David A. Wenger

Macular cherry-red spots and myoclonus with dementia: Coexistent neuraminidase and β-galactosidase deficiencies

Abstract A patient was previously characterized as having a variant form of GM1 gangliosidosis based on severe deficiencies in β-galactosidase activity in both leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-galactoside and GM1 ganglioside. Reexamination of her cultured fibroblasts revealed a severe deficiency in neuraminidase activity using neuramin lactose, fetuin and 2-(3′-methoxyphenyl)-N-acetyl-D-neuraminic acid as substrates, but normal neuraminidase activity using GM3 ganglioside as a substrate. The presence of normal levels of β-galactosidase activity in leukocytes from the mother of the patient indicates that the β-galactosidase deficiency is not the primary enzyme defect in this type of patient.

Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification: A collaborative study on 70 patients

Seventy patients were selected to cover the range of variability in clinical expression of Niemann‐Pick disease group C (NP‐C). Their individual main clinical features and course of the disease (age at discovery and type of visceromegaly, age at onset and first neurological manifestation, later neurological symptoms) are schematically described. In cultured skin fibroblasts from these patients, sphingomyelinase activities measured in vitro showed decreased values only in approximately half of the cases, and when the metabolic fate of [14C]‐sphingomyelin was studied in living cell cultures, still 20% of the cases had a normal hydrolysis rate. Esterification of exogenous cholesterol was investigated in cell lines from these and 5 additional patients and in 21 of their parents. Using a non‐lipoprotein [3H]cholesterol source, very low esterification rates were obtained in more than 90% of the cases. All the numerous other pathological conditions studied, including Niemann‐Pick disease types A and B, gave normal results. A more sensitive method was elaborated, in which the cells were challenged with pure human low density lipoproteins (LDL) and the early rate of esterification studied. With the latter procedure, a pronounced deficiency could also be demonstrated in the few cases which had shown a milder impairment using a [3H]cholesterol source, and intermediate rates of esterification were obtained in heterozygotes. Discrimination of these difficult cases and of heterozygotes could also be achieved replacing LDL with total unfrozen human serum. Correlations were established between given clinical phenotypes and the severity of the biochemical lesion. Defective intracellular cholesterol esterification is further established as an intrinsic feature of NP‐C, and demonstration of this metabolic alteration appears as a major advance in diagnosing the condition.

An improved method for the identification of patients and carriers of krabbe's disease

Abstract The activity of galactosyl ceramide and lactosyl ceramide β-galactosidase was measured in leukocyte and fibroblast homogenates. New incubation conditions for this assay provide a significantly more sensitive method than reported previously. Both reactions were stimulated by optimal concentrations of sodium taurocholate and oleic acid. Citrate-phosphate buffer, pH 4.2, was found to give maximum reaction. Galactosyi ceramide and lactosyl ceramide β-galactosidase activities in leukocytes, cultured skin fibroblasts and cultured amniotic cells from controls and patients with lysosomal disease were compared to carriers and patients with Krabbes disease. Activity for lactosyl ceramide β-galactosidase was much greater than galactosyl ceramide β-galactosidase activity and may provide a new, more sensitive method for the diagnosis of Krabbes disease as well as for the identification of suspected carriers. A prenatal diagnosis has been done using this new method.

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Abstract Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.

Synthetic substrate ß-glucosidase activity in leukocytes: A reproducible method for the identification of patients and carriers of Gaucher's disease

A method is described for the identification of patients and carriers of Gauchers disease, using leukocytes from a small volume of blood. The fluorogenic substrate, 4‐methyl‐umbglliferyl‐β‐D‐glucopyranoside, was assayed in the presence of pure sodium taurocholate (2.5 mg/ml) and Triton X‐100 (2.0 mg/ml). Some commercial brands of pure sodium taurocholate were satisfactory for this purpose. The pH optimum for controls, Gaucher disease carriers and Gaucher disease patients was 5.4 using citrate‐phosphate buffer. Although leukocytes prepared from only a small amount of blood (2–8 ml) are required, there is sufficient quantity for measuring other lysosomal enzymes as controls. Using this method, 12 patients with all types of Gauchers disease and 12 obligate heterozygotes were identified. Carrier status was predicted in six other family members and ruled out in six others. Eight unaffected people married to Gaucher carriers or Gaucher patients were predicted to be non‐carriers of Gauchers disease, thereby ruling out children affected with Gauchers disease in that mating.

Deficiency of monogalactosyl diglyceride β-galactosidase activity in Krabbe's disease

Abstract Monogalactosyl diglyceride has previously been demonstrated to be intimately associated with brain white matter, especially myelin. Enzymes responsible for its biosynthesis and degradation have been reported to be present in rat and mouse brain. In the present study, the β-galactosidase responsible for the degradation of this brain specific compound was demonstrated to be extremely deficient in brain, liver and skin fibroblasts from patients who died of Krabbes disease. This deficiency is the third enzymatic block demonstrated in this disorder. The β-galactosidase activity toward galactocerebroside and psychosine is also extremely deficient. This finding provides new information about the substrate recognition pattern of this enzyme and about the possible etiology of globoid cell leukodystrophy.

Electrophoretic forms of human liver α-l-fucosidase and their relationship to fucosidosis (mucopolysaccharidosis F)

Abstract 1. Human liver α-L-fucosidase was studied by starch gel electrophoresis and isoelectric focusing both before and after treatment with neuraminidase. These studies revealed the presence of 5–6 bands on starch gels and six isoelectric forms with p I s of 6.9, 6.5, 6.1, 5.9, 5.7 and 5.5. The activity of the two most acidic forms was reduced after neuraminidase treatment. 2. Liver tissue from a patient with fucosidosis had approximately 4% of normal α- l -fucosidase activity. Starch gel electrophoresis revealed that all electrophoretic forms of the enzyme appear to be deficient. 3. Apparent Michaelis constants ( K m s) determined for both the normal and fucosidotic liver α- l -fucosidase for 4-methylumbelliferyl-α- l -fucopyranoside were found to be 0.085 mM and 0.058 mM, respectively. 4. The pH activity profile of normal and fucosidotic liver α- l -fucosidase were very similar with major pH optima at 5.4 and 5.3, respectively.

Possible misdiagnosis of Krabbe disease.

KRABBE DISEASE or globoid cell leukodystrophy is a severe genetic disease of children with onset of symptoms at about 4 to 6 months of age, although forms with later onset have been reported. Symptoms include tonic seizures, optic atrophy, convulsions, and deafness with a rapid course ending in death between 1 1/2 and 2 years of age. All cases appear to be inherited in an autosomal recessive manner? The primary defect is reported to be a deficiency of the enzyme galactosyl ceramide beta-galactosidase (galactocerebrosidase) which is necessary for the degradation of galactosyl ceramide, a major component of myelin. -~ The amount of gal-cer storage in Krabbe disease is not large when compared to Gu2-ganglioside storage in Tay-Sachs diseasel apparently due to the diminished quantity of myelin formed in these children:~; for some reason myelination appears to be shut off early. This suggests that all of the symptoms and pathologic observations cannot be explained simply on the basis of a cerebrosidase defciency. Because of this, we looked for other natural substrates in brain that could be acted on by the same enzyme. At about that time Miyatake and SuzukP reported that the same enzyme could act on psychosine, the deacylated derivative of cerebroside which is cytotoxic, owing to the free amine group. We then reported that the same enzyme could not degrade another myelin-specific compound, monogalactosyl diglyceride? Patients with Krabbe disease could not degrade this compound nor lactosyl ceramide, a glycolipid that also has a beta-linked galactosyl residue?. ~ Measurement of the degradation of lac-cer has provided a more sensitive diagnostic test for Krabbe disease, in which

Mass spectra of complex molecules. I. Chemical ionization of sphingolipids.

Abstract Methane chemical ionization spectra of acetylated and perdeutero-acetylated ceramides, cerebrosides, and ceramide dihexosides have been analyzed and compared with electron impact ionization spectra of the same compounds. Abundant fragment ions for the loss of acetic acid from the protonated molecular ion in C.I. spectra readily enable the determination of the molecular weights of the principal species of sphingolipid mixtures. Complete structures can be elucidated by the combination of E.I. and C.I. mass spectra.

Isolation of heat-stable glucocerebrosidase activators from the spleens of three variants of Gaucher's disease.

Abstract Heat-stable glycoprotein activator substances which stimulate the activity of human liver glucocerebroside:β-glucosidase have been purified extensively from the spleens of five patients representing the clinical extremes of Gauchers disease ranging from the acute neuropathic form (type 2) to the adult, chronic nonneuropathic form (type 1). Each preparation of Gaucher factor chromatographed as a single coincident peak of protein and activator activity on Sephadex G-150 and each electrophoresed as a single protein band of molecular weight approximately 10,000–12,000 on disc gels in the presence of sodium dodecyl sulfate. With the exception of one preparation, which contained 7.1% carbohydrate, the activator substances contained approximately 17% carbohydrate. The various factors have similar, but not identical, amino acid compositions and are rich in acidic and hydrophobic amino acids. The yield of activator substance ranged from 12.9 to 27.4 mg of protein per 100 g wet wt of tissue, with the highest and lowest yields being obtained from the spleens of two patients with the intermediate form of Gauchers disease. The amount of glucocerebroside:β-glucosidase-stimulating activity obtained from the spleens of adult and infantile patients was similar. In addition, the purified heat-stable factors from the Gaucher spleens were capable of stimulating the hydrolysis of the galactose-containing ganglioside, G M1 , by a partially purified preparation of human liver β-galactosidase. This study documents the presence of a factor that will stimulate the activity of glucocerebroside:β-glucosidase and β-galactosidase in Gaucher spleens but fails to indicate any correlation between the concentration or activity of the stimulatory substances and the extent of pathology or age of onset of the disease; the spleens of patients with the more severe forms of Gauchers disease do not contain the lowest amounts of stimulatory activity.