SEPARATE ANALYSES OF THE CORPUSCLES AND THE PLASMA.

BY HSIEN WU.

(From the Laboratory of Physiological Chemistry, Peking Union Medical College, Peking.)

(Received for publication, July 29, 1921.)

In spite of the varied functions of the different components of blood the value of separate analyses of the corpuscle and the plasma has been little appreciated. Unless distribution of a substance within the blood is known and the percentage of the corpuscles is taken into account, the data obtained from the whole blood analysis admit of no strictly scientific treatment. In the transport of material to and from the tissues both the corpuscles and the plasma may play a part; but t,heir actual passage into or out of the circulation is effected only through the plasma. Hence for the solution of problems in connection with absorption, excre- tion, or similar processes, a knowledge of the concentration in the blood as a whole is insufficient, and the determination of the concentration in the plasma is a necessity.

Even in clinical practice where the whole blood analysis has proved a useful method of diagnosis and prognosis, separate analyses of the corpuscles and the plasma would afford a more reliable index of certain pathological conditions. For, if the distribution of a substance within the blood is unequal, the concen- tration in the blood as a whole will fluctuate with the percentage of the corpuscles, although the concentration in the plasma and and that in the corpuscles may remain unaltered.

The distribution of the better known water-soluble non-protein constituents has been a subject of considerable investigation. It is tacitly assumed by nearly all investigators that the results of the analysis of the corpuscles and the plasma obtained in tfhe usual way truly represents the relation that exists in the circulat- ing blood. Recently, Falta and Richter-Quittner (1) advanced

21

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22 Analyses of Corpuscles and Plasma

the view that in the circulating blood the non-protein nitrogenous substances, the sugar, and the chloride occur only in the plasma, and that these substances enter the corpuscles only when they are injured by sodium oxalate, fluoride, or by ot,her manipulations prior to the separation of the corpuscles from the plasma. In explaining the unusual findings upon which they based their conclusion, they referred to t,he use of hirudin as t-he punctum saliens of their research, believing that this anticoagulant, unlike the oxalate or fluoride, did not injure the corpuscles. The experimental data presented by these authors are, however, of such a character as to indicate faulty technique,’ and their findings have failed to be confirmed by some Danish investigators (2, 3, 4). The view of Falta and Richter-Quittner cannot, therefore, be accepted at present.

The results of the study on distribution obtained by other inves- tigators are of considerable interest. The total non-protein nitrogen (5, 6, 7), the amino-acids (5, 6, 8, 9, lo), and the creatine (6, 7, 11) are localized in the corpuscles, the urea (3, 5, 6, 12, 13, 14) and the creatinine (6, 7, 11) are equally distributed, while the uric acid (15, 16) is sometimes more and sometimes less con- centrated in the plasma. The distribution of sugar (1, 17, 18) has long been a disputed question; in the case of human blood the preponderance of evidence now points to an approximately equal distribution. The chloride, the only inorganic constituent touched upon in the present paper, is more abundant in t,he plasma (19).

Unfortunately, most of the studies on distribution were con- ducted on the blood of different species of animals and the data at hand do not lend themselves to correlation. Moreover, parallel analyses were made not of the corpuscles and the plasma, but of the whole blood and the plasma, frequently neglecting to mention the percentage of the corpuscles. Where this percentage is given the concentration in the corpuscles can, of course, be computed. But it is obvious that with methods capable of only moderate degrees of accuracy slight differences between the concentration

1 For instance, they reported in some cases as low as 9 mg. of non- protein nitrogen per 100 cc. of whole blood which is lower than the lowest figure for urea N we have ever found.

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Hsien Wu

in the corpuscles and that in the plasma can be shown only by direct analyses of t~he corpuscles and the plasma.

Folin and Wu (20) developed some time ago a system of blood analysis in which the blood proteins are removed with tungstic acid, yielding a filtrate suitable for the determination of non- protein nitrogen, urea, uric acid, creatine, creatinine, and sugar, and, as shown recently by Whitehorn (21), also for the determina- tion of chloride. It is, of course, very easy to extend that system so as to include separate analyses of the corpuscles and the plasma, and by this extension systematic studies on distribution will be facilitated, and the clinical application of the knowledge gained by such studies will be encouraged.

In applying the tungstic acid to the analyses of the corpuscles and the plasma we have required that the procedure employed must permit the quantitative recovery of at least 10 mg. each of uric acid and creatinine added to 100 cc. of plasma or corpuscles and that the non-protein nitrogen figures must be comparable with those of the whole blood. The latter requirement was deemed necessary in view of the fact that the non-protein nitrogen is not a very definite quantity but depends rather on the kind and amount of the precipitant used. It was found as a matter of fact, however, that the non-protein nitrogen values of the cor- puscles and of the plasma obtained with different amounts of tungstic acid were substantially the same when the amount of this precipitant used was above a certain minimum. Table I gives the result of an experiment on this point. Table II shows that the non-protein nitrogen values for the whole blood, calculated from those of the corpuscles and the plasma, agree with those determined directly.

Instead of using tungstate and sulfuric acid solutions of dif- ferent strengths for the corpuscles and for the plasma, it has seemed convenient to use the same 10 per cent tungstate solution and the 3 N sulfuric acid and to retain the 1: 10 dilution for the corpuscles and the plasma as for the whole blood. The method is as follows.

The oxalated blood is centrifuged in graduated tubes until the volume of the corpuscles remains constant.. The length of time required for this has been determined previously. After not- ing the volume of the whole blood and that of the corpuscles,

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24 Analyses of Corpuscles and Plasma

TABLE I.

Experiment Showing Effect of Di$erent Dilutions and Different Amounts of Tungstic Acid on Non-Protein Nitrogen of Corpuscles and Plasma.

Composition of precipitated mixture.

5 cc. corpuscles + 25 cc. Hz0 + 10 cc. 10 per cent tUngsi%te + 10 cc. 8 N sul-

furic acid.. . . . . . . . . . . . . . . . . . . . . . . . . . 4 cc. corpuscles + 26 cc. Hz0 + 10 cc.

10 per cent tUngState + 10 cc. 3 N sul-

furic acid............................... 6 cc. corpuscles + 24 cc. Hz0 + 10 cc.

10 per cent tungstate + 10 cc. N sulfuric acid....................................

5 cc. corpuscles + 75 cc. Hz0 + 10 cc. 10 per cent tungstate + 10 cc. + N sulfuric

acid.................................... 73 cc. corpuscles + 223 cc. Hz0 + 10 cc.

10 per cent tungstate + 10 cc. f N sulfuric acid.....................................

10 cc. plasma + 80 cc. Hz0 + 5 cc. 10 per

cent tungstate f 5 cc. 9 N sulfuric acid. 8 cc. plasma + 82 cc. Hz0 + 5 cc. 10 per

cent tungstate + 5 cc. $ N sulfuric acid.. 12 cc. plasma + 78 cc. Hz0 + 5 cc. 10 per

cent tungstate + 5 cc. $ N sulfuric acid.

10 cc. plasma + 30 cc. H20 + 5 cc. 10 per cent tungstate + 5 cc. % N sulfuric acid.

15 cc. plasma + 25 cc. Hz0 + 5 cc. 10 per cent tungstate + 5 cc. : N sulfuric acid..

Dilu- tion.

1:lO 50

2:25 49

3:25 51

1:20 49

3:20 Incomplete coagulation.

1:lrJ

2125

3:25

1:5

3:lO

-

Non-protein nitrogen

per 100 cc.

mo.

35

34

34

34

Incomplete

coagulation.

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Hsien Wu 25

carefully pipette off the plasma without disturbing the corpuscle layer. This is best done by means of a pipette connected at the upper end with soft rubber tubing. Measure a convenient vol- ume of the plasma, dilute with 8 volumes of water, and then add 4 volume each of 10 per cent sodium tungstate solution and 3 N

sulfuric acid. Stopper the flask and shake. Remove the plasma that remains above the corpuscle layer as

completely as possible. Insert a blood pipette (20) into the cor- puscle layer and take out a convenient volume. Lake it with 5 volumes of water and after thorough rinsing of the pipette with the corpuscle solution add 2 volumes each of the tungstate solu- tion and the sulfuric acid. Stopper the flask and shake. The amount of plasma which cannot be removed from above the cor-

TABLE II.

Non-Protein Nitrogen of Corpuscles, Plasma, and Whole Blood.

SOUID?.

Sheep 1.. . . . . . . . . . . “ 2 . . . . . . . . . . . .

Chicken l... . _. . . . . 6L 2 . . . . . . . . . .

Corpuscles.

per cent T?.

20 53 26 52

38 90 11 60

Non-protein nitrogen per 100 cc.

Corpuscles. Whole blood.

Pl%SlX%. Determined. Calculated.

ml. w7. mg.

34 39 38 36 40 40

24 48 49 10 17 16

puscle layer is less than 0.1 cc., and if it is desired to economize the material the whole corpuscle layer may be used for analysis with- out appreciable error. It is then simply washed into an Erlen- meyer flask with 5 volumes of water followed by the required amounts of tungstate and sulfuric acid.

The precipitated corpuscles and plasma may be filtered im- mediately. The precipitated plasma should be poured on the filter, slowly at first to allow the wetting of the filter paper before any filtrate has passed through. If for any reason the precipita- tion is incomplete and the filtrate is turbid, the analysis can be saved by adding a few drops of normal sulfuric acid to the pre- cipitate mixture. The plasma and corpuscle filtrates, like the filt’rate of t.he whole blood, are perfectly clear, only faintly acid,

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26 Analyses of Corpuscles and Plasma

and suitable for the determination of all constituents included in the system of blood analysis.

The method described above has been applied to a number of cases of normal human blood. These were obtained from normal persons taking the Wassermann test and from patients suffering from slight external injury such as frost-bitten fingers and lacer- ated toes. The analyses included total non-protein nitrogen, urea, uric acid, amino-acids, creatine, creatinine, sugar, and chloride. The amino-acids were determined by a new method to be published shortly from the Biochemical Laboratory of the Harvard Medical School as a supplement to the system of blood analysis.

The results of the analyses, arranged in the order of increasing urea concentration of the plasma, are shown in Table III. They constitute in the main a confirmation of previous findings, and are intended primarily for illustration. They represent, however, the first series of comprehensive analyses of the corpuscles and the plasma of human blood and some discussion may therefore not be amiss.

Urea is about equally distributed, but the concentration in the plasma is sometimes a little higher than that in the corpuscles. The average value for the plasma is 19.3 mg. per 100 cc. as against 17.1 for the corpuscles. One may t’hus wonder whether in the determination of the so called Ambard coefficient or similar con- stants more consistent values would be obtained if the urea in the plasma instead of that in the whole blood were determined.

Contrary to the finding of Bornstein and Griesbach (16) we find that concentration of uric acid in the plasma is always higher than that in the corpuscles. The values of the ratio of the former to the latter are about 2 on the average. The finding of the Hamburg investigators that uric acid is sometimes higher in the corpuscles is probably an error, although we have not studied a large number of pathological cases2 to exclude that possibility.

In view of the uncertainty in the determination of creatine and creatinine in the whole blood or rather in the corpuscles, as shown by Hunter and Campbell (II), any discussion on the distribution

2 We have studied the distribution of uric acid in a number of patho-

logical cases. The results, not here reported, are similar to those in the case of normal blood.

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TABL

E III

.

Non

-Pro

tein

N

itrog

eneo

us

Xubs

tanc

es,

Suga

r, an

d .C

hlor

ide

in

Cor

pusc

les

and

Plas

ma

of

Norm

al

Hum

an

Bloo

d.

T CO

P To

tal

pUSC

leS

non-

prote

in N.

‘lasm

a

per

cent

1

33

53

53

50

52

39

48

48

48

52

Cor-

,llS&S

m7.

44

39

53

45

49

w7.

w.

mg.

m

g.

mg.

20

12

13

1.2

3.0

23

12

14

1.7

3.4

23

13

15

1.8

4.0

25

13

15

1.5

3.4

30

18

18

1.7

3.6

52

26

18

18

3.2

4.8

7.2

51

29

18

19

2.5

4.0

6.2

54

33

18

19

1.4

2.3

8.8

47

26

15

19

2.2

3.6

8.4

45

26

18

19

1.5

4.5

9.0

54

53

57

50

47

. _

40

38

47

48

45

.

47.8

-

47

30

17

20

1.6

2.7

9.7

48

29

18

20

1.7

3.7

9.6

46

35

16

21

1.7

3.8

9.3

45

29

18

21

2.0

4.5

7.9

48

29

16

21

3.8

5.7

10.2

56

31

22

22

2.5

5.0

9.0

61

27

21

22

2.0

4.8

8.5

55

35

21

23

1.4

3.3

9.5

54

36

18

23

1.8

3.9

6.8

48

34

20

23

1.3

4.5

7.1

49.3

28

.8

Per

100

cc.

J - I

- I - I

- I - I

- I Ur

ea

N.

Uric

acid.

To

tal

creati

nine.

Pref

orm

ed

creati

nine.

Amino

-acid

N.

SU

gS%

r. NO

.

COi--

lUS&

S P

17.1

-

‘lasm

a Zl

asm

a CO

I- pu

scles

mg.

9.0

6.8 7.1

9.0

9.2

19.3

1.

93

3.92

8.

32

-1.

-T-

I.

. .

‘lasm

a

mg.

m

!J.

%7.

mg.

m

g.

ml.

mg.

1.4

9.1

4.5

109

95

330

624

1.3

11.4

5.

8 10

1 11

5 28

4 57

7

1.3

10.3

5.

4 93

83

33

7 67

6

1.4

9.1

I.6

100

105

300

575

1.3

7.7

5.5

80

76

241

560

1.5

9.6

5.0

89

95

290

580

1.4

10.8

6.

4 10

0 10

5 33

2 63

0

1.0

9.2

7.5

91

89

332

602

1.0

8.9

4.5

121

133

270

590

1.1

10.0

5.

0 91

91

34

0 67

0

1.4

9.8

6.0

109

115

337

660

1.4

9.2

5.5

103

107

312

630

1.3

9.4

6.2

100

100

313

629

1.0

8.9

6.0

87

95

292

583

1.0

10.5

5.

4 10

8 10

0 30

2 60

0

1.4

10.0

6.

6 93

10

0 34

5 67

5 1.

4 9.

0 4.

0 98

11

2 33

1 65

2 1.

4 8.

6 6.

1 10

0 11

0 32

6 61

6

0.8

8.2

5.0

110

114

313

604

0.9

9.7

5.3

115

123

270

570

1.24

9.

4 5.

52

99.!

103.

30

9.’

615.

2

‘lasm

a

mg.

1.4

1.4

1.4

1.3

1.5

1.4

1.4

1.5 1.3

1.7

1.5

1.5

1.4

1.5

2.1

1.4

1.4

1.3 1.5

1.5

1.47

cor-

pusc

let

ml.

.2.8

2.

5

2.6

2.9

2.8

3.0

2.7

2.2

2.1

2.2

2.6

2.6

2.8 1.9

1.8

3.0

2.8

2.8 1.6

2.0

2.48

6 7 8 9 10

11

12

13

14

15

16

17

18

19

20

Aver

age.

.. .

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Analyses of Corpuscles and Plasma

of these constituents would seem unwarranted. It is clear, how- ever, from Table III that the plasma is practically free from creatine. The average creatine content of the corpuscles is 5.84 mg. (as creatinine) per 100 cc., whereas that of the plasma is only 0.23 mg. This striking difference brought out by direct analysis of the corpuscles confirms and supplements the conclu- sion reached by Hunter and Campbell and by Wilson and Plass (7) that the creatine is chieily contained in the corpuscles.

It may be of interest to point out also that the apparent creat- inine content of the corpuscles is just twice that of the plasma.

The amino-acid content of the corpuscles averages almost twice that of the plasma. Similar distribution has been found in dogs (9), but, so far as we are aware no figure has been reported for human blood.

The concentration of sugar in the plasma is usually a little higher than in the corpuscles, but at times the reverse may be true. However, the difference in concentration in such cases is often so small as to be within the limits of error.

The concentration of chloride in the plasma is about twice that in the corpuscles.

There is another interesting point which the present study has brought out. It is probably a recognized fact that the non- protein nitrogen in the whole blood is not wholly accounted for by the known constituents. A normal human blood contains on the average:3

Per 100 cc. %7.

Urea N.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.10 Uric acid N.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.78 Creatinine N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.47

Creatine N................................................ 1.30 Amino-acid N............................................. 7.13

Total non-protein N calculated ............................ 26.78 “ “ N determined ........................... 35.60

Undetermined N .......................................... 8.80

3 These figures, with the exception of the amino-acid N, are taken from a paper by Hammett (Hammett, F. S., J. Biol. Chem., 1920, xli, 599). The amino-acid N figure is taken from Bock (10). That given by Hammett is 4.9 which seems too low.

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Hsien Wu 29

There are about 9 mg. of nitrogen not accounted for in the whole blood. This question has been discussed by some authors, but so far as we are aware no satisfactory explanation has yet been offered.

Can the undetermined nitrogen be due to the incomplete removal of proteins? It is well known that the non-protein nitrogen values obtained with different methods do vary appreciably, and there are doubtless some protein precipitants which leave traces of protein in the filtrate. But the tungstic acid filtrate gives as low non-protein nitrogen as can be obtained with any other method without losing the known constituents and it is improbable that as much as 9 mg. of protein nitrogen can escape precipitation.

TABLE IV.

Distribution of Non-Protein Nitrogenous Constituents between Corpuscles and Plasma.

Constituent. Corpuscles. P!%Sll~.

Urea N ..................................... Uric acid N ................................ Totalcreatinine N ......................... Amino-acid N ..............................

Total non-protein N calculated .......... “ “ N determined .........

. . .

. . .

. . -

. .

Undetermined N.. . . . . . . . . . . . . . . . . . . . . . . . . . .

17.10 19.30

0.64 1.31

3.10 0.55 9.47 5.52

30.31 49.3

19.0

26.68 28.8

2.1

The present study on the distribution seems to throw much light on this question. It will be noted in Table IV, which repre- sents the averages of Table III, that the non-protein nitrogen in the plasma is within limits of error4 all accounted for and that the undetermined nitrogen is all contained in the corpuscles. This localization shows that the undetermined nitrogen is not due to incomplete precipitation of the proteins, for otherwise we should expect the plasma also to contain some of it.

To what substance then can be ascribed this undetermined nitrogen which amounts to almost 20 mg. per 100 cc. of corpuscles?

4 There are, of course, traces of non-protein nitrogenous substances

other than those determined.

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30 Analyses of Corpuscles and Plasma

In the corpuscles there are, on the one hand, the amino-acids, and on the other hand, the proteins. In the living protoplasm of the corpuscles, as in all living cells, there is a metabolic equilibrium between .the amino-acids and the proteins with all the inter- mediates; namely, the peptides and peptones. The presence of pcptides and peptones in the corpuscles is indeed very probable, and pending further experimental evidence the undetermined nitro- gen in the corpuscles may be regarded as such.

Deductions Concerning Clinical Blood Analysis.-Since the undetermined nitrogen is all contained in the corpuscles and the amount of amino-acids in the corpuscles is larger than that in the plasma, the non-protein nitrogen of the whole blood will fluctuate with the content of the corpuscles in these constituents. As the latter is not known to vary with renal efficiency, the non-protein nitrogen of the whole blood would not be a reliable index of reten- tion. The determination of the non-protein nitrogen of the plasma should therefore be preferred to that of the whole blood.

The approximately equal distribution of sugar and urea justifies the usual practice of their determination in the whole blood, al- though their determination in the plasma is theoretically better.

The distribution of uric acid is so different and variable that one may wonder whether the content in the whole blood, the cor- puscles, or the plasma is the most valuable for practical purposes. In the absence of other information the concentration in the plasma would furnish the most useful data.

Hunter and Campbell (11) have shown that the only substance in the plasma capable of simulating the reaction for creatinine is glucose and its influence upon the determination of creatinine is too small to have much practical importance, whereas the cor- puscles contain other interfering substances which cause high results. Creatinine should therefore be determined in the plasma rather than in the whole blood.

There is thus abundant reason for substituting plasma analysis for whole blood analysis.

SUMMARY.

A method is described for the preparation of protein-free fil- trates of the corpuscles and the plasma suitable for all determina- tions included in the system of blood analysis of Folin and WU.

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Hsien Wu 31

Sample analyses of the corpuscles and the plasma of normal human blood are given and the points of interest brought out by these analyses are discussed.

It is recommended that plasma analysis be substituted for whole blood analysis.

BIBLIOGRAPHY.

1. Falta, W., and Richter-Quittner, M., &o&em. Z., 1919, c, 148; 1921, cxiv, 145.

2. Ege, R., Biochem. Z., 1920, cvii, 246. 3. Gad-Andresen, K. L., Biochem. Z., 1920, cvii, 250.

4. Hagedorn, H. C., Biochem. Z., 1920, cvii, 248. 5. Bang, I., Biochem. Z., 1915, lxxii, 104; 1916, lxxiv, 294.

6. Wilson, D. W., and Adolph, E. F., J. Bid. Chem., 1917, xxix, 405. (In the case of fishes they found more urea in the whole blood than in the plasma.)

7. Wilson, D. W., and Plass, E. D., J. Biol. Chem., 1917, xxix, 413. (In the case of fishes they found more creatine in the plasma than in the whole blood.)

8. Costantino, A., Biochem. Z., 1913, li, 91; Iv, 402. 9. Gyiirgy, P., and Zunz, E., J. Biol. Chem., 1915, xxi, 511.

10. Bock, J. C., J. Biol. Chem., 1917, xxix, 191.

11. Hunter, A., and Campbell, W. R., J. Biol. Chem., 1917, xxxii, 195; 1918, xxxiii, 169.

12. Hammarsten, O., Text-book of physiological chemistry, translated

by Mandel, J. A., 1915, 333. 13. Marshall, E. K., and Davis, D. M., J. BioZ. Chem., 1914, xviii, 53. 14. Karr, W. G., and Lewis, H. B., J. Am. Chem. Sot., 1916, xxxviii, 1615.

15. Benedict, S. R., J. BioZ. Chem., 1915, xx, 633. (This author found that

in ox blood the uric acid was quantitatively contained in the cor- puscles, whereas in chicken blood the corpuscles were almost free

from uric acid.) 16. Bornstein, A., and Griesbach, W., Biochsm. Z., 1920, ci, 184. (The

distribution of uric acid in human blood was found to be irregular by these authors.)

17. Wishart, M. B., J. BioZ. Chem., 1920, xliv, 563.

18. Ege, R., Biochem. Z., 1920, cxi, 189. 19. Abderhalden, E., Lehrbuch der physiologischen Chemie, Berlin,

1906, 591-593. 20. Folin, O., and Wu, H., J. BioZ. Chem., 1919, xxxviii, 81; 1920, xli, 367. 21. Whitehorn, J. C., J. BioZ. Chem., 1920-21, xlv, 449.

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Hsien WuCORPUSCLES AND THE PLASMA

SEPARATE ANALYSIS OF THE

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