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PRENATAL DIAGNOSIS OF NIEMANN-PICK DISEASE TYPE A USING CHROMOGENIC SUBSTRATE
144 PRENATAL DIAGNOSIS OF NIEMANN-PICK DISEASE TYPE A USING CHROMOGENIC SUBSTRATE
SIR,-Niemann-Pick disease can be diagnosed reliably in laboratory assay of sphingomyelinase activity in separated
the
or cultured skin fibroblasts.2 But these tests use radioactive sphingomyelin as substrate, and this has limited their easy application. Following the suggestion of Brady et al.,3 the chromogenic sphingomyelin analogue 2-hexadecanoylamino-4-nitrophenylphosphorylcholine (H.N.P.) was shown to be a convenient alternative substrate for the diagnosis of Niemann-Pick disease type A and the demonstration of the heterozygosity of their parents.4 H.N.P. would be expected to be equally reliable for prenatal diagnosis, and this has been confirmed by the detection of a Niemann-Pick heterozygote in utero.4 We have used H.N.P. to detect an affected fetus in a pregnancy of non-consanguineous Dutch parents; their first child had Niemann-Pick disease type A (Dr H. D. Bakker, Emma Children’s Hospital, Amsterdam). Amniocentesis was done in the 16th week of the second pregnancy (Dr M. G. J. Jahoda, University Hospital, Rotterdam). Amniotic-fluid cells were harvested for enzymic assay after 3½weeks of culture. The amniotic-fluid cells and fibroblasts from the index case and both parents were homogenised and assayed for hydrolytic activity with H.N.P. or sphingomyelin substrate. The results are given in the table. Cultured amniotic-fluid cells from the pregnancy at risk for Niemann-Pick disease hydrolysed H.N.P. to only 4.5% of the mean control value, similar to the deficiency found for cul-
leucocytes’
1. 2.
Kampine, J. P., Brady, R. O. Kanfer, J. N. Science, 1967, 155, 86. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. O., Fredrickson, D. S. Biochem. Biophys. Res. Commun. 1969, 34, 582. 3. Brady, R. O., Johnson, W. G., Uhlendorf, B. W. Am. J. Med. 1971, 51, 423. 4. Gal, A. E., Brady, R. O., Hibbert, S. R., Pentchev, P. G. New Engl. J. Med. 1975, 293, 632. 5. Barnholz, Y., Roitman, A., Gatt, S. J. biol. Chem. 1966, 241, 3731. HYDROLYSIS OF H.N.P. AND SPHINGOMYELIN BY CULTURED
AMNIOTIC-FLUID CELLS AND SKIN FIBROBLASTS
hydrolysis was measured4 in a mixture containing: 0.75 µmol (Koch-Light), 12.5 µmol sodium acetate pH 5.6, and 10-25 µg enzyme protein (supernatant cell extract in 0.5% sodium cholate) in a final volume of 50 µl. After incubation at 37°C for 3 h, 200 pi of 0.1 mol/1 glycine/sodium-hydroxide pH 10-4 and 350 µl of ethanol H.N.P.
H.N.P.
added and mixed. The absorbance of the clear supernatant obtained by centrifugation was measured at 410 nm. Sphingomyelin hydrolysis was measured in a mixture containing: 0.1mg 3H-sphingomyelin (10 counts/min); 0.1 mg ’Triton X-100’; 20 µmol sodium acetate, pH 5 - 2; and 100-150 µg enzyme protein (whole cell homogenate in water) in a final volume of 200 µl After incubation at 37°C for 1 h the liberated 3H-ceramide was separated’ and estimated by liquid scintillation counting. were
tured skin fibroblasts of the index case and further cases of Niemann-Pick disease type A and type B. These results, indicating an affected homozygous fetus, were substantiated by detection of sphingomyelinase deficiency. The activity of skin fibroblasts from the parents of the index case could not be differentiated from normal control values either by sphingomyelin or-H.N.P. hydrolysis. The activity of &bgr;-galactosidase, assayed for control purposes, was normal in all cell cultures examined. The parents requested an abortion which was done by prostaglandin induction in the 20th week of pregnancy (Dr Jahoda). Enzymic analysis of the cultured cells confirmed the prediction of fetal abnormality (table). The results indicate that the use of the chromogenic substrate H.N.P. offers a sensitive method for rapid prenatal diagnosis of Niemann-Pick disease, whenever the diagnosis in the index case has been made using the same substrate. of Chemical Institute of Child Health, London WC1N 1EH
Department
Pathology,
Department of Clinical Genetics, Erasmus University, Rotterdam
A. D. PATRICK ELISABETH YOUNG W. J. KLEIJER M. F. NIERMEIJER
HYPOCHLORITE STERILISING FLUID AS SOURCE OF DIETARY SODIUM IN GAVAGE-FED INFANTS
SIR,-In view of the interest in the sodium content of infant feeds, I wish to report that inadvertent addition of sodium to milk can occur, under certain circumstances, with the hypochlorite method of sterilising infant-feeding utensils. The sterilising agent ’Milton’ contains 1% sodium hypochlorite, 16-5% sodium chloride, and stabilising ions, producing a total sodium concentration of 3160 mmol/1 (our own estimates on four samples gave an average of 3380 mmol/l). When milton is diluted correctly (1/80) the sodium concentration should be around 40 mmol/l, excluding the additional sodium from tap water; expressed breast milk contains, on average, 6.4 mmol/l.1 In this hospital’s special-care baby unit premature infants are fed on pooled human milk which is measured out and drawn up, before each feed, with utensils (beaker, syringe, plastic quill) that have been soaked in milton. Although the utensils were drained, the remaining traces of milton formed a significant proportion of the small volume of feed given to premature infants. The makers of milton claim, on the basis of long-term animal and human studies,2-4 that traces of milton (e.g., 1-2 ml) left on feeding bottles used for larger infants are non-toxic and need not be removed. Low-birth-weight babies (1-2.5 kg range) were fed, 1-3 hourly, with an average feed volume of 11 ml (range 5-38 ml). In a series of nine paired estimations, the sodium concentration of the feed rose by 50%, from an average of 8.1 mmol/l before contact with milton to 12.1 mmol/1 afterwards (t test, p <0.05). The calculated average volume of milton added to each feed was 0.55 ml. The proportional sodium increase would have been less with milk formulae (with their higher sodium content), but in these babies a third of the sodium in their diet came from milton. Part of this increase in sodium in the feed was found to be due to inaccurate dilution of the stock milton solution. Eleven random samples of diluted milton made up by experienced nurses on the special-care baby unit showed an average sodium content of 79 mmol/l, which is much more than the recommended concentration. Overzealous addition of the stock milton solution probably happens because of this agent’s repu1. Department of Health and Social Security. The Composition of Human Milk. H. M. Stationery Office, 1977. 2. Dost, F. H., and others in Infant Hygiene (edited by H. Berger and R. S. Illingworth); p. 130. Stuttgart, 1971. 3. Miller, D. S., Yudkin, J. ibid. p. 135. 4. Satgé, P., Colin, J., Nobre, R. Unpublished (courtesy of Richardson Merell).
SIR,-Niemann-Pick disease can be diagnosed reliably in laboratory assay of sphingomyelinase activity in separated
the
or cultured skin fibroblasts.2 But these tests use radioactive sphingomyelin as substrate, and this has limited their easy application. Following the suggestion of Brady et al.,3 the chromogenic sphingomyelin analogue 2-hexadecanoylamino-4-nitrophenylphosphorylcholine (H.N.P.) was shown to be a convenient alternative substrate for the diagnosis of Niemann-Pick disease type A and the demonstration of the heterozygosity of their parents.4 H.N.P. would be expected to be equally reliable for prenatal diagnosis, and this has been confirmed by the detection of a Niemann-Pick heterozygote in utero.4 We have used H.N.P. to detect an affected fetus in a pregnancy of non-consanguineous Dutch parents; their first child had Niemann-Pick disease type A (Dr H. D. Bakker, Emma Children’s Hospital, Amsterdam). Amniocentesis was done in the 16th week of the second pregnancy (Dr M. G. J. Jahoda, University Hospital, Rotterdam). Amniotic-fluid cells were harvested for enzymic assay after 3½weeks of culture. The amniotic-fluid cells and fibroblasts from the index case and both parents were homogenised and assayed for hydrolytic activity with H.N.P. or sphingomyelin substrate. The results are given in the table. Cultured amniotic-fluid cells from the pregnancy at risk for Niemann-Pick disease hydrolysed H.N.P. to only 4.5% of the mean control value, similar to the deficiency found for cul-
leucocytes’
1. 2.
Kampine, J. P., Brady, R. O. Kanfer, J. N. Science, 1967, 155, 86. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. O., Fredrickson, D. S. Biochem. Biophys. Res. Commun. 1969, 34, 582. 3. Brady, R. O., Johnson, W. G., Uhlendorf, B. W. Am. J. Med. 1971, 51, 423. 4. Gal, A. E., Brady, R. O., Hibbert, S. R., Pentchev, P. G. New Engl. J. Med. 1975, 293, 632. 5. Barnholz, Y., Roitman, A., Gatt, S. J. biol. Chem. 1966, 241, 3731. HYDROLYSIS OF H.N.P. AND SPHINGOMYELIN BY CULTURED
AMNIOTIC-FLUID CELLS AND SKIN FIBROBLASTS
hydrolysis was measured4 in a mixture containing: 0.75 µmol (Koch-Light), 12.5 µmol sodium acetate pH 5.6, and 10-25 µg enzyme protein (supernatant cell extract in 0.5% sodium cholate) in a final volume of 50 µl. After incubation at 37°C for 3 h, 200 pi of 0.1 mol/1 glycine/sodium-hydroxide pH 10-4 and 350 µl of ethanol H.N.P.
H.N.P.
added and mixed. The absorbance of the clear supernatant obtained by centrifugation was measured at 410 nm. Sphingomyelin hydrolysis was measured in a mixture containing: 0.1mg 3H-sphingomyelin (10 counts/min); 0.1 mg ’Triton X-100’; 20 µmol sodium acetate, pH 5 - 2; and 100-150 µg enzyme protein (whole cell homogenate in water) in a final volume of 200 µl After incubation at 37°C for 1 h the liberated 3H-ceramide was separated’ and estimated by liquid scintillation counting. were
tured skin fibroblasts of the index case and further cases of Niemann-Pick disease type A and type B. These results, indicating an affected homozygous fetus, were substantiated by detection of sphingomyelinase deficiency. The activity of skin fibroblasts from the parents of the index case could not be differentiated from normal control values either by sphingomyelin or-H.N.P. hydrolysis. The activity of &bgr;-galactosidase, assayed for control purposes, was normal in all cell cultures examined. The parents requested an abortion which was done by prostaglandin induction in the 20th week of pregnancy (Dr Jahoda). Enzymic analysis of the cultured cells confirmed the prediction of fetal abnormality (table). The results indicate that the use of the chromogenic substrate H.N.P. offers a sensitive method for rapid prenatal diagnosis of Niemann-Pick disease, whenever the diagnosis in the index case has been made using the same substrate. of Chemical Institute of Child Health, London WC1N 1EH
Department
Pathology,
Department of Clinical Genetics, Erasmus University, Rotterdam
A. D. PATRICK ELISABETH YOUNG W. J. KLEIJER M. F. NIERMEIJER
HYPOCHLORITE STERILISING FLUID AS SOURCE OF DIETARY SODIUM IN GAVAGE-FED INFANTS
SIR,-In view of the interest in the sodium content of infant feeds, I wish to report that inadvertent addition of sodium to milk can occur, under certain circumstances, with the hypochlorite method of sterilising infant-feeding utensils. The sterilising agent ’Milton’ contains 1% sodium hypochlorite, 16-5% sodium chloride, and stabilising ions, producing a total sodium concentration of 3160 mmol/1 (our own estimates on four samples gave an average of 3380 mmol/l). When milton is diluted correctly (1/80) the sodium concentration should be around 40 mmol/l, excluding the additional sodium from tap water; expressed breast milk contains, on average, 6.4 mmol/l.1 In this hospital’s special-care baby unit premature infants are fed on pooled human milk which is measured out and drawn up, before each feed, with utensils (beaker, syringe, plastic quill) that have been soaked in milton. Although the utensils were drained, the remaining traces of milton formed a significant proportion of the small volume of feed given to premature infants. The makers of milton claim, on the basis of long-term animal and human studies,2-4 that traces of milton (e.g., 1-2 ml) left on feeding bottles used for larger infants are non-toxic and need not be removed. Low-birth-weight babies (1-2.5 kg range) were fed, 1-3 hourly, with an average feed volume of 11 ml (range 5-38 ml). In a series of nine paired estimations, the sodium concentration of the feed rose by 50%, from an average of 8.1 mmol/l before contact with milton to 12.1 mmol/1 afterwards (t test, p <0.05). The calculated average volume of milton added to each feed was 0.55 ml. The proportional sodium increase would have been less with milk formulae (with their higher sodium content), but in these babies a third of the sodium in their diet came from milton. Part of this increase in sodium in the feed was found to be due to inaccurate dilution of the stock milton solution. Eleven random samples of diluted milton made up by experienced nurses on the special-care baby unit showed an average sodium content of 79 mmol/l, which is much more than the recommended concentration. Overzealous addition of the stock milton solution probably happens because of this agent’s repu1. Department of Health and Social Security. The Composition of Human Milk. H. M. Stationery Office, 1977. 2. Dost, F. H., and others in Infant Hygiene (edited by H. Berger and R. S. Illingworth); p. 130. Stuttgart, 1971. 3. Miller, D. S., Yudkin, J. ibid. p. 135. 4. Satgé, P., Colin, J., Nobre, R. Unpublished (courtesy of Richardson Merell).