Nucleotide sugars: determination of cellular levels and discrepancies in results.

Abstract

Two important questions exist: (1) what are true cellular UDP-hexose levels, and (2) is there a difference between galactosemic patients and controls? Various laboratories have published cellular levels of uridine diphosphate galactose (UDP-gal) [1-4, 6, 7]. The discrepancies in their results caused controversy. My first assumption regarding the discrepancies was the known instability of UDP-gal. Different conditions for extraction, storage etc. may influence the final results. In our method [7] we have kept washed red cells frozen and extracted them by heating just before the analysis. However, Segal and co-workers have commented during this meeting that there were no differences in the values when different extraction methods were used. My second assumption was that the lower values produced by the spectrophotometric method [2-4, 6] may be caused by the inhibition of epimerase by NADH generated in the reaction mixture by UDP-glucose dehydrogenase. However, all groups now use the two-step procedure which would eliminate this effect [2-4, 6]. The radioisotopic method [7] used in our laboratory is a simple and sensitive procedure but has two major pitfalls: first, the inhibition of uridyltransferase by galactose-1phosphate, and second, the lack of specificity (it measures not only UDP-gal, but also other uridine diphosphate sugars such as UDP-glucose). The first difficulty was overcome by diluting the cells sufficiently, and for the latter the values should be changed to UDP-hexose instead of UDP-gal. Ng and his group [2, 4, 6], who found generally higher UDP-gal concentrations than Kirkman [3], repeatedly reported a decreased cellular UDP-ga l level in galactosemic tissues, and so did Ornstein et al. for fibroblasts [5]. In contrast, Kirkman [3] showed no difference in red cells, nor did Keevill et al. in fibroblasts [8]. In red cells, on the other hand, Holton and Segal reported some differences in the UDP-gal concentration during this meeting. Kirkman ascribed the higher UDP-gal levels obtained by Ng to impure UDP-glucose dehydrogenase [3]. It is interesting to note that Ng's normal red cell values are about 400% higher than those of Kirkman, but those of galactosemics only about 150%-200%. Furthermore, Palmieri et al. [6] found that the red cell UDP-gal concentration was higher in children than in adults, which was again in contrast with the findings of Kirkman who found somewhat lower concentration in children [3].