SUMMARY

Gel filtration *.echniques ofdetermining serum unbound calcium and magnesium have been studied. Dry and pre-swelled Sephadex (;-50 was used in procedures modelled on reported methods. Data are presented to support the conclusion that gel filtration, as a batch technique, remains unproved for the satisfactory quantitative estimation of unbound calcium and magnesium in serum. Serum calcium exists in three forms: pr,,tein-bound, free ionic, and complexed in a small molecule t. Ionic and protein-bound forms interact o produce an equilibrium influenced by pH, protein concentration, and to a lesser degree by temperature and other factors t. Free ions and complexes are identified as 'unbound, diffusible, ultra-filterable ' species. Serum magnesium is believed to have similar forms and to exhibit similar behavior. Because classic quantitative methods t-7 for serum unbound calcium and magnesium set high technical denmnds, gel filtration has been proposed s- ~ as a simple, rapid procedure. We have determined serum unbound calcium by both ultrafiltrati~m and ultracentrifugation; each method has been found reproducible and values agreed well between methods. Using gel filtration methods 8,~0, however, we have found poor reproducibility and have obtained values which did not parallel those from other methods. We therefore undertook a study of factors involved in the gel filtration techniques of SchatzS, 9 and von Hattingberg and Klaus ~°. MATERIAL AND METHODS Specimens A serum pool was used for replication experiments. Individual specimens were drawn from healthy, non-fasting staff members into Vacutainer * tubes, allowed t