Determination of lonized calcium in serum with a calcium electrode

f%IOCHEMICAL

MEDICINE

3,

?&+-?a75

Determination

of with

( 1970)

Ionized

a Calcium

Calcium

in Serum

Electrode

A. RAMAN Department

of Physiology, Faculty of Medicine, Kuula Lumpur, Malaysia Received

October

University

of Malaya,

13, 1969

Calcium exists in serum in three forms-protein-bound, complexed, and ionized; of these, only the ionized form seems to play a part in the normal physiological processes such as synaptic transmission (1) and coagulation of blood (2). Determination of the ionized calcium by the usual methods, such as ultrafiltration (3) and ultracentrifugation (4), do not actually measure the ionized fraction alone but also include the complexed form. In addition, these methods are time-consuming and require rigid precautions such as maintenance of a constant temperature, pH, and pC0,. It is mainly for these reasons that ionic calcium is not determined as a routine procedure, although this is the fraction which is regulated by the parathyroid hormone (5). It has been shown by Lloyd and Rose (6) that measurement of serum ionic calcium is a very important aid in the diagnosis of primary hyperparathyroidism as, in most cases, it is raised when the total calcium may be normal. Within the last few years, potentiometric methods using calcium-sensitive electrodes have been developed to measure the ionized form of calcium in serum and plasma (7, 8). However, in these methods, an older model of the calcium electrode was used, and this particular electrode, according to the manufacturers, is not ideal for the measurement of ionic calcium in biological fluids containing proteins. This report describes the estimation of ionic calcium in serum utilising a newer model of the calcium electrode. MATERIALS

AND

METHODS

The calcium-selective electrode employed in this study was that manufactured by Orion Research Inc., Cambridge, Massachusetts (Calcium activity flow-thru system, Model 99-20). The system consists of a calcium flow-through electrode, a flow-through reference electrode, and an infusion pump to deliver the serum directly from a syringe through the 369

370

A. RAMAN

calcium flow-through reference electrodes. The electrode potentials developed were read on the expanded millivolt scale of an Orion Specific Ion Meter ( Model 401). The principle underlying the functioning of the calcium electrode is similar to that of the conventional pH electrode. The electrode contains a liquid ion exchanger (a calcium salt of an organophosphoric acid) with a high specificity for ionized calcium, and a solution of calcium chloride. The calcium chloride is separated from the liquid ion exchanger by a porous disc. A stable potential is thus created between the calcium chloride solution and the inner aspect of the porous disc. When a solution containing ionized calcium comes in contact with the outer aspect of the disc, a potential difference is created as the ionized calcium moves toward the liquid ion exchanger-impregnated disc and this is proportional to the amount of ionic calcium present. If the electrode is first calibrated with solutions containing known concentrations of calcium chloride, it is then possible to determine the ionic calcium content of an unknown SOlution from the potential generated and reading off from a calibration curve. Preparation

of Serum

Venous blood was collected in plastic syringes from 17 healthy male volunteers, and the blood was immediately introduced into centrifuge tubes containing 1 ml of liquid paraffin. This enables one to maintain anaerobic conditions. After incubation for 1 hour at 37”, the clotted blood was centrifuged, and the serum separated and filtered to remove traces of oil. The serum was then divided into three aliquots for pH determination, ionic calcium measurement and total calcium estimation. The pH and ionic calcium were measured within 30 minutes after centrifugation while the total calcium was estimated at a later time. The pH of the serum prepared this way was read on a Radiometer pH meter (Model 27) and was found to be 7.41 t 0.02 ( &SD). Ionic Calcium Measurements The calcium electrode was first calibrated with standard calcium chloride solutions made LIP in 150 mM/liter of sodium chloride (the solutions contain 0.5, 1, and 2 mM/liter of calcium chloride and are supplied with the electrodes). The electrode potential developed with each solution was then observed, and a calibration graph of the potential generated against the calcium chloride concentration was drawn on a semilogarithmic paper. The serum sample was then passed through the electrodes and from the observed potential developed, the ionic calcium was determined using the calibration curve. Duplicate readings were made on each sample and it was found that the voltage drift between

IONIC

CALCIUM

IN

371

SERUM

identical samples was less than 0.5 mV showing good reproducibility. In order to ascertain whether storage affected the ionic calcium levels, the serum samples were read at different intervals after collection of the serum, and it was seen that the levels of ionic calcium diminished with time. After every two samples, the electrodes were recalibrated with standard solutions. Total Calcillm

Estimation

Total calcium was determined by a photometric method using glyoxal bis (2-hydroxanil) solution (GBHA) according to the method of Mager and Farese (9). The standard deviation of 85 duplicate determinations by this method in this laboratory is kO.24 mg/lOO ml. RESULTS

A typical plot is shown in Fig. Table 1. One of 100 ml) and this Reproducibility

of the electrode potential vs. log calcium concentration 1, and the results obtained from the subjects is given in the subjects had a low total calcium value (7.73 mg/ might have been due to slight haemolysis of the blood.

and Recovery

Studies

It was found necessary to calibrate the electrode with standards solutions between samples as there was noticeable electrode drift, probably due to fluctuations in room temperature, 26” f 2“ ( *SD), Further, it

5x10-4

lrlo-3 C&I>

2x10-3

CONCENTRATION Ihl ,Lk\ ILITHF,

FIG.

1

372

A. RAMAN

IONIC

Subject K(I.1;. H.T.S. .J.K. Y.T.C. -4.12. S.R. S.S. I..A.K. Y.P.H. F.E.L. I..N.F. I,.K.(‘. S.K.H. Eli. ?‘.w.c:. Ii.S.(‘. S.K. iilexn $1)

AND

TOTaL

Tot al calcium (mg/lOO ml! s.45 9.55 10.75 7.73 s.41 10 51) 10. ?!I 0.50 9.25 K.IS 10 2.5 9 06 IO.25 11.76 11.1s !, <5’:I 9 05 0 6‘3.

,I

‘I Bound calcium :t~lrdlp meaxru-ed.

11 refers

C.lLCIUM

1X

(:LINICALLY

Icnic calcium (mg/lOO ml1 4 50 4.25 4.25 4.10 4.25 5 80 5.50 4.25 4.50 4.25 4.‘“5-.. 5.00 4.25 6.30 5.50 4.85 4.50 4 i” +o.c1 to the

difference

NORMBL

hDIVlDK4LS

Bound cnlcinm” img/lOO ml) 4-7 55 GO 4i 49 45 47 S6 51 4x 59 -It5 5!) 46 51 49 50 50.76 k5.02

:; 95 5 :
6.50 3 A3 4. I6 4.T!) 4.x 5 25 4.;5 :3. 0:; G 00 4.05 6, 00 5.46 4.68 4.w 4.55 4 91 +0.x:; between

ionic

and

t,ot;tl

calcium

and

not.

was also noted that readings were not steady if the infusion rate was too rapid. In the present study, a l-ml syringe was used at an infusion rate of 1 ml/17 minutes Recovery studies were carried out by adding known quantities of calcium chloride solution ( 10 mg/lOO ml made up in 0.15 M NaCI) to pooled human serum and determining the ionic calcium with the electrode. From the work of Peterson et al. ( lo), it could be assumed that addition of Ca2+ to serum without marked changes in pH and total protein concentration would increase the serum ionized calcium mole for mole provided the amount of added Ca’+ is within the physiological range. Table 2 shows the results of these studies showing good recovery with simultaneously increasing percentage of serum and ionic calcium concentration. DISCUSSION

The values obtained in this study for the ionic fraction of serum calcium varied from 41 to 55% with a mean value of 49.2% (4.72 mg/ 100 ml). These results compare favourably with those obtained by Briscoe and

IONIC

RECOVERY

Sample no. 1 2 3 4 ‘( Assay

CALCIUM

OF IONIC

Serrlm (‘;C total) 100 80 66.67 50

20 33.33 50

-

valrle

373

SERUM

TABLE: 2 CALCIUM IN HT:M.IN

10 mg/lOO ml calcium chlcride added (c;‘, total)

valrke/calculated

IN

ASa> vallle (mg/lOO

ml)

4.15 4.96 5.50 6.30

SERUN

CMculated vallle (mg/lW ml)

Per cenl recovery’1

-

-

5.32 6.10 7.0s

93.2 90.2 90.0

x 100 = per cent, recovery.

Ragan (11) 49.6%, Robertson and Peacock (12) 50.8%, and Oreskes et al. (8) 46.4%. Many techniques utilising ultracentrifugation, ion-exchange chromatography ( 13), and spectrophotometry ( 14) have been described to determine the ionic calcium in serum. The results of these studies have been reviewed recently (12, 15) and show that the values for ionic calcium obtained by these techniques range from about 40%to 70%of the total calcium. Such a wide range is due to such factors involved in the maintenance of ionic calcium as pH, ionic strength, pC09, and temperature. The question arises as to the extent the above-mentioned factors affect the ionic calcium estimation with the calcium-sensitive electrode. From an exhaustive study using the calcium electrode, Moore ( 16) has shown that the electrode has appreciable selectivity for calcium ions; the selectivity for Ca 2+over Na+ is approximately 5000/l (on a mole basis in mixed CaCI,-NaCl solutions) and the preference for Ca?+ over Mg*+ is about 40/l. With regard to other ions, such as Cu2+ and ST”+,the selectivity of the electrode for calcium is greater. In the case of Zn”+, the electrode has a higher selectivity for Zn2+ over Ca”+ but, in the plasma zinc concentrations normally found, 0.1 mg/lOO ml, this is negligible. However, if abnormalities in zinc metabolism are suspected, the electrode might not give a true value for ionic calcium. Changes in pH affect the ionic calcium levels as well. Thus, a decrease in the pH from 7.8 to 6.8 showed an increase of 0.44 & 0.10 mM/liter in the ionic calcium level (16). Ionized calcium activities and thus the electrode potentials also depend on the total ionic strength of the sample and follows the Nernst equation, E = Ex + 2.3 RT/SF log (A Ca2+), where E is the electrode potential, Ex the millivoltage from the reference electrode, 2.3 RT/2F the Nernst potential factor for a divalent electrode, and A is the ionic activitv.

374

A.

RAMAN

The calcium flow-through electrode has been devised in such a way as to overcome these problems. Since the electrode is calibrated with solutions having the same ionic strength as that of serum, changes in ionic strength do not come into the estimation. Also, due to the fact that the serum is not exposed to the air for any considerable time, changes in pH and pC0, are minimal, and it is possible to get a truer picture of the ionic calcium level as it exists in the body. This is a very big advantage over the older model of the calcium electrode, which is a dipping electrode, and the serum has to be exposed to the atmosphere with unavoidable changes in pH and pC0,. However, in order to obtain reproducible results, estimations must be carried out with fresh serum since changes in pH and pC0, take place with aging of the serum. As the calibrations and actual measurements are carried out at room temperature, changes in temperature are also not important. Addition of heparin, citrates, or oxalates to prevent the blood from clotting lowers the level of free calcium ions so that the amount of ionic calcium in plasma compared to serum is less. It is, therefore, advisable to employ serum in the determination of ionic calcium levels. Although the electrode is specially constructed for serum ionic calcium measurements, it can also be used for determining the ionic calcium in other biologic fluids such as urine ( 17), cerebrospinal fluid ( 18)) and gastric juice ( 19). However, in measuring ionic calcium in fluids other than serum and plasma, the standardising solutions should be prepared so as to have a calcium concentration in the expected range of the unknowns and an ionic strength similar to the unknowns. Until now, determinations of ionized calcium in serum in various diseases have been few owing to the lack of simple and reliable methods. Arras (20) has mentioned a variety of diseases in which determinations of ionic calcium levels might be useful. The development of the calcium electrode has provided a most practicable and reproducible way for measuring this parameter. The technique is simple, the amount of serum required is very small, and as many as 10-15 estimations can be carried out within an hour. It is hoped that routine ionic calcium measurements in the various pathological conditions mentioned would clarify our understanding of the disorders of calcium metabolism. SUMMARY

Using a calcium-sensitive electrode, the determination of ionic calcium in human serum was investigated. Serum samples obtained from 17 healthy individuals yielded the following data: total calcium 9.63 f 1.11 mg/lOO ml; ionized calcium 4.72 t 0.61 mg/lOO ml; and bound cal-

IONIC

CALCIUhf

IN

SERUM

375

cium, 4.91 t 0.83 mg/lOO ml. The proportion of ionized and bound calcium as a percentage of total calcium was 49.24 t 5.08% and 50.76 -t 5.02%, respectively. It was concluded that the electrode method of measuring ionized calcium is probably the simplest and most practicable, ACKNOWLEDGMENTS My thanks are due to Prof. G. H. Bell of the University an d encouragement, and to the Department of Medical Malaya, for photographic work.

of Dundce Illustrations,

for his advice University of

REFERENCES 1. 2. 3. -1. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

KURII.A~IA, II., J. Physiol. 175, 211 (1964). LOVELOCK, J. E., AND PORTERFIELD, B. M., Biochem. .I. 50, 415 (1952). ROSE, G. A., Clin. Chim. Acta 2, 227 ( 1957). LOKEN, H. F., HAVEL, R. J., GORDANAND, G. S., AEZU \VHIITINGHAAI, S. L., J. Biol. Chem.235,3654 (1960). NORDIN, B. E. C., AND SMITH, D. A., in “Diagnostic Procedures in Disorders ot Calcium Metabolism,” p. 12. Churchill, London ( 1965 ). LLOYD, II. M., AND ROSE, G. A., Lancet 2, 1258 ( 1958). ARNOLD, D. E., STANSELL, M. J., AND MALVIN, H. H., Amer. J. CZin. PatZwZ. 49, 627 ( 1968 ) . ORESKES, I., HIHSH, C., DOUGLAS, K. S., AKD KUPFER, S., Clin. Chim. Act0 21, 303 (1968). MAGF:R, M., AND FAHESE, G., Clin. Chem. 12, 234 (1966). PETERSON, N. A,, FIEGEN, G. A., AND CRISMON, J. hl., Arncr. J. Ph!ysiol. 201, :I86 (1961). BHISCOE, A. M., AND RAGAN, C., J. Lab. C/in. Med. 69, 351 ( 1967). ROBEWSOK, W. G., AND PEACOCK, M., CZin. Chim. Acta 20, 313 (1968). SCHIRARDIN, H., ASD METAIS, P. A., Ann. Bid. Clin. 17, 465 (1959). LUXXR, G. A., CZin. Chim. Actu 8, 33 (1963). P~UPE, J., AI\D SAINT-MARTIN, J., Pres e &fedic& 75, 2153 (1967). hIooHE, E. W., Ann. N. Y. Acad. Sci. 148, 93 ( 1968). ROBERTSON. W. G., Ch. Chim. Acta 24, 149 (1969). MOORE, E. W., AND BLUM, A. L., J. C/in. Intzst. 47, 70a (1968). SIOORE, E. CV., ASD MAKHLOUF, G. hf., Gastroenterdogy 55, 46.5 (1968). ARHAS, hl. J., PO tgrad. Med. 45, 57 (1969).