Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues

J Chromatogr A. 2014 Jan 3;1323:82-6. doi: 10.1016/j.chroma.2013.11.004. Epub 2013 Nov 11.

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.

Keywords: Anion exchange HPLC; Boric acid; Ion-pair solid-phase column; Tissue extraction; UDP-sugar.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipose Tissue / chemistry*
  • Animals
  • Anions / chemistry
  • Borates / chemistry*
  • Brain Chemistry*
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Liver / chemistry*
  • Male
  • Myocardium / chemistry*
  • Rats
  • Uridine Diphosphate Galactose / analysis
  • Uridine Diphosphate N-Acetylglucosamine / analysis
  • Uridine Diphosphate Sugars / analysis
  • Uridine Diphosphate Sugars / isolation & purification*

Substances

  • Anions
  • Borates
  • Uridine Diphosphate Sugars
  • Uridine Diphosphate Galactose
  • Uridine Diphosphate N-Acetylglucosamine