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THE study of the diffusibility of blood serum-calcium has been an interesting development in the investigation of calcium metabolism. The finding of Rona and Takahashi [1911], that only a part of the serum-calcium was diffusible through membranes impermeable to protein, has been corroborated by many observers and by various methods. The figures for diffusibility recorded in the literature cover a wide range, but most of them lie between 45 and 70 % of the total serum-calcium. It is generally assumed that the non-diffusible fraction is bound to protein, this being suggested by the removal of a certain fraction of calcium in serum-protein precipitation [Csapo and Faubl, 1924], by the parallelism between the non-diffusible calcium and protein contents of various body fluids [Salvesen and Linder, 1923-24; Liu, 1927], by an apparent binding of a fraction of the calcium when CaCl2 is added to protein solutions, as shown by diffusion experiments [Loeb, 1928; Marrack and Thacker, 1926], and by conductivity titrations when calcium is added to serum solutions [Shear and Offner, 1931]. Furthermore, when the total serum-calcium concentration is increased the non-diffusible fraction also increases [Loeb and Nichols, 1927; Brull, 1930; Smith and Sternberger, 1932; and Benjamin and Hess, 1933]. It is natural to assume that at least a part of this process is due to a further combination of calcium with serum-protein to preserve the equilibrium between protein-bound calcium and calcium ions. In contrast to the state of serum-calcium, the inorganic phosphate of mammalian serum is generally agreed to be entirely diffusible or almost so [Rona and Takahashi, 1913; Cushny, 1919-20; Neuhausen and Pincus, 1923; Grollman, 1927; Brull, 1930; Greene and Power, 1931; Benjamin and Hess, 1933]. However, the interesting observation was made by Grollman [1927] by ultrafiltration of pig and dog sera that inorganic phosphate gradually becomes non-diffusible as the serum-calcium is raised above the normal level. He suggested the formation of a colloidal complex of Ca3(PO4)2 and protein. This finding has since been corroborated by other observers [Brull, 1930; Benjamin and Hess, 1933; Laskowski, 1933]. Thus when calcium is added to serum a part of the added calcium becomes non-diffusible. As Laskowski [1933] has stated, this may be accounted for by two different processes-firstly by a combination of calcium with protein (as occurs in phosphate-free protein solutions [Loeb, 1928]), and secondly by the formation of a calcium phosphate complex with protein (as suggested by the formation of non-diffusible phosphate along with the increase in non-diffusible

Non-diffusible serum-calcium at various calcium, phosphate and hydrogen ion concentrations