Nils E. Nordén

Urinary mannose in mannosidosis

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Isoenzymes of four acid hydrolases in human kidney and urine

Abstract The occurrence of different isoenzymes of β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-mannosidase in human urine and kidney tissue was studied by isoelectric focusing. Artificial substrates were used for the enzymatic assays. There was a predominance of isoenzymes with a low isoelectric point in the urine. In the kidney tissue isoenzymes with higher isoelectric point predominated. This difference may be due to a higher proportion of N-acetylneuraminic acid-containing enzymes in the urine than in the kidney tissue.

Gel chromatographic distribution of urinary carbohydrate compounds

Abstract Normal human urine was fractionated on a column of Sephadex G-25. Eluted fractions were assayed for mannose, galactose, glucose, fucose, xylose, and ribose by gas-liquid chromatography (GLC) and ion exchange chromatography. Good agreement between the two methods was obtained in fractions eluted with a Kav less than that of disaccharides (0.60). Inositol was assayed by GLC only. The precision was about 10% and the recovery 80–120% in the GLC assay of the most important sugars. A procedure is suggested for sugar assay of urine specimens when mannosidosis is suspected.

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D-[U-14C]mannose or N-acetyl-D-[1-14C]-glucosamine). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver alpha-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electrophoresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-D-GlcNAc (I), alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-GlcNAc (II), and alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-GlcNAc (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean alpha-mannosidase gave D-mannose and a disaccharide (containing D-mannose and N-acetyl-D-glucosamine). Thus the three main compounts observed in the fibroblasts from patients with mannosidosis are most probably identical to the oligosaccharides I--III.

Postoperative studies on parathyroid hormone secretion in patients operated on for primary hyperparathyroidism

The secretion of intact parathyroid hormone (PTH) was investigated in 11 patients operated on for parathyroid adenoma at 1 year after surgery and compared with that of seven healthy individuals and five patients operated on because of clinical and biochemical signs of primary hyperparathyroidism with equivocal diagnosis after surgery. The investigation was performed by infusing Na2EDTA and CaCl2 at constant rates. No significant difference was found in the suppressibility of PTH secretion by calcium. The set point (the calcium concentration required for half-maximal inhibition of PTH secretion) was slightly lower in patients (1.20 +/- 0.02 mmol/l) compared with healthy subjects (1.23 +/- 0.03 mmol/l; P < 0.05). During the hypocalcemic EDTA infusion, the secretion of PTH was higher in controls compared with patients (P < 0.01). By comparing the data from the infusion tests in patients operated on for parathyroid adenomas with the data obtained from the patients with equivocal diagnosis after parathyroid surgery, a good probability for the diagnosis could be obtained.

Intact parathyroid hormone assay is superior to mid region assay in the EDTA-infusion test in hyperparathyroidism

We examined the use of an intact parathyroid hormone two-site immunoradiometric assay compared with a mid region parathyroid hormone radioimmunoassay in ethylene diamine tetraacetic acid-infusion test in 15 patients with hyperparathyroidism. During the test, plasma intact parathyroid hormone levels increased by 240 +/- 43%, whereas the plasma levels of mid molecule parathyroid hormone increased by only 65 +/- 17%, which is significantly lower (P less than 0.01). Four patients had no increase in plasma mid molecule parathyroid hormone level but still a large increase in plasma intact parathyroid hormone level (P less than 0.01). Thus, plasma measurement of intact parathyroid hormone is superior to that of mid molecule parathyroid hormone in the ethylene diamine tetraacetic acid-infusion test in patients with hyperparathyroidism.

Isolation and Characterization of Oligosaccharides from Urine of Patients with Abnormal Glycoconjugate Metabolism

Diseases involving abnormal glycoconjugation are frequently diagnosed by enzymic methods of varying complexity (Table 1) and/or by analysis of abnormally excreted carbohydrate-containing material. Gas-liquid chromatography-mass spectrometry (GC-MS) has been used to detect specific compounds known to be excreted in some glycogen storage diseases and muscular dystrophies (Lennartson et at., 1976) and in mannosidosis (Lundblad et al., 1975). This method has now been extended for use as a general screening method for the detection of low molecular weight carbohydrate-containing compounds. A number of oligosaccharides are present in normal human urine and include lactose, maltose, isomaltose, fucosyl-a( 1-2)-glucose, xylosyl-a-( 1-3)-glucose, fucosyl~-(1-O)-inositol, a glucose-containing tetrasaccharide and a series of disaccharides to pentasaccharides, the presence of which depends on ABO blood group, secretor status and diet (Lundblad et al., 1973). The amounts of these compounds excreted are usually quite low ( < 20 rag/24 h). In certain diseases additional compounds are excreted in much larger amounts, for example in mannosidosis a mannose-containing trisaccharide and tetrasaccharide are excreted in quantities up to 470 and 60 mg/24 h respectively (Lundblad et al., 1975; Norddn et al., 1974) and a glucose-containing tetrasaccharide is excreted in amounts approaching 90 mg/24 h in certain glycogen storage diseases (Lennartson et al., 1976). The overall picture therefore is of a complex mixture of low molecular weight carbohydrate-containing material which can show considerable non-pathological variation, but in at least some pathological states this pattern is overshadowed by great excesses of specific compounds or series of related compounds. The basic procedure developed to analyse urinary oligosaccharides involves desalting 20 ml of ultrafiltered urine through Dowex 50 (H +) and AG-3 (OH-) resins, fbltowed by reduction of the oligosaccharides with sodium borodeuteride and methylation (Lundblad et al., 1975). The methylated and reduced oligosaccharides are then separated by gas-liquid chromatography (GLC ; Perkin-Elmer 3920 : capillary column, 25 m x 0.25 mm, wall-coated with SE-30; temperature programme 230 °C/30 min, 230 °C 330 °C at 4 °C/rain, 330 °C/30 min) and identified by mass spectrometry (Varian MAT 311A, 70 eV, 1 mA, ion source 120°C) linked to a Spectrosystem 100 computer. The resulting chromatogram contains a large number of peaks grouped in the monosaccharide and disaccharide regions, but the trisaccharide to pentasaccharide region is usually dominated by a few compounds as shown for a normal blood group B secretor individual in Figure 1. The major component is the blood group B trisaccharide (Lundblad et aI., 1973). Also shown is the glucosecontaining tetrasae.charide (Glq). By comparison, the same region of the chromatogram is shown for urine from a mannosidosis patient in Figure 2. The only significant peaks in this case are two of the mannosecontaining oligosaccharides which cannot be degraded by these patients. These oligosaccharides are characterized by their GLC retention times and by comparison of their mass spectra with those of authentic compounds (Norddn et at., t973 and 1974). This method is therefore capable of detecting 1-10 ng of oligosaccharides containing up to about five sugar residues. Chemical or enzymic pretreatment of the urine samples, improvement in GLC methodology and singleion detection will increase the scope and sensitivity of the method.