Screening for Sanfilippo disease type A by infrared spectroscopy of hair.
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Biomedical subjects
Publications and source records attributed to G Lubec.
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A 5 year old girl suffering from chronic tonsillitis and bilateral tympanic effusion developed atrophy of the right half of the tongue. With the exception of the twelfth cranial nerve (right) no neurological signs could be detected. We are reporting a case of an isolated hypoglossal nerve paralysis.
As previously shown by two dimensional thin-layer chromatography L-azetidine-2-carboxylic-acid (L-Az) is incorporated into type I skin collagen instead of proline when 3 week old mice are fed with a 0,1% solution of L-Az orally. Ultrastructural investigations did not reveal significant changes in collagen periodicity and on fibril diameter. The collagen fibrils of the upper papillary dermis seemed to be packed more densely, sometimes only one electron dense lamina was seen instead of basal lamina and plasma membrane. The glycosaminoglycane-induced fibrillogenesis was not changed in contrary to the collagen-heat-gelation fibrillogenesis at 37 degrees C, where no gel aggregation could be seen. The reconstruction of native fibres from collagen solutions was disturbed too, several finer precipitated fibrils being detectable. On infrared spectroscopy significant differences in absorption spectra were detected. Correlating with previous results of reduced tensile strength and normal melting point of L-Az collagen we can conclude that L-Az might cause rather intermolecular than intramolecular disturbances of crosslinking.
A specific and sensitive method for the quantitative determination of the stable, reduced glucose-lysine adduct, glucitollysine (GL), in plasma protein samples is described. The method uses standard amino acid ion exchange chromatography followed by reverse phase high performance liquid chromatography after derivatisation of GL to a fluorescent product. Moreover, GL was characterised and identified in plasma samples by means of mass spectroscopy. GL measured in plasma samples of eleven type I diabetics and two healthy controls showed a significant linear correlation to concomitantly determined haemoglobin AI and glucosylated plasma proteins, but did not correlate with plasma glucose levels. This method allows the estimation of non-enzymatic glucosylation in biological samples with a high degree of specificity and sensitivity down to the low nanogram range.
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Ultrasonic median values of the dimensions of the uterus and volume of the ovaries were determined in a statistically sufficiently large group of girls in the hormonally inactive stage between 1 and 14 years of age who were healthy from the paediatric-gynaecological viewpoint. The graphic presentation of the results is an essential prerequisite for routine outpatient sonography. The most striking result is an almost linear, significant growth of the uterus and ovaries even before the menarche.
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Non-enzymatic galactosylation has been investigated by in vitro incubation of red cell haemolysate, a HbAo-preparation and of GBM of healthy children. The effects of non-enzymatic galactosylation of haemoglobin has been studied by high pressure liquid chromatography, the effects of GBM galactosylation by immunoelectrophoresis. Subsequently, the occurrence of elevated values for HbAIa-c and GSP was evaluated in 14 galactosaemic children (11 transferase deficiency, 3 galactokinase deficiency), as well as urinary acid glycosaminoglycae excretion and GBM immunoelectrophoretic mobility in 6 of these 14 children measured. The results were compared to the respective values of healthy control children. After exclusion of significant non-enzymatic glucosylation by measuring postprandial blood glucose values the galactosaemic children showed significantly increased values for HbAIa-c (8.85 +/- 2.0% versus 7.7 +/- 0.3%; p less than 0.02), for GSP (0.43 +/- 0.13 mmol 5-HMF/mg protein versus 0.32 +/- 0.07 mmol 5-HMF/mg protein; p less than 0.005) as well as for urinary acid glycosaminoglycane excretion (45.3 +/- 23.4 micrograms/mg kreatinine versus 9.9 +/- 2.3 micrograms/mg Kreatinine; p less than 0.01). 3 out of the 6 children showed alpha 1-immunoelectrophoretic mobility of GBM antigens which was found also after incubation of GMB with galactose. The other 3 children had alpha 2-immobility, which was found in the healthy controls as well as in the control incubations. The impact of galactose on increased non-enzymatic glycosylation in children with galactosaemia as well as the significance of this finding for diagnostic purposes or for clarifying pathophysiological aspects of the disease remains to be studied further.
Non-enzymatic glycosylation (NEG) of alkaline phosphatase (AP) was studied after short- and long-term incubation with glucose and other carbohydrates. Glucose and amino sugars clearly inhibited the enzyme activity; this was in contrast to reducing and non-reducing disaccharides, which had an enhancing effect. After AP had been incubated with 18 nmol/l glucose for 180 minutes (short-term incubation), a subsequent extensive dialysis revealed full recovery of the enzymatic activity. This, plus the demonstration of a [3H]sodium borohydride-reducible glucose-protein adduct, indicated that initially a labile aldimine (Schiff base) had been formed. Binding experiments with [14C]glucose and failure of dialysis to achieve a recovery of enzymatic activity after long-term incubation suggested that subsequently a stable ketoamine product had been formed. This was further confirmed by the thiobarbituric acid test, which revealed 0.65 nmol 5-hydroxymethylfurfural/mg protein for glycosylated AP compared to 0.11 for the non-glycosylated control. Preliminary results further suggest that NEG of AP also occurs in vivo. Streptozotocin diabetic rats had significantly lower serum AP activities than did non-diabetic controls (mean +/- SD: 153.7 +/- 28.4 vs. 760.5 +/- 95.7 U/l; p less than 0.001). Blood glucose levels and serum AP activity, which had been determined simultaneously during an oral glucose tolerance test, showed without exception an inverse relationship in each of 32 healthy children studied. The biological significance of these findings remains to be established.
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8 mice were given L-acetidin-2-carboxylic acid orally over a period of 5 weeks. Another 8 mice served as control animals. Finally, the mice were sacrificed, their hair samples were hydrolized and applied to thin layer chromatography which revealed the incorporation of L-acetidin-2-carboxylic acid into mouse hair expressed by an additional spot. This experimental amino acid metabolic disorder served as a model for the application of infrared spectroscopy for screening hair in order to check metabolic disorders. As the multiinternal reflection-infrared spectra showed significant differences we suggest that infrared spectroscopy can be used as a noninvasive screening technique.
In order to elucidate the role of L-acetidine-2-carboxylic acid (L-ac) incorporated into collagen type I of the skin instead of proline, we looked for the mechanoelastic properties of skin. Mice were orally fed with 0, 1% solution of L-ac for 5 weeks, then sacrificed, and type I collagen was extracted. Incorporation of the proline analogue (L-ac) could be shown by two dimensional thinlayer chromatography. The melting point (Tm) of type I collagen was determined by circular dichroism: 36, 5 +/- 1 degrees C for normal collagen, 37 +/- 1 degrees C for L-ac-collagen. This insignificant difference indicates that there was no alteration of the thermal stability of the collagen helix after incorporation of L-ac. Tensile strength was examined on whole strips of skin and worked out at means = 1,00 N/mm, s = 0,20 N/mm, sigma = 0,04 N/mm for normal individuals and means = 0,62 N/mm, s = 0,24 N/mm, sigma = 0,05 N/mm for L-ac-fed animals. The considerable difference could be estimated by the Wilcoxon test (p less than 0,005). As the stability of the collagen helix (shown through melting point determination) has not decreased, the reduced mechanoelastic property of tensile strength seems to be due to intermolecular rather than to intramolecular disturbances of the cross connection of the triple helix.
Recent investigations point to the nonenzymatic glycosylation as a cause of long-term complications in diabetes mellitus. We describe an enzymatic activity that cleaves glucose from the glomerular basement membrane (GBM), present in lysosomal preparations of diabetic lymphocytes. The GBM, nonenzymatically glycosylated or obtained from rats with diabetes, were incubated with enzyme preparations, separated on Sephadex G-25 and applied for glucose measurement on gas chromatography and mass spectroscopy. The lysosomal preparation of diabetic lymphocytes cleaved from rat GBM, which were nonenzymatically glycosylated 300-500 ng glucose/mg GBM protein, from diabetic rat GBM 300 ng glucose/mg GBM protein. A lysosomal preparation of normal lymphocytes failed to do so, indicating enzyme induction in the diabetic state. Control studies with the glycosylated hemoglobin AIc confirmed this finding and showed the specificity of the enzyme, as alpha-glucosidase and beta-glucosidase failed to cleave the N-glycosidic bond between glucose and the protein. The enzymatic activity can be described formally as a N-l-deoxyfructofuranosyl-glucohydrolase, which could be responsible for a potential reversibility of diabetic GBM changes.
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In order to look for the position of amyloid P in the macromolecular connective tissue and extracellular matrix system, we performed binding studies involving affinity chromatography. Binding studies revealed the strong binding of fibronectin to amyloid P (S-AP). The fibronectin-amyloid P linkage was dissociated after elution with 2 M urea. Heparan sulfate, a major glycosaminoglycan of the extracellular matrix, showed strong binding to S-AP, which was dissociated at 3 M urea. Laminin, collagen type I and type IV, reduced and alkylated glomerular basement membranes as well as the glycosamino-glycans hyaluronic acid and chondroitin-4-sulfate failed to bind to S-AP. Our binding studies show that amyloid P can react strongly with extra cellular matrix proteins and can help to explain the presence of amyloid P in normal connective tissue.
Though there is sufficient evidence that fibronectin is an integral extracellular matrix protein of the normal human kidney, the distribution and the interaction with structural proteins of the kidney have not been resolved. There are disagreements between investigators whether fibronectin is a component of the glomerular basement membrane or linked to it. We have been examining the interaction between the glomerular basement membrane and its components type IV collagen and laminin and fibronectin. Applying affinity chromatography we detected significant interaction between collagen type IV and laminin, and fibronectin on the other hand. The noncovalent interaction between collagen type IV and fibronectin was reversible after elution with 2 M urea, the association between laminin and fibronectin was even stronger and reversible applying 3 M urea for elution. The glomerular basement membrane-fibronectin interaction reflected the laminin pattern though minor binding sites at 1 M urea and 2 M urea could be detected. These interaction studies showed the chemical and thermodynamical possibilities and probability of the glomerular basement membrane and fibronectin linkage.