PMID: 9447935Feb 3, 1998Paper

Quantitative uronic acid analysis of the otoconial membrane of the guinea pig

Hearing Research
H SuzukiT Takasaka

Abstract

Histochemical and biochemical studies have shown that the otoconial membrane, as well as the tectorial membrane and the cupula, contains glycoproteins and proteoglycans. However, uronic acids, which are essential elements of glycosaminoglycans (GAGs), have not so far been directly detected or quantitatively measured in inner ear samples. In the present study, we quantitatively analyzed the glucuronic acid (GlcA) content of the otoconial membrane of the guinea pig by methanolysis combined with reversed-phase high-performance liquid chromatography (RP-HPLC). The utricular otoconial membrane was treated with thermolysin to separate the supernatant (OM-sup; gelatinous layer) and precipitate (OM-ppt; otoconia). The samples were then subjected to methanolysis followed by brief TFA hydrolysis, incubated with 1-phenyl-3-methyl-5-pyrazolone for labeling, and then analyzed by RP-HPLC coupled with the detection of UV absorbance at 245 nm. The GlcA contents of OM-sup and OM-ppt were 2.1 and 5.8 pmol/100 ng protein, respectively. Based on this result, GlcA-containing GAGs estimated as (GlcA+monosulfo-N-acetylhexosamine)n would be 1.0% of protein in OM-sup and 2.61% of protein in OM-ppt, indicating that this type of glycoconjugate is a minor...Continue Reading

References

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