The generalized aminoaciduria seen in patients with hepatocyte nuclear factor-1alpha mutations is a feature of all patients with diabetes and is associated with glucosuria.

Hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations are the most common cause of maturity-onset diabetes of the young. HNF-1alpha homozygous knockout mice exhibit a renal Fanconi syndrome with glucosuria and generalized aminoaciduria in addition to diabetes. We investigated glucosuria and aminoaciduria in patients with HNF-1alpha mutations. Sixteen amino acids were measured in urine samples from patients with HNF-1alpha mutations, age-matched nondiabetic control subjects, and age-matched type 1 diabetic patients, type 2 diabetic patients, and patients with diabetes and chronic renal failure. The HNF-1alpha patients had glucosuria at lower glycemic control (as shown by HbA1c) than type 1 and type 2 diabetic patients, consistent with a lower renal glucose threshold. The HNF-1alpha patients had a generalized aminoaciduria with elevated levels of 14 of 16 amino acids and an increased mean Z score for all amino acids compared with control subjects (0.66 vs. 0.00; P < 0.0005). Generalized aminoaciduria was also present in type 1 diabetic (Z score, 0.80; P < 0.0001), type 2 diabetic (Z score, 0.71; P < 0.0002), and chronic renal failure (Z score, 0.65; P < 0.01) patients. Aminoaciduria was not associated with microalbuminuria or proteinuria but was associated with glucosuria (1.00 glucosuria vs. 0.19 no glucosuria; P = 0.002). In type 1 diabetic patients, urine samples taken on the same day showed significantly more aminoaciduria when glucosuria was present compared with when it was absent (P < 0.01). In conclusion, HNF-1alpha mutation carriers have a mutation-specific defect of proximal tubular glucose transport, resulting in increased glucosuria. In contrast, the generalized aminoaciduria seen in patients with HNF-1alpha mutations is a general feature of patients with diabetes and glucosuria. Glucose may depolarize and dissipate the electrical gradient of the sodium-dependent amino acid transporters in the proximal renal tubule, causing a reduction in amino acid resorption.

An ultrastructural and systemic analysis of glycosaminoglycans in thyroid-associated ophthalmopathy.

PURPOSE: To determine the ultrastructural localisation of glycosaminoglycans (GAGs) in the extraocular muscles (EOMs) of patients with thyroid-associated ophthalmopathy (TAO) and to see whether the quantity and type of GAGs present in blood and urine are markers of the disease. METHODS: Biopsies of affected EOMs were taken and studied by transmission electron microscopy (TEM). These were either fixed conventional for TEM, or in 0.5% tannic acid and others for immunogold staining. Serum hyaluronan (HA) was measured using a radioimmunoassay in patients with TAO as well as control subjects, and urinary GAG levels assessed by photometric quantitation of hexuronic acid after reaction with carbazole. The excretion pattern of the urinary GAGs was determined by discontinuous electrophoresis. RESULTS: TEM showed that there is a marked expansion of the endomysial space in TAO EOM biopsies as compared with non-TAO strabismus specimens. This is caused by an increased number of collagen fibres, interspersed with a granular amorphous material surrounding striated collagen fibres shown to be hyaluronan by immunogold staining. In contrast, serum hyaluronan concentrations were similar in TAO and control patients, although there was a statistically significant difference in the urinary GAG excretion between the two groups of patients examined. By discontinuous electrophoresis, chondroitin sulphate and heparan sulphate were present in both patients and controls. CONCLUSION: GAGs and in particularly HA are present at the EOM level in patients with recently inactive TAO. However, serum levels of HA and urinary GAGs are not sensitive indicators for their presence within the EOMs.

Haemoglobin and ferritin concentrations in infants at 8 months of age.

AIM: To identify the optimum age to screen for iron deficiency, the normal distribution of haemoglobin and ferritin in a representative population sample was investigated. METHODS: Normal values for haemoglobin and ferritin were measured from heel prick capillary samples obtained from a representative cohort of 1175 infants at 8 months old who were randomly selected from children taking part in the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC). RESULTS: Haemoglobin was normally distributed: mean (SD) 117 (11) milligrams, 95% confidence interval (CI) 116 to 118, and range 72-153 milligrams. Ferritin was log normally distributed: geometric mean 38.5 micrograms/l, 95% CI 37.0 to 39.9, range 7.1-224 micrograms/l. The 5th centile for haemoglobin was 97 milligrams and for ferritin 16.9 micrograms/l. No correlation was found between haemoglobin and ferritin. Multiple regression analysis showed ferritin concentrations to be positively related to birth weight (p < 0.0001) and the sex of the child (girls with higher concentrations) (p < 0.0001) but negatively with the child's weight at 8 months (p < 0.0001). Haemoglobin concentrations were positively related to the child's weight at 8 months (p = 0.04). Neither haemoglobin nor ferritin concentrations were related to social class as measured by maternal education level. CONCLUSION: These data define the normal range for haemoglobin and ferritin in capillary samples in the UK population, and suggest that anaemia is common in infancy. Using current recommendations, 23% of infants would be identified as anaemic. For British infants at 8 months of age, a more representative 'cut off' for anaemia would be haemoglobin concentration < 97 milligrams and for iron deficiency ferritin < 16 micrograms/l.

Urinary heparan sulphate proteoglycan excretion is abnormal in insulin dependent diabetes.

Urinary excretion of heparan sulphate proteoglycan (HSPG), the main anionic component of the glomerular basement membrane (GBM), was estimated in 30 adolescents and young adults with insulin dependent diabetes (IDDM), 10 with microalbuminuria and 20 sex matched, diabetic controls of similar age without evidence of microalbuminuria. A further 10 non-diabetic control subjects were also examined. Both groups of patients with diabetes had significantly elevated excretion of HSPG when compared to normal individuals. There was no difference in HSPG excretion between diabetic subjects with and without microalbuminuria.

Decreased sialidase activity in mononuclear leucocytes of type 1 diabetic subjects: relationship to diabetic complications and glycaemic control.

Leucocyte surface sialic acid content influences surface charge, deformability, and leucocyte-endothelial interaction. Abnormal leucocyte structure and function contributes both to microvascular damage and diabetic complications. The aim of this study was to investigate altered leucocyte SA metabolism in diabetic subjects and measure lysosomal sialidase which regulates leucocyte surface sialylation. We examined 26 Type 1 (insulin-dependent) diabetic subjects with retinopathy, 26 Type 1 diabetic subjects without complications, and 38 matched normal control subjects. Sialidase was assayed in freshly prepared sonicates of pure mononuclear leucocytes (MNLs), using the fluorometric substrate 4-methyl-umbelliferyl-N-acetylneuraminic acid. In the subjects with diabetes there was a significant negative correlation between MNL sialidase activity and both HbA1c (rs = 0.37, p = 0.007) and fructosamine (rs = -0.31, p = 0.026). MNL sialidase activity was significantly decreased in diabetic subjects with clinical evidence of complications compared to control subjects. HbA1c was significantly higher (p = 0.036) in diabetic patients with complications compared to those without. The observed decrease in MNL sialidase activity related to diabetic control may be important in the pathogenesis of vascular damage. Diabetes-associated changes in sialylation of functional cell surface glycoconjugates may have important clinical consequences.

Are frozen urine samples acceptable for estimating albumin excretion in research?

Timed urine collections from diabetic children and adolescents were assessed for urinary albumin excretion rate (microgram min-1) before and after freezing at -20 degrees C. Freezing had the effect of changing the estimation such that frozen values could differ from that of the fresh, by as much as one half to twice as much. The variation depended on the concentration defining the initial albumin excretion rate but was not influenced by the length of storage when frozen. We conclude that researchers should be aware that freezing and storing of urine samples prior to albumin concentration assessments can affect the absolute values obtained. It would appear more appropriate to analyse samples prior to freezing to be certain of obtaining true prevalence estimates of microalbuminuria.

Screening for diabetic microalbuminuria in routine clinical care: which method?

The measurement of albumin/creatinine ratios and simple albumin concentrations in early morning urine specimens were evaluated to establish which was the best screening test for those likely to have microalbuminuria by the reference standard analysis of timed overnight urine specimens. The measurement of an albumin/creatinine ratio with a cut off of > or = 2.0 mg/mmol was found to be suitable with a specificity of 93% and sensitivity of 97%.

Fructosamine and glycated haemoglobin in the assessment of long term glycaemic control in diabetes.

Fructosamine and glycated haemoglobin were measured simultaneously in 147 children with diabetes. If glycated haemoglobin is considered as the 'gold standard' for long term glycaemic control, then fructosamine is a poor indicator of actual glycated haemoglobin values, with wide 95% confidence (fiducial) limits. This shows that it is impossible to accurately predict glycated haemoglobin concentrations and therefore, by implication, longer term glycaemic control, from measurements of fructosamine. As the major studies on the prevention of microvascular complications in diabetes have used glycated haemoglobin levels to assess glycaemic control, it is suggested that this measurement should be used in all children with diabetes in preference to the measurement of fructosamine.

Freeze-stable sialidase activity in human leucocytes: substrate specificity, inhibitor susceptibility, detergent requirements and subcellular localization.

Human leucocytes contain a freeze-stable sialidase (neuraminidase; EC 3.2.1.18) activity in addition to the better-characterized lysosomal freeze-labile enzyme. In order to discriminate between the sialidase activities detected with the synthetic fluorimetric substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different tritiated sialoglycoconjugate substrates were prepared. Using this sensitive radioactive assay system, leucocyte sialidase activity towards glycoproteins was shown to be labile to repeated freeze-thawing, but a Triton-stimulated activity towards gangliosides was entirely freeze-stable. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocytes (MNLs) on Percoll-density gradients showed that this ganglioside sialidase activity was entirely associated with the plasma membrane. Study of the detergent requirements showed that MNLs also demonstrated ganglioside sialidase activity when sodium cholate was present in place of Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Studies on whole homogenates of MNLs demonstrated that the Triton-stimulated and cholate-stimulated activities showed similar acidic pH optima at < or = 3.9 and were both strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by free N-acetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate. These results suggest that human MNLs contain, in addition to the lysosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and stimulated both by Triton and by cholate.

Interaction of 1,9-dimethylmethylene blue with glycosaminoglycans.

The specific binding of 1,9-dimethylmethylene blue (DMB) with glycosaminoglycans (GAGs) was studied including molecular analysis of the GAG species using mass spectrometry. The dye solution was unstable under any storage conditions. Inorganic analysis showed that the purity of the dye was variable from different sources. False negative results could be obtained when using impure dye. Binding of the dye with GAG resulted in the formation of a complex with an absorption maximum at 525 nm. The absorbance of the complex was linearly correlated with GAG concentration up to 150 mg/L. The specific molar extinction coefficients of individual GAG molecules were calculated in relation to the molar absorbance of the GAG-dye complex and the carbon and nitrogen contents of the GAGs. The results indicated that binding of dye occurred at both the ionized sulphate and carboxyl groups on the GAG molecules. Improvements of the DMB-binding method for the measurement of GAGs are suggested.

Measurement of sialic acid in serum and urine: clinical applications and limitations.

Many recent studies have examined the sialic acid content of serum or urine in various pathological states. We have briefly reviewed the substances which contribute to the observed total sialic acid concentration, and given an overview of assay methods used. Three major areas of clinical interest in sialic acid metabolism are discussed. Serum total sialic acid, 'lipid-bound' and 'protein bound' sialic acid have all been proposed as tumour markers; but the usefulness of any of these tests is severely limited by changes due to accompanying inflammatory processes. Serum total sialic acid is not a valuable simple marker of an acute phase response. Urinary free and bound sialic acid measurements should be included in screening protocols for inherited disorders of lysosomal metabolism. Current developments in research and potential applications within the clinical biochemistry laboratory are briefly discussed.

Increases in plasma lysosomal enzymes in type 1 (insulin-dependent) diabetes mellitus: relationship to diabetic complications and glycaemic control.

Lysosomal enzymes degrade membrane glycoconjugates, and increased circulating enzyme activity may be an important mechanism in the pathogenesis of diabetic microangiopathy. We have assayed a profile of seven lysosomal enzyme activities (nmol.h-1.ml-1) in platelet-free plasma from 54 Type 1 (insulin-dependent) diabetic subjects (median age 31 years) and 42 matched normal control subjects. A significant increase in median (interquartile range) enzyme activity was measured in diabetic compared to control subjects for beta-D-glucuronidase, 121 (97.7-171) vs 88.8 (62.8-113), p less than 0.001; beta-D-Nacetylglucosaminidase, 693 (568-799) vs 568 (462-686), p less than 0.001; alpha-D-mannosidase, 23.8 (16.7-28.9) vs 14.5 (10.1-20.0), p less than 0.001; and beta-D-galactosidase, 6.94 (6.11-9.99) vs 6.66 (4.78-8.33), p less than 0.04. In contrast, alpha-L-fucosidase, alpha-D-galactosidase and beta-D-mannosidase activities were similar in diabetic and control subjects. None of the enzyme activities differed significantly (p less than 0.05) between 24 diabetic patients with clinical complications and 30 complication-free diabetic patients with similar glycaemic control which does not support the hypothesis that enzyme increases in diabetes arise simply by leakage from damaged tissues. In the diabetic subjects HbA1, median (interquartile range) 9.10 (7.40-10.60), was significantly related to beta-D-glucuronidase (rs = 0.56, p less than 0.001) and beta-D-Nacetylglucosaminidase (rs = 0.55, p less than 0.001). We have therefore demonstrated in diabetic subjects an increase in certain lysosomal glycosidases, that correlates with glycaemic control.(ABSTRACT TRUNCATED AT 250 WORDS)

Results of a quality assurance exercise for urinary glycosaminoglycan excretion.

Urine samples collected from four patients with a mucopolysaccharide storage disease (MPS) and two non-MPS patients were distributed to up to 33 laboratories as a test of their ability to detect abnormal glycosaminoglycan excretion. Seven national reference laboratories made a correct diagnostic assignment to all samples analysed. Qualitative turbidity and spot tests were shown to be unreliable. Failure to identify the excretion pattern occurred when reliance was placed on one-dimensional electrophoresis or thin layer chromatography as the sole method for glycosaminoglycan identification. Two-dimensional electrophoresis appeared to be the method of choice provided that staff had adequate experience in interpretation. Clinically unacceptable delays in analysis were common, with 80% of laboratories taking longer than 10 days to issue a report.

Carrier detection for Sanfilippo A syndrome.

Leukocytes from 21 obligate carriers in 12 Sanfilippo A families and 49 normal controls were assayed for heparan sulphamidase (EC 3.10.1.1) at 55 degrees C. At this assay temperature the results show an absolute distinction between heterozygous carriers and the normal controls.