Hyaluronan molecular weight and polydispersity in some commercial intra-articular injectable preparations and in synovial fluid
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- Mendichi, R. & Šoltés, L. Inflamm. res. (2002) 51: 115. doi:10.1007/PL00012388
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, (2001) vol 50, pp 294-299 entitled "Hyaluronan molecular weight and polydispersity in some commercial intra-articular injectable preparations and in synovial fluid" . In the introduction, the authors stated that "well-defined hyaluronan molecular weight standards are not commercially available". In Table 2 the molecular parameters for seven commercially available hyaluronan (HA) samples, determined by the authors on using a multi-angle laser-light scattering (MALLS) detector coupled to a gel-permeation chromatographic (GPC) system, are presented. By inspecting, for example, the HA sample polydispersities, the reader might assume that minimally four of the samples characterized are with regard to their Mw/Mn polydispersity values, 1.04, 1.05, 1.06, 1.08, classifiable as really very narrow - GPC standards/calibrants. Moreover, also further two HA samples would fully meet the criteria for GPC calibrants as to their Mw/Mn values, 1.15 and 1.29. Regarding their Mw parameters, these six HA samples - "GPC calibrants" - would cover the range of 2.34 × 105 - 3.49 × 106 Da. However, on assuming that the Mw and Mn values established by GPC-MALLS are correct, it might have been desirable to (re)process the chromatographic records of the HA samples detected by using a UV photometer (441 UV absorbance detector), which they used for detecting on-line the sample concentration. (Since the UV light absorbance maximum of the HA samples is at ≈ 206 nm, the setting of 214 nm though not optimal may be satisfactory.) On processing the UV photometric records with calibration dependence of the GPC apparatus [logarithm of Mpeak vs. elution volume? where the Mpeak could be approximated by (Mw× Mn)1/2], another set of the output data could have been obtained. By comparing the molecular parameters for the seven commercially available HA samples established by the two qualitatively different approaches, the authors could either accept or refuse the validity of the molecular parameters of the seven HA samples. The finding of marked differences, or on the contrary, of a too close correspondence of the values Mwi and Mni (GPC-MALLS) with those of Mwj and Mnj (GPC-UV), would have indicated whether the Superose 6 column used does or does not provide proper separation performance for the high molecular weight HA samples investigated. We would like to comment on the above mentioned problem concerning the GPC analysis validity. The biological source of all seven HA samples investigated was obtained from rooster comb or by bacterial fermentation. It can be practically excluded that the polysaccharide, produced in vivo by any of the above biological systems, might be molecularly homogenous, monodisperse. At present, on producing the (commercially available) HA samples, a whole range of physico-chemical procedures has to be applied, such as protein enzymatic digestion, HA ion-pair precipitation, membrane/molecular ultrafiltration, HA non-solvent precipitation and/or lyophilization. Of these, at least the last mentioned procedure results in degradation of high molecular weight HA samples , and in the case of monodisperse polymers, this degradation is in turn invariably associated with an increase in their Mw/Mn parameters. Thus, we suppose that the true Mw/Mn values of the HA samples must be (markedly) higher than the reported ones, i.e. 1.04, 1.05, 1.06, 1.08 . Along with a potentially improper separation performance of the Superose 6 column, another possible source of discrepancy might be the exploitation of the MALLS detector for the (on-line) estimation of the HA biopolymer Mn molecular weight values. There are namely several indications that the values of the Mw/Mn polydispersity parameters, determined by the GPC-MALLS system are frequently underestimated [3, 4]. The M Mw/Mn values generated by exploiting the GPC-MALLS arrangement would thus require a more critical evaluation.