cell line is a macrophage-like responsive cell line, these mutants can be used to examine the role of plasmalogens in cellular functions such as arachidonic acid metabolism, prostaglandin synthesis, protein secretion, and signal transduction.

Sjögren-Larsson syndrome: case reports.

Sjögren-Larsson syndrome (SLS) is a rare, autosomal recessive disorder with worldwide distribution. It consists of ichthyosis, spastic diplegia, and mental retardation. An enzymatic defect in fatty alcohol oxidation recently was identified and is thought to be responsible for the disorder. We report two siblings with SLS. In addition to the typical features of the syndrome, the sister had marked hyperkeratosis of the palms and soles, which is rarely seen to this degree. The brother had joint hyperextensibility, which has not been reported previously. Both individuals had documented deficient activity of fatty alcohol:NAD+ oxidoreductase.

Dyggve-Melchior-Clausen syndrome with increased pipecolic acid in plasma and urine.

A child with the Dyggve-Melchior-Clausen syndrome associated with elevated pipecolic acid levels in plasma and urine is described. Other studies of peroxisomal function, including phytanic acid, very long-chain fatty acids, and plasmalogen synthesis, were normal. This disorder may represent an incompletely characterized defect in peroxisomal metabolism.

Oral carnitine therapy in children with cystinosis and renal Fanconi syndrome.

11 children with either cystinosis or Lowe's syndrome had a reduced content of plasma and muscle carnitine due to renal Fanconi syndrome. After treatment with oral L-carnitine, 100 mg/kg per d divided every 6 h, plasma carnitine concentrations became normal in all subjects within 2 d. Initial plasma free fatty acid concentrations, inversely related to free carnitine concentrations, were reduced after 7-20 mo of carnitine therapy. Muscle lipid accumulation, which varied directly with duration of carnitine deficiency (r = 0.73), improved significantly in three of seven rebiopsied patients after carnitine therapy. One Lowe's syndrome patient achieved a normal muscle carnitine level after therapy. Muscle carnitine levels remained low in all cystinosis patients, even though cystinotic muscle cells in culture took up L-[3H]carnitine normally. The half-life of plasma carnitine for cystinotic children given a single oral dose approximated 6.3 h; 14% of ingested L-carnitine was excreted within 24 h. Studies in a uremic patient with cystinosis showed that her plasma carnitine was in equilibrium with some larger compartment and may have been maintained by release of carnitine from the muscle during dialysis. Because oral L-carnitine corrects plasma carnitine deficiency, lowers plasma free fatty acid concentrations, and reverses muscle lipid accumulation in some patients, its use as therapy in renal Fanconi syndrome should be considered. However, its efficacy in restoring muscle carnitine to normal, and the optimal dosage regimen, have yet to be determined.

Cysteamine therapy for children with nephropathic cystinosis.

We treated 93 children with nephropathic cystinosis with oral cysteamine (mean dose, 51.3 mg per kilogram of body weight per day) for up to 73 months. This agent is known to be effective in depleting cells of cystine. In our study, the mean cystine depletion from leukocytes was 82 percent. A historical control group of 55 children received either ascorbic acid (27 children) or placebo (28). At age six, 2 of 17 controls had a serum creatinine level less than 1.0 mg per deciliter, as compared with 17 of 27 patients treated with cysteamine for at least one year (odds ratio, 12.8; 95 percent confidence interval, 2.1 to 33.9). At the end of the study, creatinine clearance was higher in the cysteamine group than in the control group (38.5 vs. 29.7 ml per minute per 1.73 m2; 95 percent confidence limits on the difference, 1.8 and 15.8), even though the cysteamine group was on average 1.4 years older than the control group. Cysteamine also improved growth; those in the cysteamine group between two and three years of age grew at 93 percent of the normal velocity, as compared with 54 percent in the control group. Fourteen percent of the patients could not tolerate the taste and smell of cysteamine. Concurrent controls treated in a blinded fashion with a placebo were not included in this study. With this limitation in mind, we conclude that oral cysteamine, by depleting cells of cystine, helps maintain renal glomerular function, improves growth, and constitutes the current treatment of choice for nephropathic cystinosis.

Plasma and muscle free carnitine deficiency due to renal Fanconi syndrome.

Plasma and urine free and acyl carnitine were measured in 19 children with nephropathic cystinosis and renal Fanconi syndrome. Each patient exhibited a deficiency of plasma free carnitine (mean 11.7 +/- 4.0 [SD] nmol/ml) compared with normal control values (42.0 +/- 9.0 nmol/ml) (P less than 0.001). Mean plasma acyl carnitine in the cystinotic subjects was normal. Four subjects with Fanconi syndrome but not cystinosis displayed the same abnormal pattern of plasma carnitine levels; controls with acidosis or a lysosomal storage disorder (Fabry disease), but not Fanconi syndrome, had entirely normal plasma carnitine levels. Two postrenal transplant subjects with cystinosis but without Fanconi syndrome also had normal plasma carnitine levels. Absolute amounts of urinary free carnitine were elevated in cystinotic individuals with Fanconi syndrome. In all 21 subjects with several different etiologies for the Fanconi syndrome, the mean fractional excretion of free carnitine (33%) as well as acyl carnitine (26%) greatly exceeded normal values (3 and 5%, respectively). Total free carnitine excretion in Fanconi syndrome patients correlated with total amino acid excretion (r = 0.76). Two cystinotic patients fasted for 24 h and one idiopathic Fanconi syndrome patient fasted for 5 h showed normal increases in plasma beta-hydroxybutyrate and acetoacetate, which suggested that hepatic fatty acid oxidation was intact despite very low plasma free carnitine levels. Muscle biopsies from two cystinotic subjects with Fanconi syndrome and plasma carnitine deficiency had 8.5 and 13.1 nmol free carnitine per milligram of noncollagen protein, respectively (normal controls, 22.3 and 17.1); total carnitines were 11.8 and 13.3 nmol/mg noncollagen protein (controls 33.5, 20.0). One biopsy revealed a mild increase in lipid droplets. The other showed mild myopathic features with variation in muscle fiber size, small vacuoles, and an increase in lipid droplets. In renal Fanconi syndrome, failure to reabsorb free and acyl carnitine results in a secondary plasma and muscle free carnitine deficiency.

Liposome-mediated transfer of simian virus 40 DNA and minichromosome into mammalian cells.

We have investigated the use of liposomes as carriers for the transfer of simian virus 40 (SV40) DNA into mammalian cells. The amount of DNA entrapped in liposomes was dependent on the input DNA concentration and lipid composition. DNA remained intact after liposome encapsulation and was resistant to deoxyribonuclease digestion. Combined transfer to and expression of liposome-entrapped SV40 DNA in monkey kidney cells was assayed by infectious plaque formation. Negatively-charged liposomes containing phosphatidylserine were more effective in DNA transfer and expression than neutral liposomes. The inclusion of carrier salmon sperm DNA inhibited liposome-entrapped SV40 DNA infectivity. Infectivity of liposome-entrapped DNA was directly related to both liposome DNA concentration and number of vesicles added. Liposome-entrapped SV40 minichromosome was 20-fold more infective than free minichromosome, but only 20% more infective than liposome-entrapped SV40 DNA. Thus, the presence of hyperacetylated histones on the DNA failed to enhance liposome-mediated DNA transfer appreciably. Incubation of cells with various modulators of endocytosis implicated the endocytotic pathway in the mechanism of liposome-mediated DNA transfer. These studies show that liposomes are suitable carriers for the introduction of viral DNA and chromatin into mammalian cells.