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.

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)

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.

Maternal leucocyte zinc deficiency at start of third trimester as a predictor of fetal growth retardation.

Leucocyte zinc concentrations were measured in 70 mothers at the beginning of the third trimester of pregnancy and compared with the weight centiles of their subsequently delivered babies. The median maternal leucocyte zinc concentrations rose progressively with weight centile. Thus the median leucocyte zinc concentration of the mothers delivering babies weighing below the 10th centile was 112 nmol/10(9) leucocytes and that of the mothers with babies weighing above the 90th centile was 229.5 nmol/10(9) leucocytes. A maternal leucocyte zinc concentration less than 120 nmol/10(9) leucocytes strongly predicted a baby weighing below the 10th centile (positive predictive value = 71.9%, negative predictive value = 91.5%, sensitivity = 64.3%, specificity = 81.8%). These findings suggest that maternal zinc concentration might have a role in antenatal screening, but larger studies are required.

A collaborative study of the measurement of glycosylated haemoglobin by several methods in seven laboratories in the United Kingdom.

A collaborative study compared methods for measuring glycosylated haemoglobin in seven laboratories in the United Kingdom. No satisfactory standard for general use was found. Satisfactory internal quality control systems were in use for each assay which allowed the maintenance of a normal range in each participating laboratory. No satisfactory quality control system suitable for general use could be identified. Costs and convenience of the assays are reported. The technical problems with each type of assay and precautions for their use were identified, such as the need for standardization in incubation times, the practicability of automation of colorimetric assays, and the precision of pH needed for buffers in column separation methods. The relevance of the technical problems to interpretation of measurements is also considered. It is concluded that laboratories measuring glycosylated haemoglobin should maintain a normal range, use 'in-house' quality controls to monitor assay performance and keep clinical colleagues informed of the findings and of any changes in methodology that might affect the interpretation of results.