Hugo W. Moser

Preliminary observations on the occurrence of cholesterol sulfate in man

Abstract In the course of studies on the incorporation of sodium [ 35 S]sulfate into sulfatide in patients with neurological disorders, a metabolically active substance was found which resembles sulfatide both in solubility and behavior on column chromatography but is separable from it on thin-layer chromatography, A method was devised for the isolation of larger quantities of this material. It was found to have the same melting point and Chromatographie properties as authentic cholesterol sulfate and strongly resembles it in its infrared spectrum, Mild acid hydrolysis yielded a substance identical to cholesterol in its melting point, and infrared and nuclear magnetic spectra. A survey of human tissues revealed the presence of small amounts of cholesterol sulfate in plasma, bile, urine, liver, kidney, and larger quantities in feces. It was also found in the brain of a 4-day-old infant. Intravenously administered sodium [ 35 S]sulfate was rapidly incorporated into cholesterol sulfate. Until recently, cholesterol sulfate had not been isolated from any biological material. This study demonstrates that it is metabolically active and that it is present in a variety of human tissues and body fluids.

Solubilization and partial purification of steroid sulfatase from rat liver: Characterization of estrone sulfatase☆

Abstract Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.

Cholesterol sulfate in rat tissues. Tissue distribution, developmental change and brain subcellular localization.

1. A reliable micromethod for the determination of the tissue level of cholesterol sulfate has been developed. Cholesterol sulfate was separated from the bulk of the free cholesterol by silica gel column chromatography, and the cholesterol sulfate fraction subjected to benzoylation. A small amount of contaminating free cholesterol and other lipids remaining in this fraction were converted to benzoyl esters while the cholesterol sulfate remained unreacted. The cholesterol sulfate was then separated from the benzoylated contaminants by a second silica gel chromatography column and subjected to solvolysis. The liberated cholesterol was determined by gas-liquid chromatography. 2. The cholesterol sulfate contents of the visceral organs of 43-day-old rats were determined. Every tissue examined contained small amounts of this sulfate. Kidney contained the highest concentration of cholesterol sulfate (250-300 mug/g dry tissue weight) followed by spleen (77 mug/g), adrenal gland (50-70 mug/g) and lung (50-57 mug/g). 3. In brain, cholesterol sulfate level rises sharply from 17 mug/g dry weight in 7-day-old rats to more than 50 mug/g in 15-day-olds, then it declines rapidly to 15 mug/g in the 40-day-olds and this level is maintained to adulthood. The developmental pattern in the liver resembles that in the brain, except that the peak is somewhat flatter with the highest value (60 mug/g dry weight) occurring in the 21-day-old animal. In contrast to the above two tissues, the level of kidney cholesterol sulfate increases steadily from 15 mug/g in 7-day-olds and reaches the adult level of approx. 200 mug/g in 50-day-olds. 4. The highest level of cholesterol sulfate in subcellular fractions of rat brain occurred in a fraction rich in nerve endings. The level here was 10 times higher than that in the mitochondrial fraction, which contained the lowest levels of this steroid sulfate.

BRAIN LIPIDS, PROTEOLIPIDS, AND FREE AMINO ACIDS IN MAPLE SYRUP URINE DISEASE*

MAPLE syrup urine disease (MSUD), an inborn error of amino acid metabolism, was first described by MENKES, HURST and CRAIG (1954), and so named because the urine odour was reminiscent of that of maple syrup. The illness generally begins during the first week of life and is manifested clinically by difficulty in feeding, irritability, rigidity, convulsions and stupor. Death may occur in the first weeks of life or the illness may be prolonged over many months with older infants exhibiting mental and motor retardation. It has been established that patients with MSUD have an excess of branched-chain amino acids in plasma and urine (DANCIS, LEVITZ, MILLER and WESTALL, 1959), and an excess of branched-chain keto acids in urine (MENKES, 1959). A deficiency of branched-chain ketodecarboxylase was demonstrated in their white blood cells (DANCIS, HUTZLER and LEVITZ, 1960). The clinical manifestations can be reversed by use of diets low in leucine, isoleucine, and valine early in the course of the disease. Post-mortem examination of the central nervous system directed attention to the white matter (MENKES et al., 1954; SILBERMAN, DANCIS and FEIGIN, 1961; CROME, DUTTON and ROSS, 1961). Myelin stains showed excessive pallor for age and, often, this was accompanied by a spongy, oedematous appearance of the white matter and an increase in the number and size of astrocytic nuclei. The observation that sudanophilic substances and lipid-containing macrophages are not prominent led to the conclusion that the formation of myelin is delayed. This is in contrast to many of the other white matter diseases of childhood in which normally or abnormally formed myelin is broken down (SILBERMAN et al., 1961; CROME et al., 1961; ALVORD, STEVENSON, VOGEL and ENGLE, 1950). Three studies have been published concerning the brain lipids in children who have died of maple syrup urine disease. The patient reported by WOOLF (1962) died at 17 days of age. He found a moderate increase in water content and a tenfold reduction in cerebrosides. A second patient, dying at 12 days of age, was described by MENKES, PHILIPPART and FIOL (1965). They noted no significant changes in the brain lipids

Farber's lipogranulomatosis. Report of a case and demonstration of an excess of free ceramide and ganglioside.

Abstract The tenth case of Farbers lipogranulomatosis is reported. The clinical and pathologic features closely resemble those previously described except that in this patient the macula was also involved, there was nerve cell loss and neuronal storage in the cerebral cortex, and there was a strong family history of rheumatoid arthritis. The foam cells within the granulomas as well as certain of the neurons contained periodic acid-Schiff positive cytoplasmic material with the histochemical properties of an acidic glycolipid. Biochemical studies demonstrated an excess of gangliosides, roughly in proportion to the accumulation of the periodic acid-Schiff-positive material. The excess gangliosides were found to consist mainly of hematoside, the visceral ganglioside. Lymph nodes, liver, kidney and lung contained a ten- to sixtyfold excess of free ceramide, and similar very high levels of free ceramide were present in a subcutaneous nodule. In rats, subcutaneous injection of ceramide produced granulomas which resembled those found in the patients with the lipogranulomatosis syndrome. It is suggested that the lipogranulomatosis syndrome represents an inborn error of lipid metabolism. Its enzymatic basis is as yet undetermined.

Argininosuccinase deficiency in fibroblasts cultured from patients with argininosuccinic aciduria

Fibroblasts cultured from the skin of four mentally retarded patients with argininosuccinic aciduria were markedly deficient in argininosuccinase activity.

Ceramide and Ganglioside Accumulation in Farber's Lipogranulomatosis.

Summary An analysis of the lipids of various organs of an 11-month-old female dying of Farbers Disease indicated a marked increase in ceramide varying from 8 times normal in the kidney to 66 times normal in the liver. Ceramide made up 13% of the liver lipids. There was also a slight increase in other lipids in the liver, but aside from the ceramide, only the gangliosides were increased in all organs tested. The increased ganglioside was more marked in subcutaneous nodule and varied proportionally with the number of foam cells seen in the tissue. The mechanism by which these lipids are stored in Farbers Disease remains to be determined.

3-Ketosphingolipids: Application to the determination of sphingolipids which contain 4-sphingenine

1. Ceramides and cerebrosides, both with 2-hydroxy or non-hydroxy fatty acids, and sphingomyelins were converted quantitatively to the corresponding 3-keto derivatives with the oxidative reagent 2,3-dichloro-5,6-dicyanobenzoquinone. Sulfatides were converted to 3-ketocerebrosides. 2. The conversion of these sphingolipids to the alpha,beta-unsaturated ketone permits three methods of quantitation: in Method I, the absorption of the reaction product is measured at 230 nm; Method II utilizes high performance liquid chromatography and an ultraviolet light detector; in Method III, the product is reduced with NaB3H4 and the radioactivity of the reduced products is determined. All three methods can be used to measure sphingolipids in nanomole quantities. 3. Methods I and II have been applied to the determination of cerebroside and sulfatide content of normal and metachromatic leukodystrophy brains. In the white matter of the pathological brains, there was a greater accumulation of sulfatides containing non-hydroxy fatty acids than of those containing 2-hydroxy fatty acids. The ceramide levels of the cerebellum in a Farbers disease patient and a control were determined by Method II. Method III was used to determine the content of free ceramides in human sera.

Detection of the carrier state of Hurler's syndrome by assay of α-l-iduronidase in leukocytes

Abstract The mean specific activity of α- l -iduronidase in leukocytes from six obligate heterozygotes for Hurlers syndrome was found to be slightly less than one-half of the mean in normal controls; no overlap of normal and known heterozygote values was encountered. The assay has been applied with success to six potential heterozygotes, siblings of a child with Hurlers syndrome. Thus heterozygote detection of Hurlers syndrome is clearly possible; this finding, as well as the ready availability of leukocytes for screening tests, recommends their use for examination of potential carrier status in this disorder.

STRUCTURAL STUDIES ON SULPHATIDES IN METACHROMATIC LEUCODYSTROPHY

Sulphatides have been isolated by a newly developed method from the brain white matter of normal human adult and child, and of two patients with metachromatic leucodystrophy.