Christopher M. Starr

Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates

Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.

Erratum to: Fluorophore-assisted electrophoresis of urinary carbohydrates for the identification of patients with oligosaccharidosis- and mucopolysaccharidosis-type lysosomal storage diseases

Lysosomal storage diseases result from defects in the activity of the lysosomal enzymes that break down macromolecules in the cell. These enzyme defects contribute to over 30 separate storage diseases that result in nearomuscular and intellectual impairment and, in some cases, early childhood death. This report describes a new method for identifying defects in the lysosomal enzymes and in the metabolic pathway that functions in the degradation of complex carbohydrates. The method involves identifying ‘abnormal’ carbohydrates in the urine of affected patients using fluorescent carbohydrate tags and polyacrylamide gel electrophoresis. Currently, the method can be used as a simple screen for the identification of at least 12 different lysosomal storage diseases using a single electrophoretic procedure. Both oligosaccharidoses and mucopolysaccharidoses (MPS) can be identified, and in many cases the MPS subtype can be determined. In addition, the method can be used to confirm enzymatically the results of the initial screening test. We believe that this method will become extremely useful not only in the diagnosis of these diseases but in the management of patients on therapy.

Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and α-L-iduronidase substrates

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexo-saminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure α-L-idopyranosyluronic acid (1→3)-α,β-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian α-L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.

The Different Faces of Disease

Carbohydrates have important biologic roles, and they participate in a number of biologic reactions including stabilizing the structure and function of proteins, as receptors for soluble effectors, microorganisms and intracellular recognition, as biological catalysts for protein enzymes, and as primary or secondary messengers for a variety of intracellular processes (Varki, 1993). Because carbohydrates are vital to biological processes, their presence or absence can be associated with disease making them excellent candidate diagnostic markers. Measurement of carbohydrate analytes are used to predict disease, and several are approved by the US Food and Drug Administration (FDA) for use by physicians. These include the measurement of advanced glycation end-products in diabetes mellitus (Bucala, 1992) and the measurement of oncodevelopmental carbohydrate antigens in cancer (Sell, 1990, Laferte, 1994). Other carbohydrates being measured experimentally to determine their usefulness as predictors of disease include the measurement of free oligosaccharides in body fluids of patients with lysosomal storage diseases (LSD)(Hommes, 1991, Wappner, 1993), unique carbohydrates in mycobacterial infections (Hunter, 1986, Honda, 1993), abnormal glycosylation of proteins in cancer (Aoyagi, 1993), and liver disease (Martinez, 1987, Stibler, 1991, Yamauchi, 1993, Tsusumi, 1994). Additionally, as more carbohydrate pharmaceuticals emerge and enter the clinic, ways to measure and monitor their levels in patients will become important.

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that func tional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in pa tients receiving anticoagulant therapy. This creates the possi bility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore assisted carbohydrate electrophoresis (FACE) to directly mea sure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct bio chemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is prac tical and useful for studies of the correlation of biochemical and biological activities.