title: Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.
abstract: In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.
authors.affiliation: Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland; Institute of Dentistry, University of Eastern Finland, Kuopio, Finland. Electronic address: sanna.oikari@uef.fi.
authors.forename: Sanna
authors.initials: S
authors.lastname: Oikari
authors.affiliation: Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
authors.forename: Tuula
authors.initials: T
authors.lastname: Venäläinen
authors.affiliation: Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Tammi
mesh_terms: D000273:Adipose Tissue / Q000737:chemistry*; D000818:Animals; D000838:Anions / Q000737:chemistry; D001881:Borates / Q000737:chemistry*; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008099:Liver / Q000737:chemistry*; D008297:Male; D009206:Myocardium / Q000737:chemistry*; D051381:Rats; D014531:Uridine Diphosphate Galactose / Q000032:analysis; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000032:analysis; D014539:Uridine Diphosphate Sugars / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D000838:Anions; D001881:Borates; D014539:Uridine Diphosphate Sugars; D014531:Uridine Diphosphate Galactose; D014537:Uridine Diphosphate N-Acetylglucosamine
keywords: Anion exchange HPLC; Boric acid; Ion-pair solid-phase column; Tissue extraction; UDP-sugar