COLLAGEN EVALUATION 329 Radiometer PHM240 instrument (Copenhagen, Denmark). Conductivity was deter­ mined using a Radiometer CDM3 instrument. Water regain. Collagen samples were dried by lyophilization in preweighed bottles with stoppers and then held over silica gel for more then 72 h. Samples were then stoppered and reweighed. They were then transferred and opened in an environment of 32% constant relative humidity, maintained over saturated CaC12 solution. After equilibra­ tion for more than 48 h, the samples were stoppered and reweighed. Water regain by the dried samples was determined, and expressed as a percentage of the dry weight. Spectroscopy. For UV/visible spectroscopy, spectra were collected from 400 nm to 650 nm on a Shimadzu UV-265 recording spectrometer using cells of 10-mm path length on collagen solutions as supplied or after dilution to equal concentrations of 1 mg/ml with milliQ water. For IR spectroscopy, collagen samples were lyophilized and then examined using a Perkin Elmer 2000 Autolmage spectrometer with the ATR accessary. Alterna­ tively, samples may be analyzed as KBr disks. Electrophoresis. Collagen samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12), using 5% (w/v) running gels. Prior to analysis, collagen samples in sample buffer were neutralized, if necessary, with 2 M Tris and then heated at 100°C for 2 min. Separation of the al(I) and al(III) chains was by reduction with 2-mercaptoethanol during interrupted electrophoresis (13 ). Collagen samples were also examined by a non-denaturing, lactic acid buffer system, pH 3 .1 (14), using 4% running gels. Estimation of the isoelectric point, pl, was by examination of the direction of electrophoretic mobility in gel electrophoresis in 5% running gels in a Tris/Borate pH 8.9 system (15). Amino acid analysis. Lyophilized collagen samples were hydrolyzed in vacuo by constant boiling (5.8 M) of HCl containing 0.1 % phenol in a Waters PicoTag system at 108°C for 20 h. After drying in vacuoJ hydrolysates were examined on a Waters HPLC system with ninhydrin detection. Differential scanning calorimetry. Collagen melting temperatures (T m ) were determined by DSC using a Mettler Toledo DSC 3000 instrument (Mettler, Schwerzenbach, Switzer­ land). Collagen samples were prepared at about 5 mg/ml, and equilibrated in 50 mM acetic acid, pH 2.8, by dialysis. Samples, about 100 mg of solution, were examined at a temperature increase of 1 ° C/min. Collagen T m values are given as the temperature at the mid-point of the thermal transition. Scanning electron microscopy. Collagen samples as provided, or after dilution to equal concentrations of 3 mg/ml with milliQ water, were coated on clean glass coverslips and air dried at room temperature in the clean air flow of a laminar flow hood. Dried samples were then gold coated and examined in a Philips XL-30 microscope. RESULTS AND DISCUSSION GENERAL SOLUTION PROPER TIES There are a wide variety of solution measurements that can be readily made on collagen preparations (Table I). These include analysis for toxic metal components such as arsenic and lead, whose presence would make a preparation unsuitable for cosmetic application.
330 JOURNAL OF COSMETIC SCIENCE Table I Analysis Results on Three Samples of Collagen Suitable for Cosmetic Applications Test AteloHelogen ® CLR Collagen® Collasol® Protein content (%w/v) 1.05% 0.28% 4.00% pH 4.7 3.8 4.2 pl 8.9 8.9 8.9 Arsenic 1 ppm 1 ppm 1 ppm Heavy metals 5 ppm 5 ppm 5 ppm Dry weight 1.15% 5.07% 5.25% Conductivity 0.45 ms 19.0 ms 42.0 ms Ash content 0.1% 1.2% 1.1% Hydration regain 21% 3% 7% In the present study, the levels of these components were below the detection limit for all three samples. An important feature of a collagen product is its collagen protein concentration. For fully soluble and essentially pure collagen preparations with concentrations above about 2-3 mg/ml (or solutions after clarification by centrifugation), a Biuret assay (16) against appropriate collagen standards is simple and rapid. However, non-collagenous impuri­ ties could interfere with this determination, leading to higher values. Total nitrogen content can also be readily determined by Kjeldahl analysis. This value allows the collagen concentration of the sample to be estimated, using a conversion factor based on the known structure of collagens (17). This factor is dependent on the collagen type and the species of origin. Again, this method can give erroneous values for collagen content if any non-collagenous proteins are present. Hydroxyproline is found as an amino acid that is characteristic of collagenous proteins, and therefore provides a specific method for determination of collagen content, separate from any other proteins that may be present. Hydroxyproline content and the total amino acid composition can be determined by amino acid analysis (see below). Hydroxyproline content can also be determined by a specific colorimetric assay (18). The use of dry weight is not suitable to determine collagen content, as some preparations contain a significant salt content, as can be seen from the ash content of samples (Table I). The conductivity of a sample can confirm the presence of salts (Table I). For example, AteloHelogen® shows a negligible ash content, and has a very low conductivity, whereas both CLR Collagen® and Collasol® both have a significant ash content and significant conductivities, although these measures are not necessarily linked, as they depend on the nature of the salts present (Table I). The pH values for the preparations examined in this study varied (Table I). These values may be important in developing formulations. The presence of salts may affect the pH, as they may be buffering the collagen solution (Table I). Samples of collagen that contain oligomers are more soluble under low pH conditions-at higher pH the collagen oligo­ mers become less soluble and may precipitate (10). On the other hand, at a higher pH, such as neutral pH, a minimum salt content of around 0.15 M NaCl is needed to ensure collagen solubility (10). Thus, the higher pH of the AteloHelogen® collagen with a very low salt content (Table I) indicates a highly monomeric collagen preparation. WATER REGAIN The intrinsic water-binding property of collagen is critical to its excellent performance
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