Comparison of CPC and CETAB extractive procedures for quantification and characterization of human urinary glycosaminoglycans

Background. Glycosaminoglycans (GAGs) are natural, complex polysaccharides having great importance in several pathological processes. Urinary GAGs have long been investigated for their possible modifications in many pathological conditions and, in some cases, results useful for diagnosis have been observed. As a result, the determination of GAGs in the urine is gradually gaining importance in the literature. Cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CETAB) are generally used to extract urinary GAGs before analyses. In this study we evaluated the extraction of human urinary GAGs by using CPC in comparison with CETAB. Methods. Extracted urinary GAGs were qualitatively and quantitatively analysed by agarose-gel electrophoresis in the presence of sequential staining and densitometric scanning, able to give more reproducible and reliable results for urinary GAGs, and HPLC for the evaluation of chondroitin sulfate (CS) disaccharides.Results. Differences were observed between CPC and CETAB extractive protocols. The absolute amount of CS evaluated by electrophoresis was found 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 are the quantitative expression of a greater recovery of HS by using CPC protocol than CETAB. No significant differences were found between CS quantified by agarose-gel and HPLC. Furthermore, no differences were observed for the CS disaccharide composition purified by using CPC or CETAB, and quite similar results were observed for 4s/6s disaccharides ratio and charge density values.Conclusions. Extractive procedures of urinary GAGs using CPC or CETAB are able to recover same amounts of CS quantified by agarose-gel electrophoresis and HPLC, and same CS structural composition determined as pattern of disaccharides. However, CPC produces a great recovery of HS than CETAB protocol, an increase of approximately 3.3 times as evaluated by electrophoresis. This different capacity in HS extraction between CPC and CETAB should be carefully kept in mind when urinary GAGs of subjects affected by various diseases and related pharmacological treatments are considered or meta-analysis is determined by comparing various studies and trials performed under different experimental conditions.