Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.

Frequency of carriers of chronic (type I) Gaucher disease in Ashkenazi Jews.

In this study we estimate the frequency of carriers of chronic (type I) Gaucher disease among Ashkenazi Jews by examining the glucocerebrosidase activity in leukocytes in a population of 635 blood donors (441 Ashkenazi) and 57 obligatory heterozygotes. Estimation using the defect in the enzyme glucocerebrosidase (beta-glucosidase) in leukocytes is complicated by the existence of considerable overlap between enzyme activity in normals and in heterozygotes. The assay was carried out with a natural substrate labeled with 14C. Discriminant analysis was used to establish an optimal cutoff point between the obligatory heterozygotes and normal (non-Ashkenazi) subjects for the purpose of estimating frequency of carriers. Applied to the Ashkenazi group, the cutoff point identified 3.17% as heterozygotes. Corrected for errors in classification, the carrier rate was estimated as 4.67%. This figure is in good agreement with a carrier rate of 4% estimated from the number of known cases of clinical Gaucher disease ascertained in Israel.

Sphingomyelinase activity levels in human peripheral blood leukocytes, using [3H]sphingomyelin as substrate: study of heterozygotes and homozygotes for Niemann-Pick disease variants.

Patients and heterozygous carriers of Niemann-Pick disease types A and B as well as the primary (genetic) sea-blue histiocyte syndrome were investigated for their leukocyte sphingomyelinase activity. In parallel, glucocerebrosidase activity was determined in all cases studied. [3H]Sphingomyelin and [14C]glucocerebroside served as substrates for sphingomyelinase and glucocerebrosidase activity measurements, respectively. Conditions for these enzymes' assays are discussed. Sphingomyelinase activity was completely absent in three cases of Niemann-Pick disease type A and significantly diminished in one patient with Niemann-Pick disease type B and two with the sea-blue histiocyte syndrome. Sphingomyelinase activity in obligatory heterozygotes of all variants investigated represented about 40 to 70% of normal activity. Nevertheless, some overlapping with normal values occasionally occurred. Interestingly, glucocerbrosidase activity was elevated in patients with Niemann-Pick disease variants.

Prenatal diagnosis of Gaucher's and Niemann-Pick diseases. Assays of glucocerebrosidase and sphingomyelinase in tissue cultures using natural substrates.

Prenatal diagnosis has been successfully achieved by enzyme assays in cultured amniocytes in three high risk pregnancies for Gaucher's disease and three for Niemann-Pick disease type A. [14C]Stearic acid glucocerebroside and [3H]-dihydrosphingomyelin were used as substrates for glucocerebrosidase and sphingomyelinase activity measurements, respectively. Values for the above two enzyme levels in cultured amniotic fluid cells of normal controls and in the pregnancies at risk are presented. The diagnosis made in utero in one fetus affected with Niemann-Pick disease was subsequently confirmed following abortion (at 19 weeks gestation), by the specific biochemical and pathological features of various organs of the afflicted fetus (to be published). Confirmation of diagnoses made in utero for heterozygous and healthy fetuses was obtained by post-partum examination of glucocerebrosidase and spingomyelinase activity levels in leukocytes of the appropriate infants.

Chemical and biochemical studies in human fetuses affected with Niemann-Pick disease type A.

Chemical and biochemical studies were performed on two unrelated fetuses affected with Niemann-Pick disease type A, following abortion at about the 19th week of gestation. Abortion was performed as a consequence of previous findings, in amniotic fluid cell cultures, that sphingomyelinase activity was completely absent. Phospholipid analyses of various organs of the fetuses revealed an excess of sphingomyelin in all viscera as compared to control nonaffected fetuses. Spleen and liver were the organs mostly affected. Interestingly enough considerable accumulation of sphingomyelin was found in the placenta. The brain was the only organ in which sphingomyelin storage could not be proved. In addition to sphingomyelin a slight accumulation of cholesterol was noticed. Deficiency of sphingomyelinase activity measured at pH 5.0 was the general characteristics of the affected tissues. It could be concluded that the accumulation of sphingomyelin in various organs throughout the body of fetuses affected with Niemann-Pick disease, was suggestive of the essential role of the enzyme sphingomyelinase and its biochemical maturation, even during the early stages of gestation.

Accumulation of lactosyl ceramide in leukocytes of patients with adult Gaucher's disease.

Glycosphingolipids were isolated from the leukocytes of 10 patients with Type I, chronic nonneuronopathic (adult) Gaucher's disease and 12 normal subjects, by silicic acid column chromatography and thin-layer chromatography. Quantitation of the individual glycosyl ceramides was achieved by the determination of hexose and sphingosine content, using colorimetric and fluorometric procedures. Lactosyl ceramide, which is the main glycolipid in leukocytes of normal subjects, was significantly increased in the leukocytes of patients with Gaucher's disease. On the other hand, the amount of glycosyl ceramide, which is the main glycolipid accumulating in the reticuloendothelial cells of patients with Gaucher's disease, was similar in Gaucher and in control leukocytes.

Clinical and biochemical study of a child with the non-neuronopathic-Type B form of Niemann-Pick disease.

A child with Niemann-Pick disease type B, diagnosed at the age of two years and followed up for three years is described. Despite extensive visceral involvement--as deduced from marked hepatosplenomegaly and pulmonary infiltration--the child is in general good health and has no apparent neurologic abnormality. Biochemical studies revealed a tenfold increase of sphingomyelin content in his liver biopsy specimen and a markedly reduced sphingomyelinase activity in peripheral blood leukocytes. His parents were found to be heterozygous carriers of the disease, showing sphingomyelinase activity values intermediate between those of patient and healthy subjects. The relevant literature, concerning patients with the non-neuronopathic type B form, is reviewed.

Coexistence of factor XI (plasma thromboplastin antecedent) deficiency and Gaucher's disease.

The findings of Factor XI (plasma thromboplastin antecedent) deficiency in a patient with Gaucher's disease was investigated. A family study, which included measurements of leukocyte glucocerebrosidase activity and Factor XI levels, revealed that the two genetic disorders segregated independently. One of 12 additional unrelated patients with Gaucher's disease showed a diminished Factor XI level and two of seven unrelated Factor XI-deficient patients showed decreased glucocerebrosidase activity. It is possible that the common occurrence of both genetic disorders results from a high gene frequency of both defects in Ashkenazic Jews.

Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom.

Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).

Genetic heterogeneity in Gaucher disease.

Considerable clinical variability occurs in adult Gaucher disease type I and three main subtypes may be delineated: a very mild form, a severe form, and a moderate form which itself presents various clinical manifestations. A study based on 25 families from our clinic and a review of published reports showed that when both parents were heterozygous and more than one child was affected with Gaucher disease type I, there was always intrafamilial similarity concerning the three subtypes. In families where one parent and at least one child were affected, variability in the clinical subtype of Gaucher disease type I might occur among the affected members of the family. We propose that the three different clinical subtypes of this disease reflect the genetic heterogeneity of two alleles, G1a and G1b and the three corresponding genotypes represent the three different subtypes of the disease.

Biochemical, genetic and ultrastructural study of a family with the sea-blue histiocyte syndrome/chronic non-neuronopathic Niemann-Pick disease.

Deficient leucocyte sphingomyelinase activity has been demonstrated in a patient with the sea-blue histiocyte syndrome. Family studies revealed that two other cases previously diagnosed on clinical and histochemical criteria also had a pronounced diminution of sphingomyelinase activity. Both parents of the affected individuals were carriers of the disease as indicated by sphingomyelinase activity intermediate between normal and diseased subjects. Additional heteroxygous carriers were found among the siblings and other relatives of the patients. This family study supports further the hypothesis that the sea-blue histiocyte syndrome and chronic Niemann-Pick (Type B) disease are the same.