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).

The total serum calcium is composed of two major fractions: the non-diffusible or protein bound calcium, and the diffusible or ultrafiltrable calcium. The latter, containing predominantly ionic calcium, theoretically traverses capillary membranes in vivo and participates in metabolic processes at the cellular level.In a previous publication, a new simple method for the ultrafiltration of human serum was described (1). It was found that in normal adults, between 60 and 70 per cent of the calcium present in serum was ultrafiltrable. These findings were compared with the variable values reported by other authors for the normal percentage of ultrafiltrable calcium. It was pointed out that virtually all previous workers had failed to control both the pH and the temperature during their ultrafiltration procedures. Our data indicated that these variables had a considerable effect on the ultrafiltrability of serum calcium, thus offering an explanation for the lack of agreement, particularly among the early workers (2), in the study of normal and abnormal sera. Only the recent data of Hopkins, Connor, and Howard (3) show reasonable consistency for specific disease states. They used the Lavietes mercury-pressure ultrafiltration apparatus, controlled pH, and carried out the ultrafiltration procedure at room temperature.Utilizing the controlled technique previously described (1) METHODSPatients on the general medical wards of the Strong Memorial Hospital with abnormal total serum calcium values were selected for the study of the distribution of serum calcium in disease states. A definite diagnosis was established clinically in all cases. Blood was collected by venipuncture in the non-fasting state and serum ultrafiltered the same day. Another patient, who was being studied on the metabolism ward of the Department of Medicine, served as the subject for the detailed investigation of the effect of experimentally induced conditions on the ultrafiltrability of serum calcium. He was a 75 year old white man with chronic lymphatic leukemia and an annular carcinoma of the rectosigmoid. In spite of the two malignant processes, he appeared well nourished (body weight, 63 Kg.) and was generally asymptomatic but did have slight hypoproteinemia. No evidence of bone involvement with cancer was apparent clinically, by X-ray or balance study. 2 The higher values for ultrafiltrable calcium in the studies of Hopkins, Connor, and Howard (3) probably resulted because these authors carried out their ultrafiltration procedures at room temperature instead of 370 C. 87

Plant monoterpenes have no HDL-elevating activity of potential value for coronary artery disease prevention.

A routine procedure for the determination of calcium in the serum from abnormal and normal human patients has been devised. The calcium, separated as the oxalate from a deproteinized solution, is measured in a flame photometer. The flame emission is