John C. Klock

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.