Comparison of cetylpyridinium chloride and cetyltrimethylammonium bromide extractive procedures for quantification and characterization of human urinary glycosaminoglycans

Abstract

Background: Glycosaminoglycans (GAGs) are natural complex polysaccharides that are important in several pathological processes. Urinary GAGs have long been investigated for their possible modifications in many pathological conditions. In some cases, they have been found to have diagnostic utility. As a result, the measurement of GAGs in urine is gradually gaining importance. Cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CETAB) are generally used to extract urinary GAGs prior to analysis. In this study, we evaluated the extraction of human urinary GAGs using CPC in comparison with CETAB.

Methods: Extracted urinary GAGs were qualitatively and quantitatively analyzed by agarose-gel electrophoresis in the presence of sequential staining and densitometric scanning. This procedure was able to give more reproducible and reliable results for urinary GAGs, and high performance liquid chromatography (HPLC) was used for the evaluation of chondroitin sulfate (CS) disaccharides.

Results: Differences were observed between CPC and CETAB extract protocols. The absolute amount of CS evaluated by electrophoresis was found to be similar for the two protocols. However, the heparan sulfate (HS) concentration was calculated to be approximately 3.3 times greater for CPC than CETAB. When calculated in relative percentage, 33.6% HS was determined for CPC and 10.0% for CETAB. These results show a quantitative expression for greater recovery of HS by using CPC protocol than CETAB. No significant differences were found between CS quantified by agarose-gel and HPLC. In addition, no differences were observed for the CS disaccharide composition purified by using CPC or CETAB, and quite similar results were observed for 4s/6s disaccharide ratios and charge density values.

Conclusions: Extract procedures for urinary GAGs using CPC or CETAB are able to recover similar amounts of CS quantified by agarose-gel electrophoresis and HPLC. However, CPC yields greater recovery of HS than the CETAB protocol; an increase of approximately 3.3 times as evaluated by electrophoresis. This different capacity of HS extraction between CPC and CETAB should be considered when urinary GAGs of subjects affected by various diseases and related pharmacological treatments are considered, or meta-analysis is performed comparing various studies and trials performed under different experimental conditions.