THE PRECIPITATION OF BLOOD CALCIUM BY LEAD.

BY FRITZ BISCHOFF AND L. C. MAXWELL.

(From the Chemical Laboratory of the Potter Metabolic Clinic and the Department of Cancer Research, Santa Barbara Cottage Hospital,

Santa Barbara.)

(Received for publication, June 19, 1928.)

INTRODUCTION.

The treatment of inoperable cancer by the intravenous injection of suspensions of lead has stimulated interest in the fate of this metal in the blood stream. The original metallic colloid of Bell (1) is very sensitive to oxygen, and by in vitro experiments Brooks (2) and Bischoff and Blather- wick (3) showed that there was little likelihood of any of the metallic lead reaching the cancer as such. Aub and Reznikoff (4) had shown that the phosphates and carbonates were the only two of nine constituents normally found in blood which would neutralize the damaging effect of ionic lead to red cells, the phosphate ions being many times as effective as the carbonate. Fairhall and Shaw (5) found that the lead deposited in bones in lead poison- ing is present as phosphate and not as carbonate or albuminate, and from a critical study of the solubilities of lead carbonate, dilead phosphate, and trilead phosphate, concluded that trilead phosphate was the form in which lead was transported in the blood in lead poisoning. Bischoff (3, 6) pre-

pared various colloidal suspensions of lead with the inorganic constituents of the blood and found from rabbit tests that colloidal metallic lead, ionic lead and lead hydroxide were about equally damaging to the red blood cells. The oxycarbonate was less damaging and the phosphate without effect. Ionic lead, buffered with serum or red blood cells containing sufficient inorganic phosphorus to convert all the lead to phosphate, was also found to be non-toxic. The colloidal phosphate prepared for the rabbit experiments was then tried clinically by Ullmann (7), Pulford (8), Soiland, Costolow, and Meland (9), and others,

Brooks (2), experimenting with the Bell metallic colloid and ionic lead, found that when these substances were added to serum, the amount of phosphorus precipitated from the serum (determined by analyses of ultra- filtrates), was higher than the theoretical amount required to convert the lead to the triphosphate and corresponded more nearly to the diphosphate. Brooks explained his results by assuming a delayed equilibrium in which the dilead phosphate was first formed. At the end of 70 hours, the ratio of lead to phosphorus still corresponded to the dilead phosphate. Bischoff

5

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Calcium Precipitation by Lead

(6) showed that when ionic lead is added to the blood, the amount circulat- ing after 2 hours is nil. On the basis of Brooks’ interpretation, there would be little chance of any trilead phosphate being formed from the metallic colloid by the time the lead reached the tumor, and the experimental evi- dence for the clinical use of the trilead phosphate would be materially weakened. From his 1ead:phosphorus ratios, Brooks (2) concluded that in a phosphate buffer, lead acetate and colloidal metallic lead formed the triphosphate; in Ringer’s solution the former formed the triphosphate and the latter the diphosphate, and in blood serum they both formed the di- phosphate. These results suggested to us that calcium might be thrown out with the lead in the cases where the ratio pointed to the diphosphate, and that no dilead phosphate but a precipitation of lead and calcium triphosphates or a double triphosphate salt took place. Brooks’ analytical results would be in complete harmony with this explanation.

3PbXz + 2NazHPOa = Pb8(PO& + 4NaX + 2HX 2PbXz + CaX2 + 2Na2HP04 = Pb&a(PO&. + 4NaX + 2HX

His results with Ringer’s solution made this explanation seem likely, since this solution becomes highly supersaturated with calcium when the pH is alkaline. In the case of lead acetate in which he obtained the ratio for the tri-salt the Ringer’s solution would become more acid. For the colloidal lead, where the ratio for the di-salt was obtained, it would become more alkaline.

3Pb(Ac)z + Na2HP04 + NaH2P04 = Pb3(PO& + 3NaAc -I- 3HAc 3Pb(OH)z + NaSHPOd f NaH2POG = Pb3(P04)z + 3NaOH + 3HzO

Object.

The experiments recorded in this paper were taken up with the idea of establishing whether or not calcium was involved in the reaction of lead ions with blood serum. A few preliminary ex- periments showed that ultrafiltrable calcium was thrown out of solution. The amounts of phosphorus and calcium removed from solution were greater than is required for the above equations. In order to interpret these results in the light of recent investiga- tions as to the state of calcium in the blood, complete data for pH, C02-combining power, inorganic phosphorus, and diffusible and non-diffusible calcium were taken.

Analytical Methods.

Inorganic phosphorus was determined by the Fiske-Subbarow modification of the methods of Bell and Doisy and Briggs. The maximum experimental error was within 5 per cent. Calcium

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I?. Bischoff and L. C. Maxwell 7

was determined by the Clark-Collip modification of the Kramer- Tisdall method. Duplicate analyses checked well within 0.3 mg. of Ca. COz-combining power was determined by the Van Slyke gasometric method and in m3me experiments by the man- ometric method. Analyses of our ultrafiltrates showed a variation of 5 per cent in COz-combining power as compared with the value obtained for the original serums. The pH was determined calorimetrically with Clark and Lubs’ indicators and standard buffer solutions. Values were duplicated within 0.05 pH in the range of pH 6.8 to 7.4 and within 0.1 pH for the other values given.

Experiments with Ringer’s Solution and Phosphate Bufer.

The Ringer’s solution contained 8 gm. of NaCl, 0.2 gm. of KCl, 0.22 gm. of CaCh, 2.22 gm. of NaHC03, and 0.24 gm. of KHzPOI in 900 cc. The pH was adjusted to 7.2 by adding dilute HCl. To 180 cc. of this solution were added 20 cc. of a mixture containing 42 mg. of PbClz and 2.3 cc. of 0.1 N NaOH. The pH after the addition of the basic lead solution shifted to 8.2. The mixture was stirred half an hour and filtered. 6.2 mg. of Ca and 4.0 mg. of P per 100 cc. were found in the filtrates. The original Ca and P on a 100 cc. basis were 8.4 and 6.0 mg. re- spectively. The amount of phosphorus removed from solution lies between the values required for the di- and triphosphates. There is not enough phosphorus removed from solution to convert both the lead and calcium to the triphosphate. The results of the experiment in Ringer’s solution indicated that some calcium carbonate was also thrown down with the phosphate. The experiment was tried in a phosphate buffer solution containing calcium chloride. It is interesting to note that both this phos- phat,e solution and Ringer’s containing calcium chloride were supersaturated with respect to calcium phosphate. Thus a cal- cium chloride-phosphate solution was made by adding 0.24 gm. of KHZPO,, and 11 cc. of 0.1 N NaOH solution to 900 cc. of CO,- free water at 60”. To this solution 0.22 gm. of CaClz in 100 cc. of water was added. The mixture was placed in a thermostat for 1 hour at 37“ and a slight precipitate filtered off. The analysis of the filtrate gave 5.3 mg. of P and 5.0 mg. of Ca per 100 cc. The pH was 6.7. If the ionic strength is taken as 0.008 and the

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8 Calcium Precipitation by Lead

apparent solubility product at that ionic strength as pK 31.0 from Holt, La Mer, and Chown’s (10) data for the solubility product of tricalcium phosphate, at 37”:

(Ca)3 (Pod”)3 = (solubility product) X3 ((1.71 x 10-3) (6.7 x lo-‘))3 = 1.0 x 10-3’

where 1.71 X 10h3 is the concentration of phosphorus in mols per liter, and 6.7 X 10ml is the fraction present as POJ” at the given pH.

X = 9.2 X 10m6, mols Ca for saturation 1.25 X 10-3, mols Ca present

The filtrates remained perfectly clear for days. To two 100 cc. portions of such a phosphate-calcium chloride solution were added 50 mg. of lead acetate and 50 mg. of lead acetate neutral- ized with NaOH, respectively. The original pH of the buffer solution was 7.3. Adding lead acetate changed the pH below 6.0. Adding the neutralized acetate took the pH to over 9.0. 2.9 mg. of P were removed in the lead acetate experiment (theory for the triphosphate 2.75 mg.). 4.4 mg. of P were removed in the basic lead experiment (see Table I). These experiments were per- formed at room temperature, 23”. The experiment with the phosphate buffer and lead acetate was repeated at 37” with the same results. An experiment with Ringer’s solution under CO2 tension was also performed at 37”. In the experiments with phosphate buffer a considerable shift in pH was brought about by the liberation of acetic acid, because the concentration of phosphate (about the same as in blood) was low. In Ringer’s solution the relatively high concentration of bicarbonate lessened this effect. The results of this experiment were particularly interesting. An amount of phosphorus corresponding to trilead phosphate was thrown out of solution, confirming a similar exper- iment of Brooks; at the same time all the calcium down to a concentration of 0.2 mg. per 100 cc. was removed after standing 1 hour. After the solution had stood 30 hours the amount remain- ing was not titratable. In the analysis for calcium 5 times the volume of solution usually taken was used to increase the accuracy; t,his was necessary because of the small amounts of calcium remain- ing in solution. The experiment shows that at the pH of the

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TABL

E I.

Effe

ct

of

Lead

Io

ns

upon

Ri

nger

’s So

lutio

n,

Calci

um

Chlor

ide-P

hosp

hate

So

lutio

n,

and

Bloo

d Se

rum

.

I Be

fore

lead.

l-

Med

ium.

Ring

er’s

solu

tion

+ Pb

(Ac)

z..

. 7.

7 “

“ +

Pb(O

H)z..

7.

2 Ph

osph

ate-

CM&

+ Pb

(Ac)

z..

. . .

7.3

“

+ “

6.7

I‘

Seru

m.

d .o !A

g 23

-- - m

g.

z at3

7 1.

59

60

54.!

53

54.!

+ Pb

(OH)

z..

. 7.

3

d 2 P I A 2 n _ ng

. pe

r 1.

18.F

84

79

50

79

ml.

per

1.

338

156

275

275

275

7.5:

8.

2 :6

.0

4.9

>9.0

Expe

rimen

t I..

. .

. . .

. . .

. . .

. . .

8.

3 es

14

0 96

26

8 8.

3

I‘ II.

...

......

. “

III.

......

...

“ IV

...

......

. I‘

v ...

......

...

Seru

m

and

oxala

te.

Expe

rimen

t VI

...

......

.. “

VII.

......

...

. .

8.2

. . .

7.3

. .

. 7

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7.5

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‘5

47

71

.c ‘5

88

80

50

-

WI.

per-

2. T?

. oe

r 1.

25

39

25:

27 6

140

97

110

8.2

161

108

159

7.X

145

105

198

7.2i

11

2 90

25

9 7.

4;

9.r

? 32

14

36

34

.’

291

67(

52(

102:

12

8.1

520

7.1

28

70(

32

1O.a

394

6.9!

9

77:

I

The

figur

es

in

pare

nthe

ses

indi

cate

ca

lcula

ted

value

s.

s3 a

After

lea

d.

d ‘E

4 * .I n Zi. 0.

: 62

79

&

w?.

per

1

34

21

29

26

48

571

75

80

70 .

’ 43

20

33

35.

53

1.1

52

2.

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Phos

phoru

s ca

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T=

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x %

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r 1.

33.8

51

.0

15.6

27

.0

27.5

27

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27.5

(4

8.1)

26.8

46

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11.0

22

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15.9

30

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19.8

37

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25.9

50

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48

52

35.3

39

ml.

WI.

per

1. pe

r 2.

67.8

76

.4

34.9

40

.5

[61.

8 72

.2

60

70

27.9

33

.5

38.2

45

.5

47.5

56

.4

63.1

75

.2

;t Pt

z;

;a

bd

1.37

cc

1:3

1.34

0.97

1.

09

1.09

1.

07

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10 Calcium Precipitation by Lead

blood, lead acetate t,hrows out both the calcium and the phos- phorus, but that the amount of phosphorus is not sufficient for the triphosphate of both the lead and the calcium. Some car- bonate is undoubtedly removed at the same time (see Table I). The data will be discussed again in connection with the experi- ments with blood serum.

These experiments showed that the difference in results obtained by Brooks in a phosphate buffer and in Ringer’s solution could be accounted for by precipitation of calcium, and did not indicate the formation of lead diphosphate. It will be noted that basic lead suspensions, prepared just before use, were taken instead of metallic colloidal lead solutions. This was done because colloidal lead solutions always contain appreciable amounts of lead car- bonate (10 per cent) and since it has been shown that the colloidal lead is rapidly oxidized to lead hydroxide when added to a solution saturated with air.

Experiments with Blood Serum.

The apparatus described by Brooks (2) was used in the experi- ments with blood serum. The collodion bags were made from du Pont parlodion dissolved in the amounts of alcohol-ether recom- mended for surgical collodion. Filtration was conducted under a pressure of 200 to 300 mm. of Hg. A known volume of serum was introduced into the bag, and a sufficient quantity ultrafiltered to determine the pH, calcium, phosphorus, and bicarbonate, and to make a biuret test. In the first three experiments the lead acetate peptized in 2 cc. of serum was added to the serum in the bag. After thorough mixing, the ultrafiltration was continued, the first few cc. being discarded. In the fourth experiment, the lead acetate was not added to the serum remaining in the bag, but to a fresh sample of the same serum. In the fifth experiment the lead acetate was added directly to the total volume remaining in the bag. It was thought that the way in which the lead was added might affect the results. Alveolar air was used in the CO2 tension experiments. The apparatus was first swept out with this gas mixture, which was also bubbled through the serum several minutes before the serum was introduced int.0 the bag. A stream of the mixture was also kept bubbling through the serum while the lead acetate was being peptized and mixed with the serum. In

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F. Bischoff and L. C. Maxwell

the first four experiments rabbit serum was used; in the fifth pig serum. The ratio of diffusible to total calcium for the four rabbit serums was fairly constant, varying from 67 to 72.5 per cent. The total calcium value is about 3 mg. higher in the rabbit serum than the value given for human serum. There was sometimes a slight difference between the inorganic P and always a difference between the bicarbonate values for the serum and its ultrafiltrates. The values for the ultrafiltrates were used in the calculations. The serums were preserved with phenol. One serum was used without the addition of a preservative.

The preliminary experiments with blood serum were performed without COz tension. The experiments with phosphate buffer containing calcium chloride had shown that the precipitation of the calcium depended upon the pH of the medium, lead acetate, which produced a greater acidity, throwing down no calcium. Brooks had shown that in serum there was no difference in the phosphorus removed from solutions whether one used lead acetate or colloidal lead. Our experiments were confined to lead acetate. From the solubility product theorem, the calcium in the serum should become more supersaturated as the alkalinity is increased. With use of lead acetate this condition would not arise.

The results of our experiments showed t,hat calcium was thrown out of solution whether one worked at a pH above 8 without CO, tension or at a pH of 7.4 with COz tension. The amount of cal- cium thrown out of solution was equivalent to the amount of lead added. The amount of phosphorus removed from solution corresponded to the triphosphates of lead and calcium. It will be noted that the phosphorus removed from solution is higher than most of the values reported by Brooks and that the calcium is twice as high as would be required if one interpreted Brooks’ findings on the basis of a mixed tricalcium lead phosphate instead of dilead phosphate. Brooks gives two values which are 10 per cent, and one which is 5 per cent higher than the theoretical value of his diphosphate. His other values are 5 to 10 per cent lower. In determining the phosphate value, the difference between two analytical values which may be in error by 5 per cent, is taken. The same applies to the calcium, for while the analytical values for calcium are probably more accurate than those for phosphorus, the difference between the calcium values is a smaller per cent

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12 Calcium Precipitation by Lead

than the difference between the phosphate values. The higher vaIues for phosphorus that we obtained in our experiments may be due to a difference in technique. The constancy of the ratio for added lead to precipitated calcium is well within the analytical error.

The amount of acid liberated by the reaction of the lead acetate with the phosphate in the serum is not great enough to shift the pH more than 0.2. Thus in Experiment III, the magnitude of the concentration of the various compounds expressed in mols per liter, was as follows:

7.65 X 10m4 lead added. 6.45 X IO+ acetic acid liberated.

1 Calculated from the MgHP04:

9.08 X lo-” sodium acetate liberated. MHzPOd ratio at the initial 4.5 X lo-4 inorganic phosphorus left. pH. 1.65 X lo+ bicarbonate.

The acetic acid liberated is equivalent to but 5 per cent of the bicarbonate content.

TABLE II.

Apparent Solubility Products for CaC03 and Cat(PO& in Serum after Lead.

The concentrations are expressed in mols per liter.

__--- 8.3 1.43 8.2 1.88 0.67 7.15 2.00 1.5 7.25 1.76 1.27 7.45 1.08 2.43

l~~o~~ XL [Ca x ‘po)4J b !&& c,& ~~2!? 8:

x lo-= (P04)r -------

0.36 23.2 15.7 23.8 20.8 1.03 61.8 39.0 6.41 236 22.6

3.23 0.45 1.51 6.46 7.19 0.182 25.7 3.38 1.16 5.39 5.47 7.23 1.58 24.8

10.4 1.13 9.9 11.2 6.95 1.23 24.9

Mechanism of the Calcium Precipitation.

The work of Halt, La Mer, and Chown and Hastings, Murray, and Sendroy (11) has shown that the amount of calcium in blood serum greatly exceeds the amount which should be present, cal- culated from the solubility product of calcium carbonate or calcium phosphate as determined in a salt mixture of the same ionic strength as serum. Whether this calcium is in a super- saturated, a colloidal, or a non-ionizable state does not appear to be definitely established. In order to interpret our data in the

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F. Bischoff and L. C. Maxwell 13

light of the solubility product conception, the apparent solubility product for calcium carbonate and calcium phosphate has been calculated from the analytical data of our serum ultrafiltrates (see Table II). Since the calcium in these filtrates has passed through a membrane which holds back proteins, it is obviously not in a colloidal state nor bound in a non-diffusible form to protein. The COZ ion and PO4 ion concentrations are calculated from the tables furnished by Hastings, Murray, and Sendroy (11) for carbonate, and Holt, La Mer, and Chown (10) for phosphate. In the calculation of Hastings, Murray, and Sendroy, the ioniza- tion constant is corrected for ionic strength. This was not done by Holt et al. Since our data are calculated for comparative and not absolute values, this difference is not essential to the interpretation of the results.

One might suppose that when calcium phosphate is thrown out of serum by the addition of lead acetate, an equilibrium would be established between the calcium phosphate in the solid and solu- tion phases, and a constant solubility product would be obtained. Our data show that this is not the case (see Table II). The appar- ent solubility product for Cas(PO& varies in each experiment. An error of 10 per cent in calcium and 0.1 in pH would affect the pK,.,.Ca3(P0& by 0.35 and the pK,.,.CaCOa by 0.16. In these experiments, the ultrafiltrates obtained 1 hour and 24 hours after the addition of lead were analyzed with no change in the results, so that equilibrium had apparently been reached. Since the solubility of calcium salts is not appreciably affected by the changes of temperature, we felt justified in comparing our solu- bility products, which were obtained at 23”, with those of Hastings, Murray, and Sendroy and Holt, La Mer, and Chown at 37”. Hastings, Murray, and Sendroy give pK CaC03 as 6.40 in serum and 7.40 in salt solutions of the same ionic strength. Our pK values at pH 7.15 to 8.3 for CaC03 equal or exceed 6.40, so that at equilibrium, the solution is not supersaturated with respect to calcium carbonate. Holt, La Mer, and Chown give 26.0 as the pK Ca3(PO& in serum and 27.2 in salt solutions of the same ionic strength. All our pK values are below this figure. The serums are still supersaturated with respect to calcium phosphate after the lead has been added. Sendroy and Hastings (12) have shown that when serum is shaken with solid CaC03, no precipita-

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Calcium Precipitation by Lead

tion of calcium takes place, but that when Ca3(PO& is used, the calcium content of the serum decreases rapidly, equilibrium being reached in 5 minutes. Our results would therefore tend to show that the calcium was not precipitated as Caz(PO& but as a mixed lead-calcium phosphate. The constant ratio of lead added to calcium precipitated could better be explained on this basis. If the precipitation of this double lead-calcium phosphate is not governed by the solubility product for tricalcium phosphate, and if the double salt is more insoluble than calcium phosphate, the calcium should be precipitated from a solution which was not saturated with respect to calcium phosphate. In order to test this experimentally, over 90 per cent of the calcium was removed from a serum with sodium oxalate. The calcium remaining in solution did not exceed the amount required for saturation. Upon addition of lead acetate to this solution, all the calcium was precipitated.

EXPERIMENTAL.

Precipitation of Calcium beyond the Saturation Point of Tricalcium Phosphate.

It was found that when the amount of oxalate theoretically required to convert the calcium to oxalate was added to the serum, 10 per cent of the total calcium was not precipitated and a little less than 1 mg. per 100 cc. was still ultrafiltrable. Thus 14 mg. of sodium oxalate were added to 30 cc. of serum which analyzed 13 mg. of calcium per 100 cc. After the mixture had stood overnight the calcium oxalate was centrifuged off; 1.2 mg. per 100 cc. of calcium remained, of which 0.86 mg. per 100 cc. was ultrafiltrable. To 37 cc. of another serum were added 15.4 mg. of sodium oxalate. The total calcium was reduced from 13.2 mg. per 100 cc. to 3.2 mg. per 100 cc. The ultrafiltrable calcium was 1.04 mg. per 100 cc. Lead acetate was added to these serums under COz tension, as in the other serum experiments, and the calcium determined in the ultrafiltrates. In order to increase the accuracy of the calcium determinations, double the volume usually taken was used in the analytical determination. All the calcium, within the limits of the determination, was thrown out by the lead.

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F. Bischoff and L. C. Maxwell 15

If pK 26.0 is taken as the solubility product of calcium phos- phate in serum,

X3 ((0.94 x lo-3)(2.8 x lo-6))2 = lo-26 x = 1.1 x 10-s

or 4.4 mg. of Ca per 100 cc. required for saturation in Experiment VI with serum.

x3 ((0.29 x IO-3)(1.7 x lo-y)2 = 10-26 x = 3.5 x 10-a

or 14 mg. of Ca per 100 cc. required for saturation in Experiment VII with serum.

In these calculations, 0.94 X lo+ and 0.29 X lo+ are the concentration of P in mols per liter after the addition of the lead, and 2.8 X 10m6 and 1.7 X 1O-6 are the factors which give the amount of PO4 ion at the pH of the solution. It will be noted (Table I) that in the experiments recorded above the phosphorus removed from solution corresponds very nearly to the amount required for trilead phosphate. In the calculations given in Table I, we have corrected the results for the amount of lead which would be precipitated as oxalate. This amount is small and does not materially affect the final result.

An examination of the data for the experiment in which lead acetate was added to a Ringer’s solution with a low calcium concentration, shows that the calcium has also been removed below the saturation point for either calcium carbonate or calcium phosphate. As this experiment was performed at 37” and the solid and liquid phases were in contact for 30 hours, a direct comparison with the data of Holt, La Mer, and Chown and Hast- ings, Murray, and Sendroy may be made. Thus:

X (1.9 X lo+) (5.5 X 10-s) = 3.9 X 10-S X = 3.8 X lo-4 mols of Ca required for saturation of C&OS. X3 ((8.1 X 10-4) (1 X 10-b))* = 6.3 x lo+* X = 2.1 X 10e4 mols of Ca required for saturation of Caa(P04)~.

These figures are equivalent to 1.52 and 0.84 mg. of Ca per 100 cc. of solution. The amounts found were 0.2 mg. after 1 hour and a trace after 30 hours. In this experiment there must have been some carbonate thrown down as well as phosphate. (The data

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16 Calcium Precipitation by Lead

show that carbonate was also thrown down when Ringer’s solution was treated with lead hydroxide.) The significant fact is that calcium may be precipitated by lead below the saturation point in salt solution as well as in serum. An analogous example is the precipitation of radium with barium. The interpretation of this phenomenon by Lind (13) in Taylor’s “A Treatise on Physical Chemistry” is quoted :

“The co-precipitation of radium and barium presents some highly inter- esting features. It seems anomalous that radium should be precipitated together with barium at a concentration far below that corresponding to

the solubility of pure radium sulphate (about & that of BaSOa). This

action is exactly that to be expected if radium and barium were truly isotopic, but they have, of course, atomic numbers widely different, and can readily be separated by fractional crystallization of the halides. More- over, since they are not isotopic, one would expect the theoretically unstable condition established by the co-precipitation to revert to equilib- rium by the return of radium into solution. Such is not the case. On the other hand, the degree of co-precipitation is not so rigid as with true isotopes.”

We are not inclined to regard the calcium precipitation as one involving specific adsorption, because of the large amount of calcium removed from solution. It would, indeed, be unusual for a precipitate to adsorb a molecular equivalent of another salt. Fairhall (14) has worked out a method for the quantitative determination of lead in urine, in which the calcium is precipitated as the phosphate in ammoniacal solution with the quantitative entrainment of lead. The mechanism of this react.ion is readily explained on the basis of our findings. It would appear that the action of the parathyroid hormone, which is as specific for lead as for calcium, is also in harmony with the double lead-calcium salt conception.

SUMMARY.

Lead acetate added to serum with or without CO2 tension precipitates a molecular equivalent of ultrafiltrable calcium and an amount of inorganic phosphorus required for the formation of trilead and tricalcium phosphate. Since the amount of calcium thrown out is independent of the pH (6.9 to 8.3) and of the concen-

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F. Bischoff and L. C. Maxwell 17

tration of phosphate, carbonate, and calcium ions, the reaction is not immediately concerned with the solubility product of calcium carbonate or calcium phosphate. The reaction is a specific lead effect.

BIBLIOGRAPHY.

1. Bell, W. B., Lance& 1924, 1, 267. 2. Brooks, J., Biochem. J., 1927, xxi, 766. 3. Bischoff, F., and Blatherwick, N., J. Pharmacol. and Exp. Therap.,

1927, xxxi, 361. 4. Aub, J. C., and Reznikoff, P., J. Ezp. Med., 1924, xl, 189. 5. Fairhall, L. T., and Shaw, C. P., J. Ind. Hyg., 1924, vi, 159. 6. Bischoff, F., Maxwell, L., Evans, R., and Nuzum, F., J. Pharmacol.

and Ezp. Therap., 1928, in press, 7. Ullmann, H. J., Radiology, 1927, viii, 461; J. Am. Med. Assn., 1927,

Ixxxix, 1218. 8. Pulford, D. S., Calif. and West. Med., 1927, xxvii, 18. 9. Soiland, A., Costolow, W., andMeland, O., Radiology, 1927, viii, 469.

10. Holt, L. E., Jr., La Mer, V. K., and Chown, H. B., J. BioZ. Chem., 1925, lxiv, 509.

11. Hastings, A. B., Murray, C. D., and Sendroy, J., Jr., J. BioZ. Chem., 1926-27, lxxi, 723.

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