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Cerebroside Synthesis in Gaucher's Disease

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Cerebroside Synthesis in Gaucher's Disease CEREBROSIDE SYNTHESIS IN GAUCHER'S DISEASE Eberhard G. Trams, Roscoe O. Brady J Clin Invest. 1960;39(10):1546-1550. https://doi.org/10.1172/JCI104175. Research Article Find the latest version: https://jci.me/104175/pdf CEREBROSIDE SYNTHESIS IN GAUCHER'S DISEASE* By EBERHARD G. TRAMS AND ROSCOE 0. BRADY (From the National Institute of Neurological Diseases and Blindness, Bethesda, Md.) (Submitted for publication April 12, 1960; accepted May 6, 1960) Familial lipodystrophic conditions, such as nectomized to arrest the pancytopenia of hyper- Gaucher's, Niemann-Pick, and Tay-Sachs dis- splenism. It was not possible to obtain control ease, are characterized by the intracellular ac- specimens of splenic tissue suitable for metabolic cumulation of abnormally large quantities of studies from normal children of the same age as sphingolipids. In Gaucher's disease, the offend- the patients with Gaucher's disease. Therefore, ing lipids are cerebrosides, while Niemann-Pick the results obtained in spleens from Gaucher's and Tay-Sachs diseases are characterized by the patients were compared with those obtained in accumulation of sphingomyelin and gangliosides, two children with Niemann-Pick disease and in respectively. In Gaucher's and Niemann-Pick one adult case of idiopathic thrombocytopenic disease, involvement of the reticuloendothelial sys- purpura. tem is extensive, and splenomegaly and hepato- Experiments were designed to explore several megaly are frequently observed. Lieb in 1924 factors that might be thought to contribute to the ( 1 ) identified the lipid stored in reticuloendothelial pathogenesis of Gaucher's disease. Enzymic dis- cells in a case of Gaucher's disease as a cerebro- turbances conceivably could lead to an alteration side. His findings were supported by other in- of normal processes, causing a replacement of a vestigators (2, 3), and eventually Halliday, Deuel, large proportion of cerebroside galactose by glu- Tragerman and Ward (4) and Rosenberg and cose. The metabolism of such an "abnormal" Chargaff (5) presented evidence that the ac- cerebroside might be imagined to proceed at a cumulated cerebrosides in Gaucher's disease con- slower rate than the characteristic galacto-cere- tained predominantly glucose instead of the high broside. Also, attention was directed to the rate content of galactose usually present in cerebrosides. of formation of the total cerebroside molecule com- It was felt that techniques developed in this pared with the rate observed in tissue samples ob- laboratory for studies of the biosynthesis of tained from patients who did not show signs of sphingosine and cerebrosides might be useful for cerebroside accumulation. investigations of cerebroside formation in tissue samples obtained from patients with Gaucher's EXPERIMENTAL METHODS disease. The data obtained by Brady and Koval The spleens used in this study were refrigerated fol- (6), Burton, Sodd and Brady (7), and Cleland lowing splenectomy and processed within 15 to 30 min- and Kennedy (8) indicate that cerebroside syn- utes. Slices measuring 10 X 10 X 0.5 mm were pre- thesis probably occurs in the following fashion: pared in the cold and incubated aerobically in Krebs- Ringer bicarbonate solution (9), pH 7.5, for 3 hours at palmitic aldehyde + serine -- sphingosine + C02; 370 C. The following substrates were added to the in- sphingosine + uridinediphosphate galactose -> cubation mixtures in amounts ranging from 0.5 to 5.0 psychosine + uridinediphosphate; psychosine + ,umoles per flask: uniformly-labeled glucose-C"4 (0.89 ,uc fatty acid -* cerebroside. Since purified enzyme per ,umole); galactose-1-C"' (0.44 ,c per ,umole) ; and systems which catalyze the biosynthesis of cere- sodium acetate-i-C4 (5.8 ,uc per ,umole). Each sub- strate was incubated with the tissue slices in triplicate. brosides were not available, tissue slices were em- Non-incubated samples also in triplicate were used as ployed in the present investigations to study the controls for determining the reliability of the isolation formation of these sphingolipids. Tissue was oc- and recovery techniques. casionally available from patients who were sple- After incubation, the cerebrosides were isolated es- sentially according to the method described by Radin, * The authors express their gratitude to Dr. Allen C. Brown and Lavin (10). The contents of the incubation Crocker of the Children's Hospital Medical Center, flasks were homogenized with an equal volume of 10 Boston, Mass., for his interest and help in providing per cent ice-cold trichloroacetic acid and centrifuged. much of the clinical material used in this study. The precipitated material was washed 3 times in the 1546 CEREBROSIDE SYNTHESIS IN GAUCHER'S DISEASE 1547 cold with 5 per cent trichloroacetic acid and once with RESULTS acetone at - 100 C. The residue was then extracted 3 times with chloroform: methanol (2:1) at 450 to 500 C. Accurate measurement of the specific activity The chloroform-methanol extract was washed 5 times of the cerebrosides obtained from the incubation with 2 vol of water, and sufficient methanol was added mixtures required that the cerebrosides be ob- to the residual chloroform phase to obtain a clear solu- tained in a reasonably pure state. This restriction tion. This solution was decolorized with activated char- coal and chromatographed on Florisil and on a mixed- entailed considerable losses in the purification bed ion exchange column as described by Radin and co- procedures which had to be determined. For this workers (10). reason the isotope dilution technique was used to The cerebrosides obtained in this manner were re- estimate total cerebroside content of samples of crystallized 3 times from methanol. Radioactivity was splenic tissue. Comparison of the data obtained measured in a proportional gas-flow counter. The con- centration of cerebrosides was determined by the an- by the isotope dilution method and colorimetric throne reaction for hexoses after hydrolyzing the sam- methods indicated that the latter measured from 31 ples for 3 hours (10). The relative amounts of glucose to 58 per cent of the actual cerebroside content. and galactose in the hexose fractions were determined by Data of the levels obtained with the isotope dilu- incubating the cerebroside hexoses with glucose oxidase. tion technique and the amount of glucose in the After chromatography on an MB-3 (Rohm and Haas cerebroside hexoses are shown in Table I. The Co.) ion exchange column the galactose was determined results indicate that considerable amounts of galac- and the amount of glucose was then calculated by the difference. Known amounts of glucose and galactose tose were present in the cerebrosides of Gaucher's were carried as internal standards throughout the whole spleens as well as in the control spleens. Be- isolation procedure and the subsequent determination of cause the entire procedure was controlled with galactose to ascertain the accuracy of these methods. added standard amounts of glucose and galactose, The total cerebroside content was estimated by the these data are thought to be relatively reliable. isotope dilution technique. A known amount of highly When the data of the incorporation of the iso- purified radioactive cerebroside obtained by tritiating, topically-labeled precursors were compared, it be- according to the method described by Wilzbach (11), came apparent that the uptake of each isotope by was to added tissue homogenates, and the cerebrosides the tissue samples was essentially the same for all were isolated by the procedures outlined above. Com- of the cases investigated in this study. The net parison of the specific activity of the recovered cerebro- incorporation of isotopes from the different pre- side with that of the added cerebroside, as well as per cursors is shown in Table II. The data presented cent recovery of the added isotope, permitted an accu- rate measurement of tissue cerebroside levels. The in the last column are expressed as specific activi- amounts of free glucose and galactose in tissue samples ties of the recovered cerebrosides. These figures were estimated in trichloroacetic acid extracts of tis- do not indicate that splenic tissue from patients sues by similar procedures with the use of labeled with Gaucher's disease synthesized cerebrosides hexoses. at a slower rate than did the control spleens. TABLE I Cerebroside contents of pathological states Cerebroside Glucose in Spleen Total content of cerebroside Disease Patient* Age Sex weight lipidst spleen hexoses yrs mos g % wd wt. % wet wt. % Gaucher's K.S.' 6 2 720 6.05 2.45 53 Gaucher's S.R.' 4 1 9 304 4.8 1.20 41 Gaucher's C. R.' 5 8 9 302 4.8 2.40 44 Gaucher's J.A., 8 10 1,300 6.40 3.54t 32 Niemann-Pick J.K.' 3 4 ci 258 12.9 1.05t 39 Niemann-Pick C.C.2 5 6 9 235 7.3 0.41 50 Idiopathic thrombo- cytopenic purpura M.R.3 69 9 73 0.18 * Superscripts indicate source of patient: 'Children's Hospital Medical Center, Boston, Mass. ; 2 Walter Reed Army Hospital, Washington, D. C.; 3 The George Washington University Hospital, Washington, D. C. t These data courtesy Dr. Allen C. Crocker, Children's Hospital Medical Center, Boston, Mass. t No analysis performed. 1548 EBERHARD G. TRAMS AND ROSCOE 0. BRADY TABLE II cordingly, the data obtained with labeled hexoses Incorporation of isotopic precursors as precursors were compared with those found into cerebrosides with labeled acetate. If the ceramide portion of Incorporation the cerebroside molecule were synthesized during Disease Patient Precursor into cerebrosides the incubation, rather than merely participating in cpm/g m/4molesl dry wt. jmole an exchange of labeled hexose, one would expect Gaucher's K.S. Glucose-C'4 19,840 0.134 a significant incorporation of labeled acetate into Galactose-C'4 17,720 0.680 Gaucher's S.R. Glucose-C'4 4,210 0.051 the sphingosine and fatty acid moieties. This in- 6.960 0.163 Galactose-C'4 deed was found to be the case as was indicated by Gaucher's C.R.
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