Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis.
There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.
- MeSH
- cesty eliminace léčiva MeSH
- chrupavka metabolismus MeSH
- cyklická ADP-ribosa metabolismus MeSH
- intravenózní podání MeSH
- izotopové značení metody MeSH
- izotopy uhlíku chemie metabolismus farmakokinetika MeSH
- kosti a kostní tkáň metabolismus MeSH
- kyselina hyaluronová chemie metabolismus farmakokinetika MeSH
- molekulová hmotnost MeSH
- myši inbrední BALB C MeSH
- tkáňová distribuce MeSH
- uridindifosfát-N-acetylglukosamin metabolismus MeSH
- uridindifosfátglukosa metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this paper is to review chromatographic and mass-spectrometric methods and underline the best analytical approaches for successful analysis of various hyaluronic acid species in different types of samples. Hyaluronan-degrading enzymes and chemical depolymerization produce di- or oligosaccharides suitable for hyaluronan quantification or structural characterization of hyaluronan derivatives. Efficient purification and pre-column derivatization of hyaluronan disaccharides by reductive amination allow subnanogram quantification in biological samples. The chromatographic separation is capable to distinguish all glycosaminoglycans disaccharides and to resolve hyaluronan fragments with 2-40 monomers. Using electrospray ionization or matrix assisted laser desorption ionization, hyaluronan fragments up to 8 kDa or 41 kDa, respectively, can be observed. One- or two-dimensional chromatographic separation with higly sensitive mass-spectrometric detection is an indispensable tool for revealing substituent position, extent of modification and substitution patterns of chemically modified hyaluronan derivatives. It is essential for studying structure-biological function relationships of hyaluronan and its derivatives.
