Frederick S. Wusteman

Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium.

Purified bovine milk lipoprotein lipase was shown to bind to intact porcine aortic endothelium in a specific, saturable fashion. The binding was reversed by exogenous heparin. A single class of binding sites was involved and at saturation 1.24 x 10(11) molecules of lipoprotein lipase/cm2 were bound. This represents 0.51 x 10(6) enzyme molecules per endothelial cell at a density of 1.2 x 10(3) molecules/micrometers 2. The enzyme binding was reduced by prior trypsinisation of the endothelial surface under conditions that removed cell surface glycosaminoglycan chains. The porcine endothelium was shown to have available at its surface 5.4 x 10(11) chains of heparan sulphate plus heparin-like glycosaminoglycans/cm2. Such an excess suggests that lipoprotein lipase may interact with approximately one in four of the available heparan sulphate chains.

The effects of silica dust and alveolar macrophages on lung fibroblasts grown in vitro

Abstract The effect of silica (min-u-sil) on lung fibroblasts, with and without the mediation of alveolar macrophages, has been studied by measuring DNA, protein, collagen, lysosomal enzymes and secreted glycosaminoglycans. Intact macrophages were found to stimulate collagen production whether they had first been pretreated with silica or not. The dust has a direct effect on fibroblasts, an effect dependent on silica concentration and the stage of fibroblast growth. The possible relationships of the above effects to both fibrosis and emphysema are discussed.

An improved assay for iduronate 2-sulphate sulphatase in serum and its use in the detection of carriers of the Hunter syndrome.

A more sensitive assay procedure has been developed for the enzyme iduronate 2-sulphate sulphatase which is deficient in the Hunter syndrome. The substrate is the same as previously described by Lim et al. [1], O-(alpha-L-idopyranosyluronic acid 2-sulphate)-(1leads to 4)-2,5 anhydro-D-[3H-1]mannitol 6-sulphate, but, after incubation, it is separated from the product by ion-exchange chromatography on a micro-column of Dowex 1 x 2 (Cl-1) instead of high voltage electrophoresis or ECTEOLA cellulose chromatography. Since the blank correction is then much smaller, a shorter incubation time can be used and conversion of the substrate reduced from approximately 50% down to levels where complications resulting from substrate depletion and product inhibition are minimal. Using whole serum the apparent Km for the substrate is 0.2 mmol/l. With an incubation time of 20 min, sera from heterozygotes exhibited approximately 35% of the normal levels of iduronate 2-sulphate sulphatase (0.11-0.61, mean 0.34 nmol.h-1.mg-1 protein for carriers; 0.24-2.35, mean 0.94 nmol.h-1.mg-1 protein for 37 normal females). Serum analyses can thus be used to supplement those on hair roots in the detection of carriers of the Hunter syndrome.

Measuring blood volume with fluorescent-labeled hydroxyethyl starch.

Objectives: To develop and evaluate a method for measuring blood volume using the dilution of a fluorescent‐labeled hydroxyethyl starch. Design: Laboratory and clinical investigation. Setting: Biochemistry laboratory at the University of Cardiff. Hematology clinic, surgical ward and intensive care unit of the University Hospital of Wales. Patients: Seventeen patients with suspected polycythemia. Eight patients who had undergone major surgery and/or were receiving intensive postoperative care. Interventions: All surgical and postoperative care was provided by clinicians not involved in the study. Patients with suspected polycythemia were referred for blood volume measurement using labeled albumin and red blood cells. Measurement and Main Results: A proprietary brand of hydroxyethyl starch (Elohaes) was labeled with fluorescein isothiocyanate. Dilution of this compound in vivo was used for measuring blood volume, and the results were compared with those obtained using radiolabeled albumin and the considered criterion, radiolabeled red cells. The elimination of the labeled starch follows the same progress as that of the parent compound, indicating that the fluorescent tag is stable in vivo. The volume of distribution of the labeled starch is 2.5 mL/kg lower than that for labeled albumin (p = .05). Blood volume, measured from the dilution of fluorescent starch, is lower (4.9 mL/kg) than that measured with albumin (p = .048) but higher (6.61 mL/kg) than that measured with red blood cells (p = .0007). This latter difference may be even smaller at marginally higher doses of the fluorescent starch. Conclusion: These data support the view that hydroxyethyl starch provides a valid alternative to red cell labels as a means of calculating blood volume in patients. Labeling the starch with a fluorescent marker makes the assay procedure more sensitive and infinitely easier. The dose required is not high enough to affect the hemodynamic status of the patient.

Multiple forms of iduronate 2-sulphate sulphatase in human tissues and body fluids

Iduronate 2-sulphate sulphatase (EC 3.1.6.-) was found in human placenta in three forms which could be separated by elution from DEAE-cellulose using an NaCl gradient. Form C, most firmly bound to DEAE-cellulose, was 40% larger than the other two (forms A and B in order of ease of elution from the ion exchanger). Forms B and C contained sialic acid which could be removed by neuraminidase digestion. After removal of sialic acid form B became indistinguishable from form A. The enzyme forms found in placenta were compared with those from other human tissues and fluids by means of DEAE-cellulose chromatography and gel chromatography. Serum and amniotic fluid contained only form C, urine and cultured fibroblasts contained the less-anionic forms as well, and kidney contained appreciable amounts only of form A. Pre- and post-natal diagnosis of the Hunter syndrome both involve measurements on the enzyme which is present in form C. This is not accompanied by less-anionic forms which constitute the bulk of the enzyme as it is isolated from easily available sources such as urine.

The use of ‘normal’ rats in studies on the acid mucopolysaccharides of lung

Abstract The acid mucopolysaccharide content of rat lungs from apparently healthy animals bred under normal laboratory conditions was compared with those from a caesarian-derived colony of the same strain and age range. For all but the youngest animals acid mucopolysaccharide levels per de-fatted dry weight or per connective tissue protein were elevated in the ordinary animals. These observations suggest that lungs from animals kept under conventional conditions are unsuitable for studies on mucopolysaccharide metabolism or related processes due to the persistent inflammation resulting from spontaneous pulmonary disease.

Detection of female carriers of hunter's syndrome: Comparison of serum and hair-root analysis

Hunters syndrome (McKusick 30990) results from a defect in the enzyme iduronate 2-sulphate sulphatase (EC 3.1.6.-) and is inherited as an X-linked recessive disorder. The detection of female carriers has been approached by hair-root analysis (Nwokoro and Neufeld, 1979) and by estimating enzyme levels in sera (Archer et al., 1981). In this study, the ability to predict female carriers by hairroot analysis and by serum enzyme levels have been compared.