Guy T.N. Besley

Sphingomyelinase defect in niemann-pick disease, type C, fibroblasts

Niemann-Pick disease is inherited in at least five different phenotypes [l] all of which exhibit degrees of sphingomyelin accumulation, particularly in the reticuloendothelial system. The diseases differ with respect to age of onset, degree of neurological involvement and clinical course of the disorder. In NiemannPick diseases types A and B, where the lipidosis is most severe, sphingomyelinase activity is profoundly deficient. However, in types C, D and E disease, sphingomyelinase activity is normal [ 1,2] or only partially reduced [2,3]; the biochemical basis for these conditions has yet to be firmly established. Recently, Callahan et al. [4] have indicated that a specific sphingomyelinase component, identifiable by isoelectric focusing, is deficient in Niemann-Pick type C (NPC) liver and brain [ 51 and in Niemann-Pick type E fibroblasts [6]. These authors did not study NPC fibroblasts but suggested that these might also be deficient in this sphingomyelinase component. Recent work [7] has indicated that fibroblast sphingomyelinase probably exists in a bound state but that the various enzyme components may be separated by isoelectric focusing in the presence of Triton X-l 00. This technique has been applied to the separation of sphingomyelinases in NPC fibroblasts in order to identify any missing component. The resulting enzyme profile indicated that NPC cells were profoundly deficient in two cathodic forms of sphingomyelinase.

Recognition of abnormal lysosomal enzyme patterns in childhood leukaemia by isoelectric focusing, with special reference to some properties of abnormally expressed components.

Abstract Isoelectric focusing profiles of six lysosomal enzymes, β-hexosaminidase, α-fucosidase, α-mannosidase, β-glucuronidase, acid phosphatase and α-galactosidase, were studied in lymphoid cells derived from childhood leukaemic patients and control subjects. Altered β-hexosaminidase patterns were observed in nine out of eleven patients with common ALL, in one patient with null-cell ALL and two with acute undifferentiated leukaemia. These changes were not confined to β-hexosaminidase, for other enzymes including α-fucosidase, α-mannosidase, β-glucuronidase and acid phosphatase were commonly involved. Altered enzyme patterns returned to normal with remission. With the exception of acid phosphatase abnormally expressed enzymes appeared to represent more anodic forms of the parent enzymes. For β-hexosaminidase the leukaemic enzymes unlike the normal forms were found to be neuraminidase-sensitive whereas other properties of the enzymes were found to be similar to their native forms. It is suggested that studies on lysosomal enzymes may be valuable in the differentiation of sub-types of leukaemia.

Studies on sphingomyelinase and β-glucosidase activities in Niemann-Pick disease variants: Phosphodiesterase activities measured with natural and artificial substrates

Cultured fibroblasts were studied from 12 cases of Niemann-Pick disease group C. In 11, sphingomyelinase and glucocerebrosidase (and beta-glucosidase) activities were reduced to around 50% of those of controls. On isoelectric focusing, all 12 strains lacked sphingomyelinase activity in the major cathodic region (pI 8.0). The defect was also demonstrated with the artificial phosphodiester substrates bis(4-methylumbelliferyl) phosphate and 4-methylumbelliferyl pyrophosphate diester. In control fibroblasts and those heterozygous for types A or B or group C Niemann-Pick disease, the major sphingomyelinase peak electrofocused at pI 8.0. No direct interaction could be demonstrated by mixing experiments between group C Niemann-Pick extracts and those of type A disease or Gaucher disease. Profiles for beta-glucosidase activity appeared normal in Niemann-Pick group C fibroblasts. No reduction of sphingomyelinase or glucocerebrosidase activities was found in Niemann-Pick group C liver, nor any attenuation of cathodic sphingomyelinase activity in the affected tissue. Results suggest that sphingomyelinase expression differs in fibroblasts and liver. Enzyme defects associated with Niemann-Pick disease group C were only observed in cultured cells.

Cholesterol ester storage disease in an adult presenting with sea-blue histiocytosis

An adult patient is described with hepatomegaly and sea‐blue histiocytes in the bone marrow. A diagnosis of cholesterol ester storage disease was established following enzyme and lipid analyses on liver biopsy and cultured skin fibroblasts. Acid esterase activity was deficient (approx. 5% of controls) in liver and fibroblasts using [14C]‐triolein or 4‐methylumbelliferyl palmitate as substrates. Cholesterol ester levels were raised about 70‐fold in liver, whereas triglyceride levels were only marginally raised. Marked accumulation of cholesterol esters was also demonstrated in cultured fibroblasts. Clinically, the patient responded favourably to phenobarbitone treatment. However, this was not reflected in liver acid esterase or lipid levels.

Diagnosis of niemann-pick disease using a simple and sensitive fluorimetric assay of sphingomyelinase activity

Phosphodiesterase activity of cultured cells was determined with bis-(4-methylumbelliferyl) phosphate as substrate. In the presence of Triton X-100 an acid component was evident and results indicated that this enzyme was identical with sphingomyelinase. Acid phosphodiesterase activity was specifically inhibited by sphingomyelin. In fibroblasts from patients with Niemann-Pick diseases types A, B and C, acid phosphodiesterase activity was deficient whereas neutral activity was normal. Neutral activity could, however, be removed by acid precipitation or by binding to DEAE-cellulose. Hence a simple and sensitive fluorimetric method is described for the assay of sphingomyelinase activity in the diagnosis of Niemann-Pick disease.

Spectrophotometric and fluorimetric assays of galactocerebrosidase activity, their use in the diagnosis of Krabbe's disease

Derivatives of galactocerebroside were prepared containing coloured (w-2,4,6-trinitrophenylaminolauric acid) or fluorescent (11-(9-anthroyloxy) undecanoic acid) fatty acid moieties.. These cerebrosides were used as substrates for galactocerebrosidase activity. By overcoming problems associated with the radioactively labelled substrates normally used, yet retaining good enzyme-substrate specificity, these derivatives provided useful and reliable alternative substrates for galactocerebrosidase activity. Enzyme activities in whole extracts of brain, liver, fibroblasts and cultured amniotic fluid cells were compared, using as substrates the novel cerebrosides as well as [3H]galactocerebroside. Good correlation of activities was obtained. In extracts derived from patients with Krabbes disease marked deficiency of galactocerebrosidase activity was observed with each substrate, whereas extracts from heterozygous carriers exhibited a partial reduction in enzyme activity. The results show that these coloured and fluorescent galactocerebrosides may be used with confidence in the diagnosis and carrier detection of Krabbes disease.

Effect of Triton X-100 on the isoelectric focusing profile of fibroblast sphingomyelinase

Sphingomyelinase is the only mammalian enzyme known to hydrolyse sphingomyelin. Hence, the inherited deficiency of this enzyme in Niemann-Pick disease results in the characteristic sphingomyelin lipidosis. The disease is, however, expressed in at least five different phenotypes [l] of which only types A and B lack sphingomyelinase activity. In types C, D and E where the lipidosis is less severe,‘ as also is the course of the disease, normal enzyme activities have been measured. Recently, however, Callahan et al. [2-41 have demonstrated multiple forms of sphingomyelinase following isoelectric focusing of extracts of brain, liver and cultured skin fibroblasts. Of particular interest was their finding that a specific sphingomyelinase component was absent in Niemann-Pick diseases types C and E. In view of the importance of this finding, the present work is reported in order to extend these observations on the behaviour of fibroblast sphingomyelinase during isoelectric focusing. The results suggest that in the original study the enzyme was probably in a bound state but when solubilized with Triton X-l 00, it moves to a much higher p1

Use of a chromogenic substrate for the diagnosis of Krabbe's disease, with special reference to its application in prenatal diagnosis

A chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, has recently been described for the diagnosis of Krabbes disease. Hydrolysis of this substrate by extracts of cultured cells and tissues was compared with the activities of lactocerebrosidase I and non-specific beta-galactosidase. Under appropriate conditions, hydrolysis of the chromogenic analogue was markedly reduced in extracts of cultured amniotic fluid cells and skin fibroblasts derived from cases of Krabbes disease. Activity was also markedly deficient in extracts of Krabbes brain, although only a partial reduction was measured in liver extracts. Generally activities were higher in tissues of fetal origion. Unfortunately, the new analogue proved less specific and less sensitive than the natural substrates used to diagnose Krabbes disease. Consequently, the analogue does not provide a satisfactory alternative substrate for the prenatal diagnosis of Krabbes disease.

Heterogeneity for mutations in medium chain acyl-CoA dehydrogenase deficiency in the UK population.

MCAD is the commonest inherited disorder of fatty acid oxidation. We have sought for and studied 21 affected children from 18 families within the UK. In 14 families the children are homozygous for the G985 mutation. In three families the children are compound heterozygotes for G985 and thus carry another and unknown mutation. In one family the child does not carry the G985 mutation on either allele. The carrier incidence of the G985 mutation is 1 in 68, which suggests that the natural history of MCAD deficiency deserves further study.

Studies on pyrophosphate diesterase activity in cultured human fibroblasts: A deficiency in Niemann-Pick disease

Fibroblast phosphodiesterase activity was studied using 4-methylumbelliferyl pyrophosphate diester as substrate. Release of the fluorogen, 4-methylumbelliferone, was found to be dependent on acid phosphatase activity, normally present in excess in crude cell extracts. Phosphodiesterase activity had an acid pH optimum, was deficient in Niemann-Pick disease fibroblasts, and, when assayed in the presence of exogenous acid phosphatase, had an identical electrofocusing profile to that of sphingomyelinase. These findings suggest that 4-methylumbelliferyl pyrophosphate diesterase and acid sphingomyelinase activities are dependent on the same enzyme.