the determination of metals in blood serum by atomic ... determination of metals in blood serum by...

Spectrochimica Aeta, 1960, Vol. 16, pp. 259 to 272. Pergamon Press Ltd. Printed in Northern Ireland

The determination of metals in blood serum by atomic absorption spectroscopy-I

Calcium

J. B. WILLIS Division of Chemica.1 Physics, CSIRO, Chemical Research Laboratories

Melbourne, Australia

(Received 7 November 1959)

Abstm&-The celcium content of blood mrum cm be accurately determined on 0.1-0.25 ml samples by atomic absorption measurements in an air-acetylene flame after removal of the proteins by coagulation and centrifuging. A fairly accurate value can be obtained more quickly from direct measurements on serum diluted with water, which may with advantage contain about 1 per cent of the disodium salt of ethylenediaminetetracetic acid.

Serum calcium values obtained by atomic absorption spectroscopy are signifkantly higher than those obtained by the standard oxalate-permanganate procedure and support the conclusions of MACINTYRE [3] on the shortcomings of the chemical method.

IIltroduction

THE existing methods for the clinically important determination of calcium in blood serum are all to some extent unsatisfactory. The classical method is that of KRAMER and TISDALL [l] as modified by CLARK and COLLIP [2], and involves precipitation of the calcium as oxalate followed by repeated centrifuging, draining and washing; a solution of the precipitate in sulphuric acid is then titrated with 0.01 N permanganate solution. This method is tedious, the reproducibility of the results depends on the exact way in which the operations are performed, and owing to the appreciable solubility of calcium oxalate in the wash liquid the results tend to be too low [3]. Methods involving titration with ethylenediaminetetracetic acid or with murexide yield results which are lower than those obtained by the oxalate-permanganate method [4, 51.

Most of the flame photometric methods require: prior removal of the protein by precipitation [3, 6, 71 or ashing [S, 91; or separation of calcium by precipitation as its oxalate [lo]. A few methods have been devised for measurement on directly diluted serum [ll-131, but most of these involve use of the oxyacetylene flame, which is very noisy and in unskilled hands can be dangerous. The accuracy of these

B. KRAMER and F. F. TISDALL, J. Biol. Chem. 47, 475 (1921). E. P. CLARK and J. B. COLLIP, J. Biol. Chem. 63. 461 (1925). I. MACINTYRE, Rec. trav. chim. 74, 498 (1955); &oche&. J. ‘6’7, 164 (1957). G. HUNTER, Nature 182, 263 (1958); Analyst 84, 24 (1959). T. A. HARPER, Nature 183, 232 (1959). J. W. SEVERIN~HAUS and J. W. FERREBEE, J. Biol. Chem. 187, 621 (1950). F. L. HUMOLLER and J. R. WALSH, J. Lab. Clin. Med. 48, 127 (1956). R. E. MOSHER, M. ITANO, A. J. BOYLE, G. B. MYERS and L. T. ISHERI, Am. J. Cl&. Pathol. 21, 75 (1951).

[9] V. KAPUSCINSKI, N. Moss, B. ZAK and A. J. BOYLE, Am. J. CZin. Puthol. 22, 687 (1952). IO] F. J. N. POWELL, J. Cl&. Pathol. 6, 286 (1953). 111 C. F. ROTHE and L. A. SAPIRSTEIN, Am. J. CZin Pathal. $35, 1076 (1956). 121 A. D. WINER and D. M. KUHNS, Am. J. Cl&. Pathol. 28, 1259 (1953). 131 P. S. CREN and T. Y. TORIBARA, Anal. Chem. 25, 1642 (1953).

J. B. WILLIS

direct methods has been questioned by TELOH [14], who has developed a method in which interferences are largely eliminated by measuring the emission of diluted serum, in an oxyhydrogen or oxyacetylene flame, relative to that of a serum whose calcium content has been determined by some other method [15]. The accuracy of TELOH’S method is likely to be impaired, however, if the sodium, magnesium or phosphate concentrations differ markedly in the unknown and reference sera. Flame photometric methods in general yield results about 4 per cent higher than those obtained by the oxalate-permanganate method [4, 151.

It seems then that there is need for a completely new approach to the problem of the determination of calcium in serum, and the following points were considered in developing the present method:

1. Speed. Ashing or prior precipitation of calcium by precipitation should be avoided. Measurements should be made on directly diluted serum, or where that is not possible on deproteinized serum.

2. Accuracy. An accuracy of the order of f2 per cent is desirable. 3. Amount of serum required. Most of the existing methods require O-2-2 ml

of serum, so any proposed new method should use no more than this amount. 4. Applicability. It is desirable that the method should also be applicable

to other biological fluids, such as urine, which are more variable in composition than is blood serum and for which many of the existing methods fail.

5. Expense. Any new instrument required should be reasonably inexpensive to construct and if possible be useful for other analyses also.

It seemed worthwhile to investigate the possibility of applying atomic absorption spectroscopy [ 16, 171 to this problem, since this technique has been shown to be far less subject to cationic and anionic interference than emission flame photometry [18, 191.

Apparatus The apparatus used was that developed by Box and WALSH [20]. Light from

a calcium hollow-cathode tube [21], modulated at 100 c/s, was focused by a quartz lens to a reduced image at the centre of the flame into which the sample to be analysed was aspirated, and was then re-focused by a second lens on to the entrance slit of a Beckman DU monochromator set to pass the Ca resonance line at 4227 8. The signal from a lP28 photomultiplier tube behind the exit slit was amplified by a simple a.c. amplifier, rectified, and read on a meter. By adjusting the amplifier gain so that a reading of 100 divisions was obtained when distilled water was aspirated into the flame the percentage transmission when the sample was atomized could be read off directly. Fig. 1 shows a view of the whole assembly.

The initial experiments were carried out with oxyhydrogen and oxyacetylene flames using Beckman burners, but for most of the work an air-acetylene flame of the Lundegardh type was used. The atomizer and spray chamber, shown in

[14] H. A. TELOH, Clinical Plawae Photometry pp. ‘76-77. Charles C. Thomas, Springfield, Illinois (1959). [15] H. A. TELOH, A.M.A. Arch. Pathol. 66, 474 (1958). [16] A. WALSH, Spctrochim. Acta 7, 108 (1955). [17] B. J. RUSSELL, J. P. SHELTON and A. WALSH, Spectrochim. Acta 8, 317 (1957). [18] J. E. ALLAN, Analyst 88, 466 (1958). [19] D. J. DAVID, Analyst 84, 536 (1959). [20] G. F. Box and A. WALSH, &wctrochkm. Acta 16, 255 (1960). [Zl] W. G. JONES and A. WALSH, Spectrochim. Acta 16, 249 (1960).

260

Fig. 1. Atomic absorption spectrophotometer for estimation of calcium in blood serum.


Fig. 2, were modified from those supplied with the EEL flame photometer (Evans Electroselenium Ltd., London). The atomizer was constructed with a wider bore capillary tube (O-66 mm internal diameter) both to avoid clogging by serum solutions and to enable low air pressures (10-20 lb/in2) to be used with a 10 cm long burner. This burner was made of stainless steel and the slot through which

Fig. 2. Atomizer, spray chamber and burner.

the acetylene-air mixture emerged was about 0.5 mm wide: acetylene at 1*5- 6 lb/in2 was introduced into the spray chamber through a small jet (diameter 0.45 mm), but coal gas direct from the town supply could be used instead of acetylene, if required, by removing the jet. The flow rates corresponding to the

above pressures were: air 4.35-5.4 l/min, acetylene 0.9-l-6 l/min, coal gas 3 l/min. Liquid consumption was about 9-15 ml/min, of which quantity about 10 per cent was vaporized while the remainder ran back into the spray chamber and down the waste tube.

Materials Experimental procedure

Although some workers [3, 41 have found it necessary to USC de-ionized water for calcium determinations, glass-distilled water was found to be perfectly satisfactory in the present work. Glassware was cleaned with chromic acid, and rinsed

261

J. B. WILLIS

repeatedly with distilled water. Solutions to be stored for more than a few hours were kept in polythene bottles. Serum was measured out with pipettes graduated to 0.01 ml.

All reagents were of analytical quality, and except for hydrochloric acid, which was distilled before use, were not further purified. Standard calcium solutions were made up by dilution from a stock solution containing 1000 p.p.m. of calcium, made by dissolving oven-dried calcium carbonate in the minimum quantity of hydrochloric acid and diluting to volume.

Preparation and measurement of serum solutions

(a) Xeparation of calcium by precipitation as oxalate. Serum (0.2-0.5 ml) was pipetted into a 10 ml centrifuge tube, and 1.5 times its volume of an oxalate buffer (10 ml of O-1 M oxalic acid + 190 ml of O-1 M ammonium oxalate) was added. The tube was shaken and allowed to stand for 1 hr, after which it was centrifuged for 10 min, the supernatant liquid poured off and the tube allowed to drain for 5 min. After dissolving the precipitate of calcium oxalate by warming it with one drop of hydrochloric acid, 0.25 ml of strontium chloride solution (50,000 p.p.m. Sr) was added*, and the solution transferred to a 5-ml volumetric flask and made up with water. The solution was measured relative to standard calcium solutions containing strontium chloride (2500 p.p.m. Sr).

(b) Deproteinixation by trichloroacetic acid. Serum (0.5 ml) was pipetted into a 10 ml centrifuge tube, 0.25 ml of strontium chloride solution (50,000 p.p.m. Sr) added, followed by 3.25 ml of water. While stirring the solution 1.00 ml of trichloroacetic acid was added and the mixture allowed to stand for 5 min, after which it was centrifuged for 5 min. The supernatant liquid was measured relative to standard calcium solutions containing sodium chloride (300 p.p.m. Na), strontium chloride (2500 p.p.m. Sr) and trichloroacetic acid (4 per cent).

(c) Direct dilution with water. Serum (0.2-0.5 ml) was pipetted into a &ml volumetric flask and made up with water. It was measured relative to standard calcium solutions containing sodium chloride (300 p.p.m. Na).

(d) Direct dilution with EDTA solution. Serum (0.2-0.5 ml) was pipetted into a 5-ml volumetric flask containing 2.5 ml of a solution of 20,000 p.p.m. of the disodium salt of ethylenediaminetetracetic acid (Sequestrol), and was made up with water. The solution was measured relative to standard calcium solutions containing 10,000 p.p.m. of Sequestrol.

Technique of making measurements

Standard solutions were prepared to cover the range of calcium concentrations expected (usually 01-5 p.p.m.). The standards were measured first, then the unknowns, followed by a repetition of this procedure, and the transmission values for each solution were averaged and converted to optical density (this latter step is quite unnecessary in routine work). From the calibration curve showing optical density vs. concentration (Fig. 3) the concentrations of the unknown solutions were read off by interpolation. The amount of solution needed for one reading

* The strontium chloride is added to prevent interference from any phosphate which may be co-precipitated or adhere to the calcium oxalate precipitate.

262

The determination of metals in blood eerum by atomic absorption spectroscopy-I

.6-

,4 -

,2 -

L 0 5 IO

CO, P p.m. Fig. 3. Typical calibration curve for calcium in a 10 cm air-acetylene flame.

was about l-2 ml, so that 2.5 ml would suffice for duplicate readings. However, for reasons of accuracy and convenience, the solutions were normally made up in &ml volumetric flasks. On this volume of solution duplicate measurements could be made directly for both calcium and magnesium (see Part II)*. Each absorption reading took about 7 sec.

Interfering substances Discussion of interferences

Experience with emission flame photometry applied to samples of serum suggests that in the determination of calcium by emission methods there are four major interferences due to:

(1) Sodium, and to a lesser extent potassium, which augments the calcium emission.

(2) Magnesium, which depresses calcium emission. (3) Phosphorus, which depresses calcium emission by removing some of the

calcium atoms as calcium phosphate or phosphide. (4) Protein, which may partially compensate for the depression caused by

phosphorus [ 131.

In the present work it was found that in atomic absorption spectroscopy the effect of sodium and potassium, at the concentrations in which they occur in serum, is to increase the calcium absorption by a small amount (about 2 per cent at concentrations corresponding to a ten-fold dilution of the serum). This effect may be allowed for by adding sodium chloride to the reference solutions. Clearly,

* J. B. WILLIS, Spectrochim. Acto 10, 273 (1960).

263

J. B. WILLIS

variations in the sodium or potassium content of the serum will have no significant influence on the results of the calcium determination.

Magnesium, even in quantities far in excess of its normal concentration in serum, was found to be completely without influence on the calcium absorption.

The influence of phosphorus and protein required fuller investigation, and since interference by these materials is strongly dependent on the type of flame used, it is appropriate to discuss at this point the suitability of different flames for the determination of calcium by atomic absorption.

Flames

Since most workers have found the oxyacetylene or oxyhydrogen flame to be necessary in the flame-photometric determination of calcium, the use of these flames in atomic absorption was first investigated. However, the concentric type of burner, which was the only one available, tended to clog rather readily when aspirating protein-containing solutions. To achieve reasonably high sensitivity in absorption it was necessary to run these burners at 20-30 lb/in2 air pressure, under which conditions the oxyacetylene flame in particular was intolerably noisy. Furthermore the shape of the flame was far from ideal for absorption work, and since experiments showed that phosphorus interference could not readily be completely overcome, work with these flames was discontinued.

It was found that when calcium was aspirated in the air-coal-gas flame the concentration of free calcium atoms was low in the upper parts of the flame and was highest just above the zone of unburnt gas, so that to obtain the highest sensitivity the absorption had to be measured in the lowest part of the flame. In order to obtain the highest concentration of calcium atoms a fairly rich gas-air mixture had to be used, which suggested that a reducing atmosphere was necessary to prevent the complete oxidation of calcium to calcium oxide or hydroxide. However, it was found that in the air-coal-gas flame the calcium absorption was very sensitive to suppression by phosphorus and that the suppression was difficult to overcome, so that experiments with this flame were abandoned.

The air-acetylene flame, which has proved useful for atomic absorption measurements on magnesium [18], was found to be suitable for use with calcium also and was studied in detail. As with the air-coal-gas flame the concentration of calcium atoms was highest near the base of the flame and for optimum sensitivity a fairly rich flame was necessary. In practice the hollow-cathode radiation was focused at a point about 4 mm above the centre of the burner, and with the air- pressure held constant at about 12 lb/in2 the acetylene pressure was adjusted to give maximum absorption for a test solution containing 10 p.p.m. of calcium. This occurred when the top of the blue zone of unburnt gas became hazy, and any further increase of acetylene pressure above this point caused a luminous region to appear without increasing the calcium absorption appreciably.

Phosphorus interference and its control

Fig. 4 shows the effect of the addition of phosphorus (as H,PO,) on the absorption of calcium in the air-acetylene flame. The fall in calcium absorption with increasing phosphorus concentration corresponds to the fall noted by STRASHEIM

264


0.3 Co. I0pp.m

L

z.. C e 0” 0.2

= 2 8

0.1 0 25 50 75 100

p. wm Fig. 4. Effect of phosphorus on calcium absorption in a lo-cm air-acetylene flame.

and NELL [22] in the emission of the 4227 A line of calcium in the air-acetylene flame on the addition of phosphorus. Two approaches were tried to overcome this interference:

(1) Addition of a large concentration of an ion such as Sr2+ or La3+ which can compete with the calcium for combination with the phosphorus. Fig. 5 shows

0.3

0 500 1000 1500 2000 2500

Sr. oDm

Fig. 5. Effect of the addition of strontium chloride on the suppression by phosphorus of calcium absorption in a lo-cm air-acetylene flame.

the usefulness of this method when dealing with calcium and phosphorus in low concentrations, and Fig. 6 demonstrates its success in suppressing phosphorus interference at higher concentrations.

(2) Addition of some material which can combine chemically with the calcium and prevent it reacting with the phosphorus, since ALKEMADE and VOORHUIS [23]

have shown that solutions of phosphate introduced into a flame separately from a calcium solution do not interfere with the emission of the latter. The disodium salt of ethylenediaminetetracetic acid was chosen for trial because of its ready solubility and its great tendency to form complexes with metal ions. Fig. 7 shows

[22] A. STRASEEIM and J. P. NELL, J. 8. African Chewa. In&. 7, 79 (1954). [23] C. T. J. ALKEMADE and M. H. VOORHUIS, 2. And. Chetn. 168, 91 (1958).

2 265

J. B. WILLIS

the effectiveness of this material in overcoming the suppression by phosphorus of the calcium absorption.

Both techniques were found satisfactory, and numerous atomic absorption estimations of calcium in the presence of large amounts of phosphorus, as in plant ash, have been carried out in this Laboratory. The calibrating solutions were made

Co 25ppm P K)Oppm

Concentration of additive, PP m.

3

Fig. 6. Effect of the addition of strontium and lanthanum on the suppression by phosphorus of calcium absorption in a Z-cm air-acetylene flame.

up to contain the same amount of “suppressor” as was added to the unknown.

Where acid solutions are to be measured use of the strontium or lanthanum technique is more convenient, as under these conditions free ethylenediaminetetracetic acid tends to be precipitated.

0.50

0.40

0a201 0 5000 10,000

EDTA, p.p.m.

Fig. 7. Effect of EDTA on the suppression by phosphorus of calcium absorption in a lo-cm air-acetylene flame.

Protein interference

Experiments with calcium solutions containing known amounts of serum albumen showed that the effect of the protein varied with the composition of the


air-acetylene mixture and with the height in the flame at which the absorption was measured. In rich flames the protein appeared to depress the calcium abaorp- tion slightly, while in leaner flames, and particularly when the absorption was measured higher in the flame, the calcium absorption was enhanced. Phosphorus, when added as phosphoric acid in concentrations corresponding to that of the inorganic phosphate in blood serum, had little effect on the calcium signal in the presence of protein, particularly when the absorption was measured in a rich

Table 1. Effect of protein and phosphate on calcium determination

Solution Reference solutions

I ____- (1) Synthetic mixture

Ca, 9.8 p.p.m., albumen 80,000 p.p.m.

Ca 9.8 p.p.m., albumen 80,000 p.p.m., P 6 p.p.m.

Ca 9.8 P 5 p.p.m., p.p.m.

(2) Serum 2 Diluted 1 : 10

Diluted 1 : 10 + P 5 p.p.m.

Crt 11 p.p.m. + P 5 p.p.m.

(3) Serum 2A Diluted 1 : 10

Diluted 1 : 10 + P 5 p.p.m.

Diluted 1 : 10 + P 10 p.p.m.

Ca 8.5 p.p.m. + P 10 p.p.m.

/ , Ca, alone

Ca + Na, 300 p.p.m.

Ca + Ne, 300 p.p.m.

Rich flame Lean flame (air 8 lb/in2 (air 12 lb/in2

C,H, 2 lb/in2) C,H, 1.8 lb/in2)

(p.p.m.) (p.p.m.)

9.1

9.1

6.4

8.65 8.85

8.6 8.7

8.6 8.7

4.6 4.7

10.1

9.4

7.0

12.2

11.3

4.2

flame. Table 1 shows this “buffering” action of protein on the phosphorus interference both for synthetic solutions of calcium, phosphorus and albumen, and also for two specimens of serum.

The present work suggests that, provided a rich acetyleneair mixture is used, the effects of protein and phosphorus interference largely nullify each other, and thus it should be possible to make calcium determinations on serum directly diluted with water.

267

J. B. WILLIS

In order to avoid the need for careful control of the flame conditions, however, it seemed desirable to try to find a method of overcoming both protein and phosphorus interference. The use of EDTA, which was shown to control phosphorus interference, might also be expected to overcome protein interference by providing a competitor for the protein with respect to its combination with the calcium. As shown in Table 2 the use of this material does give results which seem to approximate closely to the correct ones (see Discussion) and which are less dependent on flame conditions than those obtained by direct dilution with water.

Table 2. Comparison of the results of different methods for estimation of calcium in serum (mg/lOO ml)

serum no. Type

1*

2t 2AS 3 4 6 6 7 8 9

10

II§ 12 13 14 15 16 17 18 19 20 21 22

Bovine Equine Equine Normal human

Pathol. ‘human

Method (a) Method (b) (Ca separated (serum de-

as oxalate) proteinized

11.15 10.8 8.5

10.0 -

9.8 10.5

7.75 8.65 6.9 9.15

(2.1) 8.1

10.0 8.6 7.05 -

11.35

- - -

- -

-

8.95 10.25 10.4 9.85

10.9 7.85 8.7 6.95 9.5 3.1 8.2 9.95 8.3 6.85 9.35

IO.35 8.85 9.35 9.7 9.05

10.3

T

Method (c) (direct dilution

with water)

11.0 -

8.6 -

10.2 9.35 - - - - - - - - - -

10.1 9.75 8.6 -

9.9 9.05

10.4

Method (d) (direct di-

lution with EDTA)

11.1 10.65

8.6 9.8

10.05 9.35 9.9 7.7 8.35 5.95 9.1 3.0 8.05 9.65 8.55 6.55 9.65 9.5 9.05 8.65 9.35 8.5 9.6

f -

!-

Oxalate permanganate

method

11.3 -

9.1 9.3 9.9 - - - - - - - - - - - - - -

8.8 - - -

* Freeze-dried serum (“Chemtrol,” supplied by Clinton Laboratories, Los Aangeles, California). The calcium content, obtained by the Clark-Collip method, was given by the suppliers as 11.1 mg/lOO ml.

t An independent analysis by atomic absorption after ashing the serum was made by Mr. D. J. DAVID, using the techniques developed by him [19] for suppression of the phosphorus interference, and yielded a figure of 10.7 mg/lOO ml.

$ This serum had stood in the refrigerator for several months. It was centrifuged to remove the precipitate which had formed, and which contained some of the calcium. Similar observations have been made on aged sera by CHEN and TORIBARA [24].

9 This serum, though fresh, deposited a precipitate which was centrifuged off before measurement of the supernatant liquid.

Results One difficulty in assessing the results of a new method of determining calcium

in serum is that there is no method of unquestioned accuracy for comparison. For this reason, although opportunity was taken to compare the atomic absorption

[24] P. S. CHEN and T. Y. TORIBARA, Anal. Chem. 26, 1967 (1954).

268


results with those obtained by the Clark-Collip method wherever sufficient quantities of serum were available, more importance was placed on the criteria of internal consistency. These may be summarized thus:

(1) The result of the analysis must be independent of the amount of sample taken.

(2) Recovery of calcium added to the sample in known amounts must be quantitative to within the limit of accuracy of the method.

(3) Different variations of the method should give the same results. The way in which the atomic absorption method meets criteria (1) and (2) can

be assessed from Tables 3 and 4, while a comparison of different variations of the

Table 3. Effect of dilution on calcium determination

(Ca, mg/lOO ml serum)

Dilution of

I

Method (c) Method (d)

Dilution with water I Dilution with EDTA solution

Serum Serum Serum Serum ’ Serum Serum Serum Serum Serum

1 2A 24 1 3 7 10 23

I:7 - - - - - 9.05 8.1 1 : 10 11.0 8.65 12.05 10.85 9.8 7.7 - 7.9

1 : 15 1 - - - - - 7.35 - -

1 : 20 11.4 8.5 12.0 11.05 9.8 7.35 9.1 8.0

method is shown in Table 2. The reproducibility of the results obtained by atomic absorption measurements is illustrated in Table 5, which shows the results of calcium determinations on replicate samples of serum, using methods (a) and (d). These results show the reproducibility of a single reading, and include errors in the pipetting of the serum, but do not of course show the effect of any errors in the calibration procedure.

Discussion It will be seen from Tables 3 and 4 that in terms of criteria (1) and (2) all four

variations of the atomic absorption method, except perhaps (a), are acceptable. This underlines the difficulty of judging the relative merits of different methods, when each is apparently self-consistent but gives a different result for the amount of calcium in a specimen of serum. When the results obtained by the different variations of the atomic absorption method are compared, however, several interesting facts emerge.

Firstly, the results obtained by the methods involving (a) separation of calcium by precipitation and (b) removal of protein by coagulation give results which are in good agreement, but in almost every case the calcium content obtained by method (a) is slightly less than that by method (b). The average difference between the results of the two methods is 1.8 per cent, which is about the amount expected from the operation of two contributory factors:

(1) Failure of the oxalate precipitation to remove the last 0*8-2.0 per cent of the calcium in serum [3].

269’

J. B. WILLIS

Table 4. Results of recovery experiments

Concentration of Ca in swum Ca tided Total Ca Recovered

solution (p.p.m.) (p.p.m.) (p-p-m.) (p.p.m.)

Method (LX) : calcium separated as oxalate

Serum

1 10.7 5.0 15-7 15.4 98-l 2 5.75 5.0 10.75 10.6 98.6

Av. 98.35

2A 8.95 2A 8.56 15 5.3

I I I Method (b) : mwn deproteirkzed

I I 5.0 13.95

6.0 13.55 5.0 10.3

13.95 100.0 13.45 99.3 10.15 98.5

Av. 99.3 n thod (c): serun ‘I Gluted with wat er

1 5.7 5.0 10.7 IO.5 98.1 2A 4.3 5.0 9.3 9.3 100.0 4 5.2 5.0 10.2 10.2 100-O 5 7.5 5.0 12.5 12.15 97.2 21 8.2 6.0 13.2 13.25 100.4 22 9.55 6.0 14.55 14.45 99.3 25 8.0 7.0 15.0 15.15 101.0

Av. 99-4 Method (d): serwm diluted with EDTA solution

1 11.05 5.0 1 5.6 5.0 2A 4.25 6.0 2A 4.4 5.0 3 4.9 6.0 4 5.0 5.0 19 4.3 5.0 20 4.7 5.0 21 4.25 5.0

16.05 16.0 99.7 10.5 10.4 99.0 10.25 IO-2 99.6

9.4 9.65 102.7 10.9 10.65 97.7 10.0 10.15 101.5 9.3 9.6 103.2 9.7 9.6 99.0 9.25 9.1 98.4

- - -

-

7-

-

Recovery

(%)

Av. 100.0

(2) The O-5 per cent decrease in the volume of the serum solution after de- proteinization in method (b).

Table 4 shows that in the present work recovery of added calcium from serum by the precipitation method was significantly less than 100 per cent and six blank experiments on the precipitation of calcium from a standard solution containing 10 mg/lOO ml yielded recoveries ranging from 97-100 per cent, with an average of 984 per cent.

270


It is considered that the results obtained by method (b) (deproteinizing the solutions with trichloroacetic acid) are the most nearly correct of all those obtained by atomic absorption. Methods (c) and (d), which do not involve removal of protein, yield results which on the average are slightly lower than those of method (b). The mean deviation from the result obtained by method (b) is 0.33 mg/lOO ml for method (c) and 0.43 mg/lOO ml for method (d).

While the use of method (b), involving coagulation of the proteins, is desirable for the most accurate determinations of calcium in serum, either of the direct dilution methods (c) and (d) may be used where an approximate figure will suffice.

Table 5. Results of replicate determinations of calcium in serum

I

Method (a) Method (d)

(separation of calcium a8 j (dilution with EDTA oxalate)

I solution) ,

0.50 ml of serum 2 I 0.50 ml of serum 4

(mg/lOO ml) I diluted to 5 ml

I (mg/lOO ml)

1043 10.8 10.8

10.03 9.88 9.97

10.03 IO.03 IO.20 10.13

Mean IO.8 Mean 10.04 s.d. f 0.10

From the experience gained here it seems that while dilution with water alone gives an answer slightly closer to that obtained by method (b), more careful control of the air-acetylene mixture and height of the flame is required than with method (d).

The major discrepancies between the results of the direct dilution methods and those of method (b) occurred with serum specimens which had been stored for some time. The reason for this is not obvious, but the results suggest that measurements on aged sera should be made by method (b) rather than by methods (c) and (d).

Although no detailed study of the Clark-Collip method was made during the present investigation, the figures obtained by atomic absorption are significantly higher than those obtained chemically but are in agreement with those obtained by the flame photometric methods of MCINTYRE and othera [3, 151.

In Table 6 are shown the results of the analysis of six sera by the atomic absorption method and by the EDTA titration method of WILKINSON [25]. As

[25] R. H. WILKINSON, J. Clin. P&d 10, 126 (1957).

271

J. B. WILLIS

expected, the results of the latter method are slightly lower than those obtained by atomic absorption measurements.

Table 6. Comparison of atomic absorption and EDTA titration methods for calcium in serum

(Ca, mg/lOO ml serum)

I

Serum no. Type Method (b)

Atomic absorption

Method (c) Method (d) EDTA titration

I I 26 Human 27 Human 28 Human 29 Human 30 Human 31 Human

- - 10.7 10.6 - - 7.75 7.45 8.7 - 8.7 8.2

10.05 - 10.3 9.25 8.7 9.35 8.8 8.35 9.9 10.6 9.8 9.8

Conclusions

The atomic absorption method is suitable for the determination of the calcium content of blood serum, using samples of 0.1-0.5 ml. Removal of protein by coagulation is desirable for the most accurate work, but for rapid routine purposes measurements can be made on solutions of serum diluted directly with water. Since the interference by phosphate can be completely overcome by the addition of a suitable suppressor, and since cation interferences are small, the method should also be suitable for determination of calcium in other biological fluids.

Some problems remain, but the atomic absorption method is at least as accurate as any other and has the advantage of speed and economy of material.

Acknowledgements-The writer wishes to thank Mr. W. G. JONES for the construction of the hollow-cathode tubes, and Messrs G. BRYANT and R. VENN for the construction of atomizers, spray chambers and burners. He is also indebted to Messrs. M. HINTON and E. MASON of the Commonwealth Serum Laboratories for supplies of serum albumen and of horse serum, and to Miss B. SPLAYS and her staff at the Biochemistry Department, Royal Melbourne Hospital, for supplying the specimens of human sera and for carrying out some of the oxalate-permanganate titrations. Dr. D. B. CHEEK, of the Royal Children’s Hospital, Melbourne, kindly supplied sera nos. 26-31 and measured their calcium contents by titration with EDTA.

The constant interest and encouragement of Mr A. WALSH is gratefully acknowledged.

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the determination of metals in blood serum by atomic ... determination of metals in blood serum by...

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