A.C. Pollard

A rapid method for the estimation of β-galacto-cerebrosidase, β-glucocerebrosidase and sphingomyelinase activities in leukocytes

Abstract A method for the assay of leukocyte (β-galactocerebrosidase, β-glucocere-brosidase and sphingomyelinase activities has been developed, based on the separation of the tritiated sphingolipid substrates from their corresponding radioactive hydrophobic product (ceramide) by thin-layer chromatography on Silica gel H coated microscope slides. For the determination of β-galactocerebrosidase and β-glucocerebrosidase activities the silica gel is impregnated with sodium tetraborate. Each chromatogram is easily divided into two distinct zones and the radioactivity content of each is determined by liquid scintillation counting. The technique described, is rapid, less costly than conventional methods and provides an accurate assessment of sphingolipid hydrolase activity. It is suggested that it should be of considerable value in those areas which require the rapid analysis of large numbers of samples, such as in screening for the sphingo-lipidoses or for enzyme purification studies.

A rapid and sensitive gas-liquid chromatographic procedure for the micro determination of sodium valproate (sodium di-N-propylacetate) in plasma or serum.

A gas-liquid chromatographic procedure for the micro determination of sodium valproate is described. Valproic acid is extracted from acidified plasma or serum into n-heptane containing an internal standard (octanoic acid) and after phase separation an aliquot is analysed by gas-liquid chromatography. The procedure is rapid, reliable, sensitive and specific. It requires 25--50 microliter sample for a single estimation, has a detection threshold of less than 10 mumol/l, and is suitable for routine clinical use.

Can Head Injury Influence the Site of Demyelination in Adrenoleuko dystrophy

Adrenoleukodystrophy is an inherited disorder, in which there is degeneration of white matter of the central nervous system. The disorder presents classically in males during the first decade, and although early clinical features may be asymmetrical, radiological and pathological features are essentially generalised. Two boys are reported with proven adrenoleukodystrophy, who at first had focal clinical and radiological features, with the foci in the region of local trauma. This raises the possibility that a local insult may have precipitated or hastened the degenerative process.

A rapid ultraviolet spectrophotometric procedure for the microdetermination of theophylline (1,3-dimethylxanthine) in plasma or serum.

An ultraviolet spectrophotometric procedure for the microdetermination of theophylline in plasma is described. Theophylline is extracted at pH 5 with chloroform. An aliquot of the solvent is shaken with molar hydrochloric acid and the absorbance of the acid layer determined at 267 nm. The resulting procedure is rapid, reliable, sensitive and virtually specific. It requires 250 microliters sample for a single estimation and has a detection threshold of less than 5 mumol/l.

A rapid and sensitive spectrophotometric procedure for the micro determination of carbamazepine in blood

An ultraviolet spectrophotometric procedure for the micro determination of carbamazepine in blood is described which is based on the original 9-methyl-acridine method proposed by Beyer, K.H. and Klinge, K. (1969) (Arzneim-Forsch. 19, 1759--1760). Carbamazepine is extracted from blood with dichloromethane, which is then washed with alkali and acid. An aliquot of the extractant is evaporated to dryness and the residue heated briefly with hydrochloric acid at 150 degrees C. Following removal of non-specific interference with n-heptane, the absorbance of the acid catalysed rearrangement product (9-methylacridine) is determined at 258 nm. The resulting procedure is rapid, reliable, sensitive and specific. It requires 100-200 mul sample for a single estimation and has a detection threshold of less than 0.1 mg/100 ml. It is concluded that the method is suitable for routine clinical use.

A rapid spectrophotometric procedure for the simultaneous micro determination of carbamazepine and 5, 5-diphenyl-hydantoin in blood.

Carbamazepine and 5,5-diphenylhydantoin are simultaneously extracted from 100-200 mul blood with 1,2-dichloroethane. 5,5-Diphenylhydantoin is removed by a one-step wash into alkali. The dichloroethane is further washed with acid and then evaporated to dryness. 5,5-Diphenylhydantoin is determined in the alkali washing by a benzophenone procedure; carbamazepine is determined in the dried residue by the 9-methylacridine procedure earlier described. The combined method is rapid, reliable and has a detection threshold of less than 0.1 mg/100 ml for each drug.

A micro-radiochemical assay for α-1,4-glucosidase and its use in the assessment of type II glycogenosis (pompe's disease)

An assay for alpha-1,4-glucosidase (acid maltase) activity which is deficient in Pompes disease is described. The assay can be used to measure the enzyme in cultured skin fibroblasts, cultured amniotic cells and peripheral blood leucocytes. [U-14 C]Maltose is used as the substrate in a total assay volume of 8 microliter. The product, [U-14C]glucose, is separated from the substrate by cellulose thin-layer chromatography. The procedure permits replicate assays from 400 microliter whole blood and from amniotic cells in primary culture. Discrimination of the heterozygous Pompe state appears to be facilitated.

The magnesium content of duodenal mucosa in man.

The magnesium content of duodenal mucosa was estimated in biopsy specimens from 50 patients, 43 of which had normal mucosal histopathology. There was no significant difference in the mean mucosal magnesium content of those speciments with normal and abnormal histopathology and overall the mean value was 30.9 mmol/kg dry weight of tissue, but there was a wide spread of values. The concentration of magnesium in plasma and duodenal fluid was also determined. There was no significant correlation between duodenal mucosal magnesium content and plasma magnesium concentration. However, there was a weak but statistically significant correlation between magnesium content in duodenal fluid and duodenal mucosa (r = 0.54, p less than 0.001).

Prenatal diagnosis of lysosomal storage diseases: A review of 23 cases

During the previous 2 yr, 23 pregnancies were monitored for the presence of 9 different lysosomal storage diseases. There were 3 possible cases of GM1 gangliosidosis, 4 of GM2 gangliosidosis Type I and one of Type II, 4 of glucosylceramidosis, 3 of sulphatide lipidosis, one of galactosylceramidosis, 2 of glycogen storage disease Type II, 2 of mucolipidosis Type II and 3 of nephropathic cystinosis. Twelve foetuses were predicted to be homozygous affected and these pregnancies were terminated. Analysis of tissues (cultured fibroblasts, brain, liver, spleen) from 11 of these foetuses confirmed the prenatal diagnosis: one foetus was lost to biochemical follow-up. Of the remaining 11 pregnancies, one terminated spontaneously (4 wk after amniocentesis). Five children have been born and considered to be unaffected by the disease for which they were at risk both by clinical and biochemical evaluation: 5 further children have not yet been tested. In 22 cases cultured amniotic cells were used for the diagnosis and sufficient cells were grown to perform the analysis before the 20th wk of gestation. In one case of mucolipidosis Type II, cells failed to proliferate in culture and cell free amniotic fluid was used for assay. It is concluded that the techniques currently employed for the above tests are diagnostic. Even so, a multidirectional approach to each diagnosis, bringing to bear all available technology in our laboratory, is still used to ensure that diagnostic errors are not made.

Two cases of mucopolysaccharidosis

Case 1 An obese 6-yr-old boy was rejected from the Paeroa School for Intellectually Handicapped Children because of the development of severe mental deficiency. Facial fullness and wedging of the first lumbar vertebra prompted the forwarding of urine to the University of Auckland. The urinary mucopolysaccharide output was reported to be 15 times greater than normal, with an excess of dermatan sulphate over heparan sulphate. A fibroblast skin biopsy was sent to the Adelaide Children’s Hospital in 1977. This was reported as showing zero Iduronosyl sulphate sulphatase levels consistent with Hunter’s syndrome (Mucopolysaccharidosis type II). Currently, frozen leucocytes have been sent to Adelaide from female relatives for enzyme studies. Case 2 Beaking of the first and second lumbar vertebrae was noted in a Maori boy of 5 mth in 1974. He was being investigated at Waikato Hospital for persistent pulmonary infection. The urinary mucopolysaccharides were found to be largely heparan. In 1976 a fibroblast skin biopsy was studied at the Department of Community Health, Auckland, by cross correction studies. In 1978 a further skin biopsy was taken and forwarded to the Adelaide Children’s Hospital for enzyme studies. With heparan sulphamidase virtually absent the case has been defined as Sanfilippo type A (Mucopolysaccharidosis type IIIA).