Abstract
The authors studied a total of 392 cryofiltration (CF) treatments on twenty-four patients with rheumatoid arthritis (RA) and analyzed removal substances (cryogel) by three different methods: high performance liquid chromatography (HPLC), ouchterlony test and dynamic light scattering method (DLS).
(1) Analysis by HPLC. Chromatographic pattern of cryogel was divided into six peaks, designated as A, B, C, D, E, and F and A peak was divided into three peaks marked A1, A2 and A3. Reproductibility was extremely good. Main components in cryogel were A3 peak. The molecular weights of these three substances were 1, 300, 000, 1, 000, 000 and 640, 000, respectively. Moreover, we analyzed cryogel obtained from first, fifth and tenth CF of the same patients and cryogel obtained from different four patients. A chromatographic pattern of each cryogel did not change. This findings suggest that components in cryogel do not change during CF therapy and between the patients. Quantitative change of cryogel is an important factor for RA therapy. Moreover, to examine the relation between cryogel and immmunoglobulins (IgG and IgM), we analyzed both solution by HPLC. The peak of IgG appeared at almost the same position as components B, C and D. However, the peak of IgM was recognized between A2 and A3.
(2) Analysis by ouchterlony test. To examine the relation between cryogel and immunoglobulins, ouchterlony test was performed. For ouchterlony test, anti-IgG, A, M, D, and E antibody were used. Immuno-precipitative reaction against cryogel were observed with anti-IgG and IgA, but not anti-IgM, D and E. Moreover, we examined the relation between main components in cryogel and immunoglobulins. Main components in cryogel were collected using fraction collector of HPLC. Immuno-reaction did not occur with anti-IgG and IgA. The results obtained by HPLC and ouchterlony test suggest that main components in cryogel are not immunoglobulins, especially native IgM.
(3) Analysis by DLS. We separated A3 peak considering main components in cryogel by fraction collector and analyzed these substances by DLS. The results suggest that the distribution of particle size is dependent on temperature. The distribution of particle size at 37°C was 60-80nm. However, as temperature decreased, the distribution of particle size increased. And temperature decreased at 4°C, it became unmeasurable. Moeeover, when the solution was warmed back to 37°C again, the distribution of particle size decreased reversibly, but not completely. These findings suggest that the cryoprecipitate phenomenon is certified in the level of biochemistry and A3 peak is chief components in cryogel quantitatively and qualitatively.
