Since the demonstration by Rona and Takahashi in 1911 (1) that a considerable portion of the calcium present in the serum is not diffusible through a semi-permeable membrane, the various calciumfractions in the blood have been extensively studied. It is now recognized that calcium exists in the serum in three distinct forms. One of these, that calcium bound to proteins, comprises the nonultrafiltrable or non-diffusible portion of the serum calcium. The other two forms, ionic calcium and calcium complexed by such small anions as citrate, phosphate, and bicarbonate, are ultrafiltrable and diffusible.Ionic calcium is generally considered to be the physiologically active component of the total serum calcium (20). Recently, however, it has been suggested (21) that the "biologically active" calcium fraction of serum is different from, and probably larger than, the total ionic calcium as determined by the frog heart technique (22), approaching the value for total ultrafiltrable calcium. In any event, a practical method for the routine measurement of actual ionic calcium has yet to be devised. Consequently, a great deal of effort has been directed toward the development of indirect methods for its determination in man. A variety of techniques has been described, but man serum using a new, simple apparatus2 which eliminates most of the disadvantages of previous methods. Using this apparatus we have investigated the various factors which influence the ultrafiltrability of calcium in human serum and have determined the normal range in healthy human subjects. A subsequent paper will describe our findings in diseased states and under experimental conditions. METHODSUltrafiltration apparatus and procedure. The apparatus used in this work has been described very briefly in a previous publication (23). It uses seamless cellophane tubing to contain the serum with a sintered glass support for the membrane and centrifugal force to supply the filtration pressure. Its principal advantages over other equipment are: 1) The atmosphere and the pH can be accurately controlled within the apparatus throughout the ultrafiltration period; 2) there is no source of metallic contamination; 3) membrane breakage is virtually eliminated; 4) the apparatus is easily constructed and can be used in an ordinary laboratory centrifuge; 5) filtration pressure can be controlled by varying centrifuge speed.The ultrafiltration procedure was carried out in the following manner:A strip of Visking Nojax Casingo (size 24/32) about 9 inches long was soaked in distilled water for 10 minutes. It was wiped dry by drawing through a folded gauze sponge repeatedly until no water was visible. One end was knotted and the tubing was doubled (Figure 1) (Corning No. 39570, 25-mm. diameter with 20-mm. disc), and adding a 6-mm. glass tube at an angle near the fritted disc (Figure 1).

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An attempt is made to correlate structure and transport function in the embryonic chorioallantoic membrane. The fine structure of the endoderm and ectoderm in the membrane was examined with particular attention given to the morphological changes that occur when transport is established,in vivo. Two distinctive cells, a granule-rich cell and a mitochondria-rich cell, appear in the endoderm at the time allantoic fluid sodium, chloride and water reabsorption commences. These are indistinguishable from the cells described in toad bladder epithelium. It is suggested that the granule-rich cell is responsible for bulk water movement and the mitochondria-rich cell is specifically engaged in active sodium transport. In the ectoderm, two distinctive cell types are also found to be associated with the onset of active calcium transport. These are referred to as the capillary-covering cell and the villus-cavity cell. The preponderate capillary-covering cell is most likely responsible for transcellular calcium transport. It is postulated that the function of the villus-cavity cell is to secrete hydrogen ions which are necessary, along with carbonic anhydrase, to mobilize Ca(++) from the insoluble calcium carbonate of the eggshell.

Calcium markedly stimulates the respiration of the isolated chick chorioallantoic membrane. This stimulation of oxygen uptake appears to be closely associated with the membrane's active transcellular calcium transport mechanism. In the presence of 1MM Ca(++) the rate of uptake increases from 9.3±0.15 to 13.0±0.2 μliters O2/cm(2)/hr, an increase of about 40%. The calcium-stimulated respiration is specific for the ectodermal layer of cells, the known location of the calcium transport mechanism, and only occurs when the calcium transport mechanism is operative. Sr(++) and Mn(++) are transported by the tissue at a lower rate than Ca(++) and cause a smaller stimulation of oxygen consumption. Mg(++) and La(3+) have no effect on tissue respiration. In the presence of Ca(++), the organic mercurialp-chloromercuribenzene sulfonate (PCMBS) inhibits calcium transport and specifically decreases the oxygen uptake of the ectoderm to a rate identical to that obtained in a calcium-free medium. Stripping the inner shell membrane away from the chorioallantoic membrane mimics these effects. The specificity and locus of action of these two inhibitors suggest that a vital component of the active transcellular calcium transport mechanism resides on or near the outer surface of the plasma membrane of the ectodermal cells and that sulfhydryl groups are important to the normal function of this component.

The chorioallantoic membrane of the developing chick embryo is an epithelium that actively transports calcium. The methodology utilized to prepare this soft tissue for calcium localization with the electron probe x-ray microanalyzer is presented in detail. The preparative procedures are evaluated according to general histochemical principles and in relationship specifically to electron probe investigations. It is shown that the method employed in these studies preserves the normal fine structure of the tissue, prevents selective loss of calcium, permits only minor losses of total calcium and appears to maintain the distribution of calcium that existed in vivo. Examples are presented of artifacts that can be induced during tissue sectioning and mounting procedures. Problems of defining electron probe resolution in biologic specimens are discussed, and the critical importance of evaluating x-ray images in association with simultaneously generated sample current images is emphasized.