hymecromone and 4-methylumbelliferyl-iduronide

The lack of methodological uniformity in enzyme assays has been a long-standing difficulty, a problem for bench researchers, for the interpretation of clinical diagnostic tests, and an issue for investigational drug review. Illustrative of the problem, α-L-iduronidase enzyme catalytic activity is frequently measured with the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide); however, final substrate concentrations used in different assays vary greatly, ranging from 25 μM to 1425 μM (Km ≈ 180 μM) making it difficult to compare results between laboratories. In this study, α-L-iduronidase was assayed with 15 different substrate concentrations. The resulting activity levels from the same specimens varied greatly with different substrate concentrations but, as a group, obeyed the expectations of Michaelis-Menten kinetics. Therefore, for the sake of improved comparability, it is proposed that α-L-iduronidase enzyme assays should be conducted either (1) under substrate saturating conditions; or (2) when concentrations are significantly below substrate saturation, with results standardized by arithmetic adjustment that considers Michaelis-Menten kinetics. The approach can be generalized to many other enzyme assays.

Topics: Calibration; Enzyme Assays; Humans; Hymecromone; Iduronidase; Kinetics; Mucopolysaccharidosis I; Quality Control

Three major clinical subgroups are usually distinguished in Mucopolysaccharidosis type I: Hurler (MPS IH, severe presentation), Hurler-Scheie (MPS IH/S, intermediate) and Scheie (MPS IS, mild). To facilitate treatment with hematopoietic stem-cell transplantation, early diagnosis is important for MPS IH patients. Although screening for MPS I in newborns would allow detection at an early age, it may be difficult to predict the phenotype on the basis of the genotype in these infants. Extra diagnostic tools are thus required. Based on the hypothesis that distinct MPS I phenotypes may result from differences in residual α-l-iduronidase (IDUA) activity, we modified the common IDUA assay using the substrate 4-methylumbelliferyl-α-l-iduronide to allow quantification of low IDUA activity in MPS I fibroblasts. Enzyme incubation was performed with high protein concentrations at different time points up to 8h. Mean residual IDUA activity was 0.18% (range 0-0.6) of the control value in MPS IH fibroblasts (n=5); against 0.27% (range 0.2-0.3) in MPS IH/S cells (n=3); and 0.79% (range 0.3-1.8) in MPS IS fibroblasts (n=5). These results suggest that residual IDUA activity and severity of the MPS I phenotype are correlated. Two MPS IS patients with rare (E276K/E276K) or indefinite (A327P/unknown) IDUA genotypes had residual IDUA activity in the MPS IS range, illustrating the usefulness of our approach. IDUA(E276K) was very unstable at 37°C, but more stable at 23°C, suggesting thermal instability. We conclude that this procedure for determining residual IDUA activity in fibroblasts of MPS I patients may be helpful to predict MPS I phenotype.

Topics: Cell Line; Early Diagnosis; Fibroblasts; Humans; Hymecromone; Iduronidase; Infant, Newborn; Mucopolysaccharidosis I; Mutation

It has been previously demonstrated that the enzyme alpha-L-iduronidase (IDUA) of patients with MPS I shows a different biochemical behavior in each of the three clinical forms of these. In heterozygotes, its biochemical behavior has been recently established in leukocyte and plasma samples, demonstrating that it is possible to distinguish individuals heterozygous for MPS I within an unselected population.. We evaluated the effect of copper chloride, EDTA and sodium chloride on the activity of the enzyme alpha-L-iduronidase in the plasma of normal individuals and of MPS I heterozygotes and observed the type of inhibition caused, the Ki, the apparent Km and the apparent Vmax for each inhibitor.. Sodium chloride inhibited the enzyme in normal individuals and in 40% of the heterozygotes evaluated and activated it in 60% of heterozygotes. The remaining compounds inhibited IDUA in both heterozygotes and normal individuals.. We detected significant differences capable of differentiating MPS I heterozygotes from normal individuals by simply adding sodium chloride, EDTA or copper chloride to the incubation medium at the time of IDUA activity determination, with a potential use in carrier detection protocols.

Topics: Biomarkers; Chelating Agents; Copper; Edetic Acid; Heterozygote; Humans; Hymecromone; Iduronidase; Indicators and Reagents; Kinetics; Mucopolysaccharidosis I; Reference Values; Sodium Chloride

Topics: Diagnosis, Differential; Heterozygote; Humans; Hymecromone; Iduronidase; Mucopolysaccharidosis I; Phenotype

Some characteristics of the human leukocyte and plasma alpha-L-iduronidase are described. The enzyme from both sources is sufficiently stable and linear in time to allow accurate determinations. The leukocyte and plasma enzyme have a low acid pH optimum at 3.5 and 4, respectively, which is in agreement with the lysosomal origin of the enzyme in the cell. Both enzymes are inhibited by phenyl-alpha-L-iduronide, heparin, and heparitin sulfate although other mucopolysaccharides also inhibit the leukocyte enzyme. When kept frozen at -20 degrees C, the leukocyte as well as the enzyme in acidified plasma are very stable. We studied the plasma enzyme in more detail. If the plasma is acidified, iduronidase is very stable between 0 and 37 degrees C. CuCl2 and Na2SO4 were very potent inhibitors at concentrations of 10 and 100 mM, respectively. The determination of iduronidase in leukocyte homogenates of patients suspected of Hurler disease together with plasma activities is useful for confirming or corroborating the diagnosis of genetic iduronidase deficiency. Further investigation is needed to determine if the plasma enzyme test would be useful in the biochemical diagnosis of Scheie disease and the Hurler-Scheie compound, two diseases which are also caused by a deficiency of alpha-L-iduronidase.

Topics: Fluorescent Dyes; Glycoside Hydrolases; Humans; Hydrogen-Ion Concentration; Hymecromone; Iduronidase; Leukocytes; Umbelliferones

1. Iduronosyl anhydro[1-3H] mannitol 6-sulphate (IMs), iduronosyl anhydro [1 3H] mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of alpha-L-iduronidase activity present in human leucocyte and cultured skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. 2. NaCl, KBr and Na2SO4 were shown to be parabolic competitive inhibitors of IMs activity, the Ki with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na2SO4 appeared to have a specific ion effect at a cationic binding site. 3. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI at pH values below 3.8 but activated the enzyme at higher incubation pH values. 4. Cu2+ was shown to be a potent non-competitive inhibitor of IMs enzyme activity with an apparent Ki of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe2+ (Ki 4 mmol/l), Hg2+ and Ag+, but has no significantly been affected by other univalent or bivalent cations. 5. The presence of solvent and salt effects on apparent Km but not the Vmax. suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.

Topics: Anions; Cations; Cells, Cultured; Fibroblasts; Glycoside Hydrolases; Humans; Hydrogen-Ion Concentration; Hymecromone; Iduronic Acid; Iduronidase; Kinetics; Leukocytes; Osmolar Concentration

Using 4-methylumbelliferyl-alpha-L-iduronide as a substrate, alpha-L-iduronidase activity was measured in leukocytes and in lymphoblastoid cells obtained from patients with alpha-L-iduronidase deficiency and from obligate heterozygotes for this disease. There was complete discrimination between alpha-L-iduronide in leukocytes and in lymphoblastoid cells from the patients and controls. However, overlap was observed between values of the activity in the obligate heterozygotes and those in the controls. 4-Methylumbelliferyl-alpha-L-iduronidase activity because of greater sensitivity, easier assay procedure and shorter incubation period.

Topics: Female; Fluorometry; Glycoside Hydrolases; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Umbelliferones

4-Methylumbelliferyl-alpha-l-iduronide provided a more sensitive method than phenyl-alpha-l-iduronide for the estimation of alpha-l-iduronidase in cultured cells and could be used to diagnose Hurler's disease. The 4-methylumbelliferyl derivative was no more useful than the phenyl derivative for the detection of heterozygotes. All ten lysosomal enzymes tested could be used as reference enzymes when cell extracts were prepared by freeze/thawing in formate buffer pH 3.5 containing 150 mmol/l sodium chloride.

Topics: Amniotic Fluid; Clinical Enzyme Tests; Female; Fibroblasts; Genetic Carrier Screening; Glycoside Hydrolases; Humans; Hydrolases; Hymecromone; Iduronic Acid; Iduronidase; Kinetics; Mucopolysaccharidosis I; Pregnancy; Skin; Substrate Specificity; Umbelliferones; Uronic Acids