THE DISTRIBUTION OF SODIUM, POTASSIUM, CALCIUM, AND MAGNESIUM BETWEEN THE CORPUSCLES

AND SERUM OF HUMAN BLOOD.

BY BENJAMIN KRAMER AND FREDERICK F. TISDALL.

(From the Department of Pediatrics, the Johns Hopkins University Baltimore.)

(Received for publication, June 22, 1922.)

A knowledge of the concentration of sodium, potassium, calcium, and magnesium in the blood is necessary in connection with a variety of problems. Methods hitherto used for the quantitative determination of these elements in blood have required large amounts of material even for a single determination and have been, in most cases, difficult to carry out. These facts have tended to discourage such studies, particularly with human blood.

The earliest recorded figuresfor the concentration of cations and anions in human blood, corpuscles, and serum, are those reported by Schmidt (1) in 1850. Wanach (2) in 1888 estimated the sodium and potassium in the blood corpuscles and serum of eight adults. Some years before this Bunge (3), a pupil of Schmidt, reported a number of complete analyses of the ash of the blood corpuscles and serum of several animals. Abderhalden (4) later published the results of a similar study on a larger series of animals. No studies on the distribut,ion of cations between the corpuscles and serum of normal human blood have appeared since those of Wanach.’

The introduction of methods for the quantitative determination of sodium, potassium, calcium, and magnesium with small amounts of serum and whole blood (5 to 9) has made such studies on human

r After this paper had been written an article appeared on the effect of changes in COz tension upon the distribution of sodium, potassium, chlorine, phosphorus, and bicarbonate between the corpuscles and plasma of defibri- nated and filtered beef blood (Doisy, E. A., and Eaton, E. P., J. Biol. Chem., 1921, xlvii, 377).

241

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242 Sa, I<, Ca, and Mg in Human Blood

subjects possible. We have investigated the concentration of these elements in the venous blood and serum of normal adults.. The determinations were made on serum in preference to plasma because the addition of an anticoagulant (usually a salt) is thereby avoided. This permits the determination of all the cations to be made on the same sample. Hemolysis occurs less frequently with serum than plasma. We have found the potassium and calcium content of titrated plasma to be pract,ically the same as that of serum. Schmidt (1) has shown that the inorganic composition of plasma is identical with that of serum.

We have repeatedly demonstrated the remarkable constancy of the concentration of the elements (sodium, potassium, calcium,

TABLE I.

Concentration of 8odiu.m in Blood Serum and Corpuscles oj Normal Adults.

Sample. PlUSma.

per cent m0. m0. m0. m0.

59 335 193 198 -12 65 335 220 218 +6 58 335 195 194 +3 57 335 186 191 -12 58 335 187 194 -17 56 335 189 187 +5 60 I 335 199 201 -5

Na per 100 cc. 8er”nl.

Na in plasma of 100 cc. of

blood.

T Nn in 100 cc. corpuscles calculated.

and magnesium), in the serum of normal adults and have there- fore assumed in Tables I and II the average of our previously reported figures for sodium and potassium as representing the concentration of these elements in the serum of the normal adult male. The concentra.tion of calcium was det,ermined directly on ashed plasma. The whole blood samples were collected in dis- tilled water and weighed and the cations determined by methods referred to above. The relative proportion of corpuscles to plasma was determined by the use of hematocrit, and the concen- tration of the various elements in the corpuscles calculated from the assumed concentration of the respective element in the serum, its concentration in whole blood, and the percentage of corpuscles.

The results of our determinations are given in Tables I to VI.

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B. Kramer and F. F. Tisdall 243

Column 2 of Table I shows the proportion of plasma in the blood samples analyzed; Column 3, the average value for sodium expressed in mg. per 100 cc. of serumforall our determinations with sera of normal adults. The figures for sodium of whole blood are given in Column 4. The number of mg. of sodium present in the amount of serum contained in 100 cc. of the given whole blood sample, is given in Column 5. It will be seen that the latter is practically the same as the sodium concentration in the whole blood of the sample; i.e., that there is no sodium in the corpuscles.

TABLE II.

Concentration of Potassium in .Blood Serum and Corpuscles of Normal Adults.

Sample.

1 2 3 4 5 6 7 8 9

10 11 12 13

Average.. . . .

Plasma.

per cent 61 60 57 68 58 57 61 62 57 59 65 65 56

K per 100 CD. serum.

K per 100 cc. corpuscles.

mu.

19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5 19.5

I I

ml. %7.

172 410 187 438 188 410 153 437 186 420 200 438 180 430 175 428 202 444 193 441 164 413 169 425 201 430

.- 60.5 19.5

I 182

I 428

Column 4 of Table II gives the figures for the number of mg. of potassium per 100 cc. of whole blood. We have calculated the concentration of the same element in 100 cc. of corpuscles and placed the results in Column 5. It isevident that the K content of the blood of the adult man varies directly as the corpuscular content. With a corpuscular content of 43 per cent (Sample 3) the K of 100 cc. of whole blood was 188 mg. whereas when the percentage of corpuscles dropped to 32 (Sample 4) the K content of the whole blood fell to 153 mg. per 100 cc. This is shown in a more striking manner if one calculates the K content of the cor- puscles. When this is done the constancy of the K content of the

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244 Na, K, Ca, and Mg in Human Blood

corpuscles becomes apparent (see Column 5). Since, as we shall see later, the corpuscles contain no calcium and only traces of magnesium and, as we have seen from results recorded in Table I, no sodium, it becomes obvious that potassium constitutes practi- cally all the fixed mineral base of the corpuscles. In this respect man differs from a number of animals, namely the dog and cat,

TABLE III.

Concentration of Calcium in Blood Serum and Corpuscles of Normal Adults.

Sample. Plasma.

per cent 5s 57 72 59 58 65 57

cap~;;mly ec. ca y;o cc. ca’ci”m in Ca in 100 cc. plasma of of corpuscles

’ 100 cc. blood. calculated.

mg. mg. mQ. mg.

10.0 5.3 5.8 -1.2 9.5 5.3 5.4 -0.2 9.5 6.7 6.8 -0.4 9.8 6.2 5.8 $1.0 9.5 5.3 5.4 -0.2 9.3 5.9 6.0 -0.3 9.7 5.5 5.5 *o.o

TABLE IV.

Concentration of Magnesium in Blood and Serum of Normal Adults.

Sample. Magnesium per 100 cc. whole blood.

mQ.

2.6 4.0 3.8 3.8 2.8 2.8 3.8 2.3

Average.............. 3.2 2.5

Magnesium per 100 cc. serum.

whose corpuscles contain potassium in practically t.he same con- centration as does their plasma. These animals make up their deficit of potassium in the corpuscles with sodium.

Table III shows that in seven consecutive instances, we analyzed the calcium of the serum and whole blood and calculated from these data and the hematocrit reading the calcium of the cor-

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B. Kramer and F. F. Tisdall

puscles. In not a single instance were we able to demonstrate, with certainty, the presence of calcium in the corpuscles.

Table IV requires no explanation.

SUMMARY.

1. We have previously shown that the concentrations of sodium, potassium, calcium, and magnesium in the sera of normal adults and children are singularly constant.

2. Table I shows that human corpuscles are practically free of sodium.

3. The concentration of potassium in human corpuscles is remarkably constant varying only from 410 to 440 mg. per 100 cc. of corpuscles. The average value found for thirteen samples was 428 mg. This is about twenty times the concentration of the same element in serum. Potassium represents practically all the fixed mineral base of human corpuscles.

4. Only about 2 to 4 mg. of magnesium are present in 100 cc. of corpuscles.

5. The magnesium content of whole blood varies from 2.3 to 4.0 mg. per 100 cc. These figures agree with those reported by earlier investigators.

6. In a study of seven consecutive normal bloods we found practically no calcium in the corpuscles.

The Presence of Cal&urn in Corpuscles.-The question of the presence of calcium in blood corpuscles has recently been the sub- ject of considerable discussion. The earlier workers (4) regularly found no calcium in the corpuscles. More recently a number of investigators have reported the finding of considerable amounts of calcium in blood corpuscles. Hamburger (10) found as much as 32 mg. of calcium per 100 cc. of corpuscles. Rona and Taka- hashi (11) were able to demonstrate the presence of only 1.0 to 2.4 mg. in an equal volume of corpuscles while Heubner .and Rona (12) found demonstrable amounts of calcium in the red blood cells of only six cats in a series of twenty-six animals studied. Cowie and Calhoun (13) maintain that considerable amounts of calcium are present in corpuscles and in more recent publications Jones and Nye, and Jones (14) insist upon the presence of calcium in cor- puscles in appreciable amounts.

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246 Xa, K, Ca, and Mg in Human Blood

Howland and Marriott (15) found human corpuscles to be free of ca.lcium. Lamers (16) concluded from his analyses of the blood of healthy women and of women suffering with a variety of diseases, that detectable amounts of calcium do not occur in human corpuscles. Richter-Quittner and Falta (17) also failed to find calcium either in human blood corpuscles or in those of animals. In a series of seven consecutive samples of blood from seven normal adults we have likewise been unable to demonstrate the presence of calcium in the corpuscles of a single individual. We shall not enter here into a description of the various controls which we’ have made to convince ourselves of the accuracy of our methods both for serum as well as for whole blood: These have been described elsewhere in detail. We are convinced, and our conviction is based upon a large number of analyses of human blood, and of the blood of a variety of animals, that the calcium of serum or plasma is remarkably constant for a large variety of normal animals (man, dog, rat, sheep, and cow), varying only from 9 to 11 mg. per 100 cc. of serum. The concentration of the same element in whole blood shows a much greater fluctuation because of the variation in the percentage of corpuscles in different samples. Nevertheless, the figure for whole blood rarely exceeds 7 mg. per 100 cc. of blood. Neither does it fall, except in cases of marked polycythemia, below 5 mg. Table III illustrates some of these statements. We have been unable to demonstrate the presence of calcium in appreciable amounts in the corpuscles of the normal adult and are inclined to attribute the finding of calcium in the corpuscles by others to errors in their calcium determinations. The sources of such errors in the determination of minute amounts of calcium have been discussed by Kramer, Tisdall, and Howland (18).

A simple example in which the figures used represent the average of a large series of determinations made by Jones and Nye (14) on the blood of normal boys and girls, will serve to illustrate the usual error in such investigations. The average calcium con- centration per 100 cc. of plasma was found by them to be 10.1 mg., that of whole blood 9.4 mg., and the percentage of corpuscles was 38.4. If there were no calcium in the corpuscles the concen- tration of calcium per 100 cc. of whole blood should have been 10.1 x 61.6 = 6.2 mg. A glance at Table III shows this to be a

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B. Kramer and F. F. Tisdall 247

normal value for the calcium concentration of whole blood. The amount actually found by Jones and Nye was 9.4 mg. They used the method of Lyman (19) for their calcium determinations. It has been pointed out elsewhere (18) that when the supernatant fluid obtained after precipitating plasma proteins with trichloro- acetic acid (as performed in the Lyman procedure) is filtered through even good grades of acid-washed filter paper, calcium, in variable, but demonstrable amounts, may enter the filtrate and be responsible for many of the high values for calcium frequently obt’ained.

Existence of Alkali Protein Cornpounds in BZood.-The con- centrations of chlorine and bicarbonate have been repeatedly determined for normal serum and plasma by many investigators. We have tabulated some of these results in Table V in grams, gram equivalents, and their acid equivalent expressed as cc. of 0.1 N

acid per liter. The concentrations of chlorine and bicarbonate in corpuscles have been determined by Fridericia (20). Means (21) and his collaborators have also studied the distribution of bicarbonate between the corpuscles and plasma of normal adults. Some of these determinations are given in Tables V and VI. The value for the concentration of inorganic phosphorus in the serum represents the average of a large number of determinat,ions by ourselves. We have accepted Bloor’s statement (22) that the inor- ganic phosphorus of corpuscles is about twice that of serum.2 De Boer (23) found the concentration of sulfate in plasma to be 0.002 M. The figures recorded for the concentration of sodium, potassium, calcium, and magnesium in corpuscles and serum are the averages of all the determinations which we have made.

If one calculates the concentrations of acid and basic equivalents found in serum and corpuscles, it is found that in each case there is an excess of base of about 16 per cent. Since the pH of normal blood is about 7.35, this base cannot be free. In considering substances that occur in normal blood and might bind fixed base one thinks of (a) proteins functioning as acids (Loeb, 24) and (b) organic acids, including lactic acid and amino-acids. In a recent review Van Slyke (25) states:

* This statement has recently been challenged by Zucker and Gutman, who maintained that the inorganic phosphorus concentration is the same both inside and outside the red blood cells (Zucker, T. F., and Gutman, M. B., Proc. Sac. Exp. Biol. and Med., 1921-22, xix, 169).

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248 Na, K, Ca, and Mg in Human Blood

“ . . . . it is practically certain that it (blood) contains no sub-

stances in considerable amount of which we do not have at least sufficient knowledge to tell whether or not they can act as buffers, i.e., whether or not they are salts of weak acids or bases. An examination of the constitu- tents (of blood) reveals among those present in amounts sufficient to have significant effect only the proteins, the bicarbonate, and the phosphate, which can be expected to act as CO2 carrying buffers.”

These and chlorine, therefore, would represent the base-binding substances of blood.

TABLE V.

Concentration of Basic and Acid Radicles in Serum.

I Per liter. Gram equivalents per liter.

0.1 i-i base.

Basic radicles.

I om. I Na ............... Ii ................ ca ................ hfg ................

Total ............

3.350 0.1460 0.200 0.0050 0.100 0.0050 0.030 0.0025

0.1585

cc.

1,460 50 50 25

1,585

Acid radicles.

-Cl .............. -HCOs ........... -HPO,. .......... -so,. ............

3.600 0.1010 1,010 1.630 0.0267 267 0.092 0.0010* IS 0.192 0.0040 40

Total. . .

Excess of base. . .

0.1327 1,335

0.0258 250

Percentage of base not combined with acid radicles, 16 per cent. * This figure is really the molar concentration of HP04 rather than the

equivalent. The difference, however, is so small as to be negligible. The equivalent of 0.1 N base has been calculated on the basis of what the ratio

NZHPO, would be for ~

NaHpPOa at pH 7.35.

According to Campbell and Poulton (26) the isoelectric point of hemoglobin is at pH 6.98. Michaelis (27) gives the isoelectric point of serum globulin as pH 5.5 and that of serum albumin as pH 4.7. The pH of normal serum is 7.35 and that of corpuscles

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B. Kramer and F. F. Tisdall 249

differs probably only slightly from this figure (28). Loeb (24) has demonstrated that at a pH greater than that of their isoelectric point proteins function as acids forming readily dissociable salts with univalent, cations. These facts make the existence of protein cation compounds in serum and corpuscles highly probable.

Bloor (22) has shown that the so called undetermined phosphorus in the corpuscles may be very high. This fraction he considers as possibly an organic acid. Since we know practically nothing as to its nature, it is idle to speculate as to the possibility of its

TABLE VI.

Concentration of Basic and Acid Radicles in Corpuscles.

I Per liter. Gram equivalents

per liter. 0.1 N base.

Basic radicles.

!3m. cc.

Ii . . . . . . . . . . . . . . 4.280 0.1097 1,097 hlg . . . . . . . . . . . 0.050 0.0040 40

Total. . . . . 0.1137 1,137

-HCOa.. . . . . . . -Cl.. . . . . . . . . -HPO(. . . . . .

Total...........

Excess of base.

Acid radicles.

1.680 0.0276 276 2.230 0.0628 62s 0.182 0.0020 36

0.0924 940

0.0213 197

Per cent of base not combined with acid radicles, 17 per cent.

forming any compounds with potassium. Ryffel (29) has shown that even normal blood may contain 0.012 per cent lactic acid. Nevertheless, the ease with which this acid is formed in vitro in biological material containing sugar raises the question of its actual existence in the circulation under normal conditions. Amino-acids might possibly bind 20 cc. of 0.1 N base in the serum and GO cc. in the corpuscles (30).

JQe may conclude, therefore, t,hat there are other substances in normal serum and corpuscles, beside the well known anions,

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250 Na, K, Ca, and Mg in Human Blood

which bind base. These substances are probably for the most part proteins functioning as acids.

Total Available Fixed Base of Blood.

Table V shows that the sodium content of 1 liter of serum is equal to 1,460 cc. of 0.1 N base. The total available base of 1 liter of serum is equal to 1,585 cc. of 0.1 N base; i.e., sodium represents 92 per cent of the mineral base of strum. The remaining 8 per cent is represented by calcium, magnesium, and potassium. It has been shown elsewhere that the magnesium concentration of serum varies but little with normal individuals, as well as with those suffering from a variety of pathological conditions. Hence for practical purposes the concentration of this element may be con- sidered as a constant. The conccn6ration of calcium is likewise a fixed quantity except with nephritis in adults and tetany in children (18). Here the calcium concentration of the serum is rarely reduced below 5 mg. per 100 cc. of serum. Furthermore, the potassium concentration is usually increased under the same circumstances (31). The increase of potassium is usually com- pensated for wholly or in part by a decrease of the calcium con- centration. A decrease of the calcium concentration to 5 mg. reduces the figure for fixed base by an amount equal tjo 25 cc. of 0.1 N base, while an increase of potassium to 30 mg., the maximum which we have found in any case, corresponds likewise to an in- crease of fixed base of 25 cc. of 0.1 N base so that if we assume the concentration of K, Ca, and Mg as unchanged and add this value expressed as 0.1 N base to the figure, expressed in similar terms, obtained by actually det,ermining the concentration of sodium in serum we obtain, within s 5 per cent, a measure of the total fixed base of the serum. The only element whose concentration must be determined is sodium and ,this can be done on 1 to 2 cc. of serum with an error that does not exceed f 3 per cent. In a similar manner the total fixed base of corpuscles can be deter- mined by finding the potassium concentration of whole blood, the proportion of corpuscles to serum, and assuming the K concentra- tion of serum as 20 mg. per 100 cc.

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B. Kramer and F. F. Tisdall 251

CONCLUSIONS.

1. The corpuscles of human blood do not contain appreciable amounts of sodium or. calcium.

2. The average concentration of potassium per 100 cc. of cor- puscles found in thirteen normal adults was 428 mg.

3. The concentration of magnesium in whole blood is slightly higher than that of serum.

4. The extent to which the concentration of sodium, potassium, calcium, and magnesium in whole blood and corpuscles may vary is indicated in the tables.

5. Evidence is presented showing that there is an excess of about 16 per cent of basic radicles over the well known acid radicles in bot.h serum and corpuscles. It is likely that the excess is in combination with proteins.

6. Sodium represents about 92 per cent of the fixed base of serum; potassium, practically all that of corpuscles.

BIBLIOGRAPHY.

1. Schmidt, C., Charakteristik der Epidemischen Cholera, Leipsic and Mitau, 1850, 29, 32.

2. Wanach, R., Jahresb. Thierchem., 1888-89, xviii, 88. 3. Bunge, G., 2. Biol., 1876, xii, 191. 4. Abderhalden, E., 2. physiol. Chem., 1898, 65. xxv, 5. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 467. 6. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 339. 7. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvii, 475. 8. Kramer, B., and Tisdall, F. F., Bull. Johns Hopkins Hosp., 1921, xxxii,

44. 9. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlviii, 223.

10. Hamburger, H. J., 2. physik. Chem., 1909, lxix, 663. 11. Rona, P., and Takahashi, D., Biochem. Z., 1911, xxxi, 337. 12. Heubner, W., and Rona, P., Biochem. Z., 1918-19, xciii, 187. 13. Cowie, D. M., and Calhoun, H. A., J. Biol. Chem., 1919, xxxvii, 505. 14. Jones, M. R., and Nye, L. L., J. Biol. Chem., 1921, xlvii, 321. Jones,

M. R., J. BioZ. Chem., 1921, xlix, 187. 15. Howland, J., and Marriott, W. McK., @art. J. Med., 1917-18, xi, 289.

Marriott, W. McK., and Howland, J., J. BioZ. Chem., 1917, xxxii, 233. 16. Lamers, A. J. M., 2. Geburtsh. u. GynBk., 1912, Ixxi, 393. 17. Richter-Quittner, M., Wien. Arch., 1921, ii, 217. Falta, W., Wien.Arch.,

1921, ii, 187. 18. Kramer, B., Tisdall, F. F., and Howland, J., Am. J. Dis. Child., 1921,

xxii, 560.

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252 Na, K, Ca, and Mg in Human Blood

19. Lyman, H., J. Biol. Chem., 1917, xxix, 169. 20. Fridericia, L. S., J. Biol. Chem., 1920, xlii, 245. 21. Smith, L. W., Means, J. H., and Woodwell, M. N., J. Biol. Chem.,

1920-21, XIV, 245. 22. Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 49. 23. De Boer, S., J. Physiol., 1917, li, 211. 24. Loeb, J., Science, 1920, Iii, 449. 25. Van Slyke, D. D., Physiol. Rev., 1921, i, 141. 26. Campbell, J. M. H., and Poulton, E. P., J. Physiol., 1920-21, liv, 152. 27. Michaelis, L., Die Wasserstoffionenkonaentration, Berlin, 1914, 57. 28. Conway, R. E., and Stephen, F. V., J. Physiol., 1922, lvi, p. xxv. 29. Ryffel, J. H., Quart. J. Med., 1909-10, iii, 221. 30. Gyiirgy, P., and Zuna, E., J. BioZ. Chem., 1915, xxi, 511. 31. Kramer, B., Tisdall, F. F., and Howland, J., Am. J. Dis. Child., 1921,

xxii, 431.

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Benjamin Kramer and Frederick F. TisdallBLOOD

HUMANCORPUSCLES AND SERUM OF MAGNESIUM BETWEEN THEPOTASSIUM, CALCIUM, AND

THE DISTRIBUTION OF SODIUM,

1922, 53:241-252.J. Biol. Chem. 

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