EVIDENCE OF ADSORPTION EXPERIMENTS ON THE FORMS OF

EVIDENCE OF ADSORPTION EXPERIMENTS ON THE FORMS OF CALCIUM AND INORGANIC PHOS-

PHORUS IN BLOOD SERUM

BY DAVID M. GREENBERG AND CLARENCE E. LARSON

(From the Division of Biochemistry, University of California &fedical School, Berkeley)

(Received for publication, July 2, 1934)

It has been known, since the pioneer investigation of Rona and Takahashi (l), that the calcium of the blood stream exists in more than one form. Starting from this investigation there have been developed well defined procedures for fractionating the serum calcium into a diffusible and a non-diffusible component either by ultrafiltration or by compensation dialysis through collodion mem- branes. Many investigators assume that the calcium is present in more than one chemical state in both the diffusible and non- diffusible fractions of the serum.

In the present communication there will be mainly considered the claims for the existence of more than one form of calcium in the diffusible fraction. In this fraction, it has been proposed by Greenwald, Sendroy and Hastings, Klinke, Brull, and others (see (2) for literature) that there is present, besides ordinary calcium ion, an unknown organic citrate-like calcium compound. Accord- ing to the work of one of us and L. D. Greenberg (2) there is no good positive evidence for the existence of such a compound. However, there also is no evidence which completely rules out its existence.

Since the appearance of the above paper, Benjamin and Hess (3-5) have published the results of a study of the adsorbent action of BaSOl on serum. From this work they conclude that calcium is present in at least four forms in normal serum, two diffusible and two non-diffusible. They found no evidence for an organic calcium compound, but instead concluded that besides calcium ion, the diffusible calcium consists of a specific calcium-phosphorus

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106 Adsorption Experiments on Blood Serum

complex which experimentally is characterized by being adsorbable. According to them, calcium ion as such is not adsorbable by BaS04. The presence of this adsorbable calcjum-phosphorus substance is not limited to blood since they state that it is spon- taneously formed in artificial salt solutions that are buffered at a pH below neutrality. Benjamin and Hess attribute important physiological functions to this adsorbable compound in normal bone calcification and believe that it is abnormally low and there- fore the cause of the faulty calcification in rickets. Because of these important implications, it is desirable carefully to examine the basis of the claims for the existence of the adsorbable calcium phosphate. Our adsorption experiments were initiated to investi- gate the claims for the citrate-like organic calcium compound. With the appearance of the above publications, the experiments were extended to cover the adsorbable calcium-phosphorus complex, The results obtained, in our estimation, do not support the claims for the existence of any special form of diffusible calcium other than ordinary calcium ion.

EXPERIMENTAL

The experiments were carried out on blood serum, the ultrafiltrates of blood serum, and on artificial serum solutions prepared from the inorganic components in the proportion in which they are present in blood. Sealed tubes containmg the liquid and adsorbent were agitated in a mechanical shaker for periods of 3 hours or longer. The adsorbents employed were BaS04, kaolin, and permutit. These were prepared in the manner given below.

BaS04 Preparation l-This was prepared, as described by Ben- jamin and Hess, from BaN03 and HzS04. The precipitate was dried in an oven at 100”.

BaX04 Preparation P-Equivalent amounts of hot. dilute solutions of Na2S04 and BaClz were mixed. The precipitate was filtered, washed with water and alcohol, then dried by washing with ether.

BuSOh Preparation S-This was prepared by mixing equivalent portions of cold concentrated NanSOl and BaCL solutions. The finely divided precipitate was washed with dilute NaCl solution by centrifugation and then dried with alcohol and ether.

BaS04 Preparation &This was prepared by pouring 1 mole of cold saturated BaClz into 2 moles of cold saturated Na2S04 solu-


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D. M. Greenberg and C. E. Larson 107

tion. This forms a coagulum which breaks up into an extremely finely divided suspension on vigorous agitation. It is necessary to wash the precipitate by decantation with dilute NaCl in order to prevent a complete colloidal dispersion which is produced if it is washed with distilled water. After being washed with large quantities of 1 per cent NaCl solution until no further test for sulfate is obtained, the adsorbent is placed on a filter and dried by washing with alcohol and ether.

BaS04 Preparation 5-This was prepared on the same principle as Preparation 4, but with the idea of building up a Ba layer on the crystal surface. In this preparation, 1 mole of Na2S04 solution is poured into 2 moles of BaClz solution. In washing this precipitate, 1 per cent NaCl solution and then alcohol were used to prevent colloidal dispersion. The preparation was then dried with alcohol and ether.

Permutit-This was washed with distilled water and dried in vacua.

Kaolin-Kaolin was prepared by washing with 1 N HCl, then water, and drying in vacua.

The analytical methods used in this investigation were Kirk and Schmidt’s (6) for calcium, Fiske and Subbarow’s (7) for phosphate, and Morgulis and Hemphill’s (8) for sulfate. pH measurements were carried out by means of the glass electrode.

The few electro-osmosis experiments to determine the electrical charge of certain of the BaSOl adsorbents were carried out in a cell consisting of a U-tube, the two arms of the tube being sepa- rated by a 4 mm. bore stop-cock. In carrying out a determina- tion the stop-cock bore was tightly packed with the BaS04 to be tested and the solution introduced to an equal height in each arm of the cell. First, however, the BaS04 was shaken with a portion of the test solution. A 110 volt direct current was led in through platinum electrodes and the direction of the water osmosis was determined by the changes in level of the solution produced in the two arms of the cell.

Evidence of Adsorption on Claims for a Citrate-Like Complex Calcium Ion in Blood Serum

The results of adsorption experiments have been offered as proof of the presence of a negatively complex calcium ion in the blood. Thus Klinke (9) states that, while such positive adsorbents as


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Ca3(P0&, bone, BaS04, and MgNH4P04 remove calcium from blood serum, the negative adsorbents, kaolin and animal charcoal, do not. This alleged evidence can be readily shown to be without foundation. BaS04 adsorbs calcium from artificially prepared salt solutions containing no organic material. This has been shown by Benjamin (4) and was readily demonstrated by us.

The claim that negatively charged adsorbents will not adsorb calcium from serum also is not correct for all of them. In Table

TABLE I

Adsorption of Calcium from 8eTUm and fkTU?n ~~tTafi~~T&S by Certain Negatively Charged Adsorbents

Adsorbent Fluid

Permutit

Kaolin (acid- washed)

-

Serum A

Ultrafiltrate of Serum A

Serum A

Ultrafiltrate of Serum A

- -

AmcYt adsorb-

ent

gm. per 100 ml.

3 10 30

3 10 30

3 10 30

3 10 30

Calcium

a 58 $ zT$ i; :sb on E .4 --

“8. mg. ,er 100 per 101 ml. ml.

10.8 6.4 10.8 8.7 10.8 9.8

7.0 5.1 7.0 6.2 7.0 6.3

10.8 0.1 10.8 1.0 10.8 3.8

7.0 0.6 7.0 1.3 7.0 2.6

T

ml. ml. ‘7 10( ,er 101 ml. ml.

6.3 0.8 6.3 2.3 6.3 3.6 6.5 0.7 6.5 1.9 6.5 3.2 6.3 0.8 6.3 1.2 6.3 1.8 6.5 0.1 6.5 0.5 6.5 1.9

-

-

:a adsorbed P adsorbed

8.0 3.8 2.7 7.3 3.3 1.9 0.1 0.8 2.1 6.0 2.6 1.3

I there are presented the adsorption results obtained on beef serum and the ultrafiltrates from the same serum. It is seen from Table I that both kaolin and permutit, which are negative adsorbents, have removed calcium from the serum and from the ultrafiltrate. Also, they adsorb phosphate. These data do not bear out the claim that only positive adsorbents remove calcium from blood serum.

To test the influence of a negatively charged complex on adsorption, some experiments were carried out with solutions containing citrate. These are given in Table II. When citrate is present in


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considerable amount, as is seen from Table II, instead of calcium being more readily adsorbed by the positive adsorbent BaSO+ no adsorption at all is obtained as long as phosphate is absent. When phosphate is added to the same solution, some adsorption of calcium takes place, but not nearly to the same extent as from citrate- less solutions.

The concentration of citrate is of importance in connection with this effect, as is shown in Table II. In this experiment the adsorption by BaSOr and kaolin was tested on an inorganic serum

TABLE II

Adsorption with BaS04 Preparation S from Inorganic Serum Solutions Containing Citrate

Inorganic serum solution

Buffered with 0.05 M citrate, pH 6.7 No phosphate or bicarbonate

Phosphate added

Buffered with bicarbonate, 1 mole citrate added per mole Ca present

No phosphate

Phosphate added

Am2”nt adsorb-

ent

5% 3:

10 30 10 30

10 30 10 30

T Calcium T Phosphate

Ad- ;orbed

Ini- tially bresenl tL

_ -

1

;Bds J

Ini- tially resent

12.6 0 0 12.6 0 0 12.6 0.7 11.1 12.6 2.0 11.1

7.2 7.2 6.8 6.8

-

2.1 0 3.2 0 3.1 8.3 4.7 8.3

T

Ad- sorbed

% 2

0 0

2.0 3.4

solution containing citrate in the proportion of 1 mole of citrate to 1 mole of calcium. Both with phosphate absent and present, BaS04 removes calcium to a lesser extent than in the absence of citrate. However, just as was noted by Greenberg and Green- berg with the electrodialysis method, detection of a small quantity of a citrate-like calcium compound in blood is not feasible with any of the above adsorbents.

Adsorbable Calcium-Phosphorus Complex of Benjamin and Hess

An analysis of the evidence for the existence of the special adsorbable calcium-phosphorus complex proposed by Benjamin and


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Hess will now be undertaken.’ Among the arguments advanced by these authors in favor of their thesis, the most important are those given below. (1) Adsorption of calcium does not take place from solutions of such simple salts as the chloride, phosphate, and gluconate of calcium, so presumably calcium ion, as such, is not adsorbable. Adsorption of calcium in the presence of phosphate does take place when the solution is buffered at an alkaline pH by such substances as bicarbonate, sodium o-nitrophenol, sodium di- ethylbarbiturate, pyridine, and aniline. (2) Barium sulfate removes an approximately constant amount of calcium and phosphate from normal serum or ultrafiltrates, leaving a residue of calcium in the ultrafiltrate which agrees with the values in the literature for the ionic calcium of the serum. (3) The residue of calcium left behind after one adsorption by barium sulfate is not adsorbed by the immediate addition of fresh adsorbent, a supposed demonstration that the adsorbable calcium phosphate has been completely removed and the ionic calcium in the residue is not adsorbable. However, aft,er a lapse of 24 hours, more of the calcium becomes adsorbable.

We do not question the experimental data of Benjamin and Hess -indeed we have confirmed most of their findings. However, to confirm their observations, it is necessary to use a BaS04 adsorbent prepared essentially according to the directions of these authors; namely, from a sulfuric acid solution. In our estimation, quite a different interpretation can be placed upon their experiments than the one which these authors give. Our view-point is based on the experimental evidence given below which indicates that the adsorptive behavior of BaSOd preparations depends upon (1) the method of preparation, (2) the nature of the anions in the solution, and (3) the pH of the solution.

In the first instance, exception may be taken to the widely prevalent view that only one-third to one-half of the diffusible calcium of the blood is present as calcium ions. A critical examina-

1 While the analysis in the text is confined to the evidence for the existence of an ultrafiltrable calcium-phosphorus complex, it appears, par- ticularly from the work of Benjamin and Hess on hypercalcemic sera (5), that no specific fractionation of the non-diffusible components is obtained by BaSOa adsorption. This view is based on the widely varying ratios of adsorbed calcium to phosphorus reported.


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D. M. Greenberg and C. E. Larson

tion of the experimental measurements offered in support of this low level for the content of calcium ions in the blood (see (2)) shows that most of the experimental methods which have been employed for this purpose are objectionable, and that the results obtained by them have little weight. Evidence which favors a level of ionic calcium nearly equivalent to the values for the diffusible calcium of biological fluids has recently been offered by Mc- Lean and Hastings (10). The method used by these authors was based on the effect of calcium on the contraction response of the isolated frog heart.

Secondly, all BaSO* preparations do not show the same activity and it is readily possible to prepare BaSO., adsorbents of varying degrees of efficiency, ranging from ones which only have a small calcium adsorptive power to others with a higher efficiency than the BaSOe of Benjamin and Hess. Coarse precipitates formed from hot dilute solutions, as might be expected from known colloidal principles, were found to be poorer adsorbents than finely divided precipitates prepared from concentrated cold solutions. Further, on similar lines, an adsorbent prepared from an excess of sulfate ion is more effective in adsorbing the cation calcium, and one prepared from barium excess is more efficient for phosphate. In this way the amounts of calcium and phosphate adsorbed can readily be altered. These factors are well illustrated by the data given in Table III.

In the adsorptive series from artificial serum solutions given in Table III, BaSOl preparations embodying all the factors just men- tioned have been employed. BaS04 Preparation ‘2, prepared from dilute hot solutions, is the poorest adsorbent on the list for both calcium and phosphate; Preparation 1, prepared according to the method of Benjamin and Hess, is very effective for both calcium and phosphate; Preparation 3, from cold concentrated solutions, is nearly as good as Preparation 1. However, Preparation 4, prepared from sulfate excess, removed calcium almost to the limit of analytical detection, but only moderately adsorbed phosphate, while Preparation 5, prepared from an excess of barium, removed the phosphate completely. The particular amount of adsorption obtained by Benjamin and Hess, it is seen from this series, does not have the significance they assume, but merely appears to be the result of a chance preparation of a particular adsorbent. From


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our list of adsorbents, the activity of BaSO., Preparation 4 and, in some instances, permutit, which removed practically all of the calcium from serum, serum ultrafiltrates, or inorganic solutions, although leaving behind a considerable proportion of the phosphate, can hardly be fitted to the Benjamin and Hess theory. To do so would require that practically none of the calcium be present as the ion in the serum or the inorganic solutions.

TABLE III

Adsorption from Inorganic Serum Sol&ions*

Adsorbent

Kaolin

Permutit BaSOl Preparation 1

BaSO( Preparation 2

BaS04 Preparation 3

BaSOl Preparation 4

BaSOa Preparation 5

*YYt sdsorb-

ent

%’ 3T 10 30 10 10 30 10 30 10 30 10 30 40 40

Calcium Phosphate

22 2 ag :z $ 3

4 -- w. %7. er 100 per 101 ml. ml.

9.0 3.8 9.0 4.8 9.4 6.9

11.0 8.7 11.0 9.7 11.0 4.0 11.0 6.5 11.0 5.8 11.0 7.5

9.8 8.4 9.8 9.4

10.2 9.8 9.9 7.4

2% 38 .% a, d

-

P P

-j

mg. mg. er 10 ,er 101 ml ml.

8.8 3.8 8.8 7.4 8.7 3.7 8.5 5.0 8.5 6.2 8.5 2.1 8.5 4.4 8.5 3.1 8.5 4.9 8.4 4.2 8.4 4.4 5.5 2.8 5.5 5.5

Ca adsorbed P adsorbed

1.0 0.6 1.9 1.7 1.6 1.9 1.5 1.9 1.5 2.0 2.1 3.4

* The inorganic serum solutions used in these experiments were prepared to contain 80 mM of N&l, 75 mM of NaHCO,, 4 mM of KCl, and 3 mM of KzSOa per liter. Calcium chloride and dipotassium phosphate were added in the amounts desired. The pH of each solution was adjusted to 7.4 by bubbling in COZ.

While with the BaS04 adsorbent prepared by the method of Benjamin and Hess no calcium is adsorbed from simple calcium salt solutions, by the principles just given, it is possible to prepare BaSOl powders that will readily adsorb calcium even from these. Such a series of experiments is given in Table IV, where the adsorption of BaS04 Preparations 1 and 4 and permutit was tested on


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dilute solutions of the chloride, lactate, gluconate, and phosphate of calcium. BaSOl Preparation 1 removed no calcium. On the other hand, both BaS04 Preparation 4 and the permutit removed the calcium from these solutions almost to completeness. Calcium ion as such, then, can be adsorbed by some BaS04 and other adsorbents.

Not only the method of preparation, but also the nature of the anions present in the solution, markedly affects the adsorption of

TABLE IV

Adsorption from Dilute Solutions of Calcium Salts

Adsorbent

BaSOl Prepara- tion 1 (40 gm. per 100 ml.)

BaSO( Prepara- tion 4 (30 gm. per 100 ml.)

Permutit (30 gm. per 100 ml.)

Salt

CaClz I< in 0.1 N NaOH (I “ 0.1 “ HZSO ‘L “ 0.1 per cent Na#O,

Ca lactate “ gluconate

CUCIZ ‘, in 0.05 M HCI

Ca lactate “ gluconate “ phosphate (CaHP04)

CaCL Ca lactate “ gluconate

- 1 %X 10.3 11.6

9.1 11.5

6.4 8.2

15.0 8.0 8.2 5.8 5.0

16.1 22.1 14.6

-

_

” ? 1

e PH 8- 3 2 8 8

.s .M

=: “a f? $

“2 EO a’;: o-3 Ld

D 2 m gal

-4 --- ng. per ‘00 ml.

10.5 6.504.00 10.0 7.7 9.6 6.7 6.574.16 8.4 6.324.18 0.2 6.508.67 1.7 0.3 6.578.5G 0.2 6.33 8.33 0.0 0.2 0.2 0.2

The BaSOa adsorbents have the following pH in distilled HzO: Prepara- tion 1, 4.0; Preparation 4, 8.62; Preparation 5, 3.96.

calcium by BaS04 and also by other adsorbents. An insight into the reason for this is offered by the work of Michaelis and Dokan (11) on the influence of ions on the electrical charge of BaS04. By electro-endosmose experiments, these authors showed that BaS04 precipitates usually carry a positive electrical charge. With the exception of hydrogen and hydroxyl ions, univalent ions influence this charge very little. Hydrogen ions markedly increase the


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positivity of the charge and hydroxyl ions are capable of reversing the sign of the charge of BaSOl from positive to negative. How- ever, ions of higher valency strongly affect the charge, and bivalent anions too are readily capable of reversing the sign of the adsorbent from positive to negative. While hydrogen ions tend to increase the degree of positiveness of the charge, their effect may be out- weighed by anions of higher valence. As an example of this, BaS04, when placed in dilute sulfuric acid solution, is found to assume a negative charge.

The adsorption of calcium by BaS04 parallels the changes produced in the electrical charge. Adding phosphate or increasing

TABLE V

Independent Adsorption with BaSOn Preparation 3 of Calcium and Phosphate from Inorganic Serum Solutions

Buffered with 0.01 M borate, pH 7.60, no phosphate or NaHC03

Phosphate added

Buffered with 0.05 M potassium acid phthalate, pH 4.0

-

-

Amount of adsorbent

Orn. ,“,“rT loo

10 30 10 30 10 30

Calcium T

hitirdly present

ml. PO 100 ml.

7.6 7.6 7.6 7.6 6.1 6.1

T _ Ad-

sorbed _ .

l;“o”d sz

0.8 1.7 2.4 3.7 0 0.9

-

Phosphate -

IGti~dl~

- mg. per 100 ml.

Ad- sorbed

mg. per * 100 ml.

6.0 2.6 6.0 3.8 8.1 0 8.1 0.1

the phosphate concentration of a solution increases the amount of calcium which is adsorbed. Ample evidence of this is offered in the work of Benjamin and Hess. Our own experiments, shown in Table IV, demonstrate that the BaS04 of Benjamin and Hess, listed here as Preparation 1, which does not adsorb calcium from aqueous solutions of simple calcium salts, does adsorb calcium from calcium chloride dissolved in dilute solution of NaOH, Na2S04, or even HJ304.

From Table V it is seen that even BaS04 preparations which are capable of adsorbing calcium from solutions containing no phosphate ions show an increase in the amount of calcium adsorbed, if phosphate is present and the solution is buffered on the alkaline side of neutrality.


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The fact that other polyvalent ions can also favor the adsorption of calcium is strong evidence that the formation of a peculiar calcium-phosphorus compound is not a necessary condition for the adsorption of calcium by any BaS04 preparation. In Table VI there are tabulated experiments which demonstrate the capacity of sulfate ion to act in this manner. In these experiments it is

TABLE VI

Injuence of Sulfate on Adsorption of Calcium Inorganic Serum Solution Containing No Phosphate, Buffered with Bicarbonate at pH 7.4

Constant sulfate content, varying amounts BaSOc Prepara- tion 1

Varying sulfate content, constant amount BaSOl Prepara- tion 1 (40 gm. per 100 ml.)

-

-_

-

Amount of adsorbent

gm. pe7 100 ml.

0

10 20 30 40 60 80

100

Sodium sulfate added to solution

per cent 0

0.10 0.20 0.40 0.50

T Calcium

Present

mg. per 100 ml.

8.0 6.2 5.8 5.8 5.6 5.3 5.0 4.2

1

TbQd 3: ;$.;r ?o$ iL?

0 47.3 0

1.8 45.2 2.1 2.2 44.9 2.4 2.2 42.4 4.9 2.4 40.4 6.9 2.7 38.1 9.2 3.0 35.1 12.2 3.8 32.2 15.1

9.5 0 0 7.8 1.7 5.6 7.8 1.7 7.0 7.9 1.6 7.0 7.8 1.7 6.8

Ad- sorbed

T- Sulfate

‘resent Ad- sorbed

shown that BaS04 Preparation 1, which does not adsorb calcium from solutions of calcium salts of univalent anions, acquires the capacity to adsorb calcium if the bivalent sulfate ion is added to the solution. It is true that the sulfate ion is not quite so effective in this respect as is the corresponding phosphate ion, probably because phosphate ion is more readily adsorbable. Paralleling the behavior of this adsorbent in phosphate-containing solutions, there is an adsorption of sulfate as well as of calcium from solutions


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in which sulfate replaces phosphate. As has already been pointed out, calcium is adsorbed by BaS04 Preparation 1 from solutions of calcium chloride containing NaOH, Na$SOb, or HzS04.

The influence of hydrogen ion is somewhat subsidiary to the other factors which have been discussed. Depending upon their preparation, the different BaS04 adsorbents show either an acid or alkaline reaction in solution. Preparation 1 changes the reaction of the solutions into which it is introduced to a greater acidity. In the solutions of the calcium salts in the experiments of Table IV, the acid shift is from about pH 6.5 to 4.0. BaSOl Preparation 5, prepared from a barium excess, also shows a residual acidity. In distilled water it developed the pH of 3.96. On the other hand,

TABLE VII Effect of Adsorption on pH of Serum and Inorg anic Serum Solutions

Solution

Beef serum........................ Inorganic serum solution. . Beef serum................. Inorganic serum solution.

LL “ ‘I Beef serum...

‘I I‘ . . . . . . . . . . . . . .

BaSO!

prepi%?n

PH

Before After adsorphon adsorption

7.51 7.44 7.60 7.42 7.48 7.35 7.57 7.48 7.60 7.46 7.48 7.52 7.51 7.13

BaS04 Preparation 4, prepared from sulfate excess, produces an alkaline shift in dilute calcium salt solutions from about pH 6.5 to 8.5.

The pH of blood serum and inorganic serum solutions is also shifted by the BaS04 adsorbents. The magnitude of this effect is illustrated in Table VII. All the BaS04 preparations, except Preparation 4, cause a shift of pH toward greater acidity in serum or inorganic serum solutions. This is true of adsorbents prepared from equivalent quantities of the neutral salts, BaClz and Na2S04, as well as the powder prepared from acid solution or an excess of BaC12. The mean change in pH produced by these adsorbents is around 0.15 unit. BaS04 Preparation 5, prepared from barium excess, produced an alteration of about 0.35 pH unit. On the other


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hand, Preparation 4, which has an alkaline reaction in water, caused a small alkaline shift in serum.

The residual acidity of the Benjamin and Hess BaS04 preparation helps to explain why it does not adsorb calcium from dissolved CaHP04. The acidity from the BaSO* Preparation 1 aids the hydrolysis of the secondary to the primary phosphate. This being a univalent anion influences the nature of the adsorption very little.

On the other hand, the favoring effect pointed out by Benjamin and Hess of bicarbonate and other alkaline buffers on the adsorption of calcium follows from the increase and the stabilization of the content of the higher valence phosphate ions, and probably also from a neutralization of hydrogen ions adsorbed on the BaS04. In solutions on the acid side of neutrality, the other BaS04 preparations tend to acquire the characteristics of Preparation 1. Thus, BaS04 Preparation 3, which readily adsorbs calcium and phosphate from solutions on the alkaline side of neutrality, adsorbs only a trace of calcium and no phosphate from a solution buffered with potassium acid pthalate at the pH of 4.0. This is shown in Table V. On the other hand, BaS04 Preparation 4, because of its excess of sulfate, readily adsorbs calcium even from 0.05 M HCl solution (see Table IV).

DISCUSSION

The following hypothesis is offered as an attempt to explain the many different features observed with the individual adsorbents in various types of solutions. The explanation will be confined to the BaS04 adsorbents, although the others can readily be fitted into a similar scheme.

From the work of Michaelis and Dokan, it would appear that monovalent ions are only very slightly adsorbed but bivalent ions and ions of higher valency are readily adsorbed by BaSOa powders. Exceptions to this are hydrogen and hydroxide ions which are also strongly adsorbed. One would expect the common ions, barium and sulfate, to be quite strongly adsorbed by BaSOa. The nature of the ions which are adsorbed determines the electric charge upon the BaS04. In the course of their formation, the BaS04 powders acquire an adsorption layer which is dependent on the type of the solution from which they are prepared. This layer


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is all important since, in all likelihood, it determines the specific properties of each adsorbent. The subsequent. behavior of each adsorbent, when equilibrated with various solutions, to a large extent, consists of an exchange between the ion components of the already adsorbed layer for other strongly adsorbed ions which may be present in a given solution. Some qualitative electro- osmosis measurements, carried out on certain of the BaSO* preparations, may be offered in favor of the above statements.

BaS04 Preparation 1, it was observed, carried a strong positive charge when it was immersed in an inorganic serum solution buffered with bicarbonate at pH 7.4, but containing no calcium

TABLE VIII Further Adsorption Obtained on Addition of Fresh BaSO, Preparation 1

(40 Gm. per 100 Ml.)

The values are given in mg. per 100 ml.

-i

Initial value.. . . . . . . After 3 hrs. shaking.. . Liquid then divided into 2 portions

Sample 1. Immediately shaken with fresh BaSO,. .

Sample 2. Kept for 24 hrs., then shaken .

Calcium Phosphate

Present

7.1 3.1

,,“r& Present --

0 6.0 4.0 4.0

2.1 1.0 Trace 1.8 1.3 “

Ad- sorbed

0 2.0

4.0 4.0

or phosphate. BaSOI Preparation 5 likewise was strongly positive under the same conditions. When phosphate equivalent to 5 mg. of P per 100 ml. was added to the solution, the charge on BaSOl Preparation 1 became negative in sign. On the other hand, BaS04 Preparation 4, in the absence of either calcium or phosphate, did not show a charge of sufficient magnitude to be detected with the apparatus which was employed.

The adsorption of calcium, it was found, is favored by the presence of phosphate or of sulfate. Adsorption of calcium ions alone leads the adsorbent to become increasingly positive in charge and so tends to inhibit the further adsorption of calcium. Similarly, adsorption of phosphate alone should lead to the inhibition of phosphate adsorption because of the increasing negativity. How-


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ever, by the adsorption of the two ions together, there is a neutralization of the charge which makes it possible to increase the amount of each which is adsorbed through the building up of extended layers, of alternatingly adsorbed calcium and phosphate.

An explanation appears from this reasoning for the last of the three important arguments that have been advanced for the existence of a specific adsorbable calcium phosphate; namely, the demonstration of a non-adsorbable residue believed presumably to consist of calcium and phosphate ions.2 When by adsorption with one portion of BaSO., Preparation 1 the phosphate is reduced to a level too low further to appreciably affect the charge, no calcium will be adsorbed by a fresh portion of BaS04 because of the in- hibiting effect of the positive charge it carries.

Biochemical SigniJicance-If the failure of the Benjamin and Hess theory of the existence of a specific adsorbable calcium-phosphorus complex is granted, then their claims for an important relationship between the amount of adsorbable calcium with normal calcification and its aberration in rickets has to be revised. The findings of Benjamin on the calcification of cartilage comes to no more than the well known experiments of Shipley, Kramer, and Howland (12) with the substitution of an adsorption explanation. Shipley, Kramer, and Howland found that when the product of calcium and phosphorus expressed in mg. per cent was 40 or above, calcification readily takes place, but below a level of about 35 it does not. Benjamin no more than repeated this observation. While we do not subscribe to the view that the Ca X P

2 We have not been able unequivocally to confirm the experimental evidence offered in this connection. With one preparation of BaSOc Preparation 1 there was obtained no further adsorption of calcium after a first adsorption either immediately or after 24 hours standing. With another preparation, however, there was obtained a further uptake of calcium of about the same magnitude on second adsorption both when carried out immediately or after a 24 hour period. The results of an experiment with the latter preparation, which was carried out on an inorganic serum solution of the composition used by Benjamin (4), are shown in Table VIII. Furthermore, the observation that calcium and phosphate are as readily adsorbed and apparently to the same extent from solutions to which calcium and phosphate have been freshly added, as from those of long standing, is difficult to reconcile with the deduction drawn by Ben- jamin that the alleged adsorbable calcium-phosphorus complex is only slowly formed.


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product is a quantitative measure of calcification, it appears to us that a true solubility rather than adsorption is involved in calcification.

The lowered adsorption of calcium from rachitic serum is also readily explainable by the small amount of phosphate ion present without any need to postulate the absence of a specific compound. Increasing the phosphate content, it has been noted, leads to an increase in the amount of calcium adsorbed and a logical explanation for this has been offered in this discussion. On this reasoning the lowered phosphate in rickets would rationally lead to a decrease in calcium adsorption.

Physicochemical States of Calcium and Phosphate in Serum- The analysis in this paper and that in the previous publication by the senior author and L. D. Greenberg (2) show that a very large part of the evidence offered for the existence of biochemically important calcium compounds in the diffusible fraction of blood serum, other than ordinary calcium ion, is unsubstantial. This holds for both a citrate-like calcium compound and a specifically adsorbable calcium-phosphorus complex. It is true that the work given in these two papers does not categorically dismiss the possi- bility of such special calcium compounds, but rather shows that their existence has in no way been actually demonstrated. How- ever, until such compounds, if they exist, are firmly established, it is more useful to attempt to explain the properties of blood on the basis of existing knowledge.

Until more substantial proof is offered for other substances, it is more useful to accept the existence of only calcium and phosphate ions in the diffusible component of blood serum. Even if low values of calcium ion activity in serum are admitted, it is quali- tatively, at least, to be ascribed to an explanation familiar in connection with modern theories of strong electrolytes; namely, as the result of the depressing action of the interionic influence of the electrolytes and proteins of the blood.

SUMMARY

Adsorption experiments with variously prepared BaS04 powders, permutit, and kaolin give no evidence for the existence of either a citrate-like calcium compound or for a specifically adsorbable calcium-phosphorus complex in blood serum. Instead, all of the


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D. M. Greenberg and C. E. Larson

adsorption features which have been attributed to these hypo- thetical substances can be obtained under the proper conditions with merely calcium and phosphate ions.

It is preferable to accept the existence of only calcium and phosphate ions in the diffusible fraction of the serum until there is good evidence to the contrary.

BIBLIOGRAPHY

1. Rona, I’., and Takahashi, D., Biochem. Z., 31,336 (1911). 2. Greenberg, D. M., and Greenberg, L. D., J. Biol. Chem., 99,l (1932-33). 3. Benjamin, H. R., and Hess, A. F., J. BiuZ. Chem., 100, 27 (1933). 4. Benjamin, H. R., J. Biol. Chem., 100, 57 (1933). 5. Benjamin, H. R., and Hess, A. F., J. Biol. Chem., 103, 629 (1933). 6. Kirk, P. L., and Schmidt, C. L. A., J. BioZ. Chem., 83,311 (1929). 7. Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 68,375 (1925). 8. Morgulis, S., and Hemphill, M., J. BioZ. Chem., 96, 573 (1932). 9. Klinke, K., Ergebn. Physiol., 26, 235 (1928).

10. McLean, F. C., and Hastings, A. B., Proc. Sot. Exp. BioZ. and Med., 30, 1344 (1933); J. BioZ. Chem., 108, 285 (1935).

11. Michaelis, L., and Dokan, S., KoZZoid-Z., 37, 67 (1925). 12. Shipley, P. G., Kramer, B., and Howland, J., Biochem. J., 20,379 (1926).


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David M. Greenberg and Clarence E. LarsonPHOSPHORUS IN BLOOD SERUM

CALCIUM AND INORGANICEXPERIMENTS ON THE FORMS OF

EVIDENCE OF ADSORPTION

1935, 109:105-121.J. Biol. Chem.

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