STUDIES ON BLOOD CELL METABOLISM.

V. THE METABOLISM OF LEUCOCYTES.

BY E. S. GUZMAN BARRON AND GEORGE A. HARROP, JR.

(From the Chemical Division of the Medical Clinic, the Johns Hopkins Hospital and University, Baltimore.)

(Received for publication, June 28, 1929.)

In previous papers (1) we have reported studies on the oxygen consumption and on the lactic acid production, under aerobic and anaerobic conditions, in various types of erythrocytes and other cells, and on the effect of methylene blue and other dyes on these processes. In the present paper we are concerned with the metabolic behavior of the leucocytes of the blood, with a com- parison of the cells of the granulocytic series with the non-granu- locytic elements, and, so far as possible, with cells of different stages of maturity. It was finally desired to determine in how far the metabolic behavior of the white cells of leucemic blood corresponds to that of cancer cells or to that of the cells of em- bryonic tissues.

It is at once evident that the colorless elements of the blood, cell for cell, have a much higher metabolic activity than do the erythrocytes. The increased rate at which leucemic blood (or blood in which a high degree of leucocytosis exists) darkens on standing, indicative of inceased oxygen utilization, is a matter of every day clinical observation.

Although the sugar-splitting power of the leucocytes was known for a long time, the production of lactic acid was clearly demonstrated only through Levene and Meyer’s (2) studies. This qualitative demon- stration was followed later by Slosse’s (3) quantitative determinations. Maclean and Weir (4) conclude that leucocytes have from 200 to 1000 times the glycolytic activity of erythrocytes. The earlier work is con- sidered in a recent review by Fleischmann (5). The metabolism of the polynuclear and the mononuclear leucocytes obtained from sterile

89

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

90 Studies on Blood Cell Metabolism. V

exudates has been studied by Bakker (6), who used the material formed after injections of various materials (Hamburger (7)) into the perito- neal cavity of rabbits. He concluded that both polynuclear leucocytes and mononuclear cells behave in their metabolism like cancer cells, inas- much as they have a high anaerobic metabolism, while the splitting of sugar into lactic acid is not materially reduced under aerobic conditions. Fleischmann and Kubowitz (8), using goose and rabbit leucocytes sus- pended in Ringer’s solution, concluded that the oxygen consumption was much higher (10 times greater) than Bakker had found it and that the fermentation also was appreciably greater. Fujita (9), in Warburg’s lab- oratory, studied leucocytes from rat blood which he received into citrate solution. The leucocytes were then removed from the upper layer after brief centrifugalization. When his experiments were prolonged, the oxygen consumption was greatly reduced, but in short experiments in which the oxygen utilization was not damaged, he found that leucocytes have a metabolism similar, not to cancer tissue, but to normal embryonic cells; that is, a large anaerobic glycolysis, which under aerobic conditions, is nearly replaced by the respiration. In Warburg’s nomenclature,r the average results of his series were as follows:

Qo, = -9, CJ;; = 1.9, Qg = 19.9

It is evidently of prime importance at the outset to determine the effects of external agents upon the metabolism of leucocytes. Of the two fundamental sources of cell energy, respiration and fermentation, the first is far more sensitive to external injuries than the second. While the fermentative process goes on as long as the cell surface and the glycolytic enzyme remain unaltered, the respiratory process is readily injured by the action of external agents that do not affect the cell surface or the respiratory ferment, if the correctness of- Warburg’s hypothesis is assumed. Early in the course of this study, in an effort to obtain a constant sdurce of “normal” material, we decided to employ peritoneal exudates from the rabbit, following closely Hamburger’s (7) methods. The results obtained were extremely irregular. In some cases the cells would respire, reduce methylene blue, and cause glycolysis both aerobically and anaerobically, but in other instances their respiratory power was lost, although glycolysis and the power to reduce methylene blue remained intact. Evidence of this loss of

1 Qo, represents the amount of oxygen consumed per unit of material and

per unit of time, Q$ the amount of lactic acid under aerobic, and Q&the

amount. of lactic acid under anaerobic conditions, per unit of material and per unit of time.

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. S. G. Barron and G. A. Harrop, Jr. 91

oxidative power was also proved by the high glycolytic quotient in aerobic glycolysis, which in some cases reached almost as high a value as during anaerobic glycolysis (Table I). We ‘then found

TABLE I.

Glycolysis of Rabbit Leucocytes.

Values for sugar and lactic acid are expressed in mM per liter.

Aerobic.

Polynuclear leucocytes. I. . . . . . . .

Lymphocytes.

Anaerobic. -

Method of produc- ing anaerobiosis.

Polynuclear leucocytes. I . . . . . . . . . . . . . . . . . . . . Nitrogen

atmosphere. II. . . . . . . . . . . . . . . 6.062.353.710.717.61~6.90~0.93 Nitrogen

atmosphere. III. . . . . . . . . . . . . . . . . ..6.344.861.480.452.792.340.79 Nitrogen

atmosphere. IV . . . . . . . . . . . . . . . . 6.283.832.450.826.155.331.08 0.002~ KCN. V . . . . . . . . . . . . . . . . . . . 6.154.391.760.934.193.260.93 0.002 “ “

Lymphocytes. Sample I.. . . . . . . . 4.734.080.650.742.03 1.29 1.00 Nitrogen

atmosphere.

that centrifugation caused this damage to the respiration and that when sodium citrate was employed to avoid clotting, according to Hamburger’s technique, cell metabolism was further impaired. We therefore abandoned this method. Shortly afterward Fujita’s

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

92 Studies on Blood Cell Metabolism. V

paper appeared confirming our observation upon the damage done by centrifugalization and showing also that change of temperature similarly has a harmful effect on respiration.

1

o-

O-

D-

.

/ !

/ / / /

A / /

/ /

PO0 1m.

l.@o,ooo

CHART 1. Variation of the oxygen consumption of leucocytes with varia- tion in their concentration. The solid line represents oxygen consumption of granulocytes (material from a patient with chronic myeloid leucemia). The broken line represents oxygen consumption of lymphocytes (material from a patient with chronic lymphatic leucemia). Oxygen consumed per c.mm. is shown on the ordinate scale.

When cells are collected from anesthetized animals, we have observed that although slight ether anesthesia (30 minutes) does not impair the respiratory process, a longer anesthesia (2 hours)

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. S. G. Barron and G. A. Harrop, Jr. 93

lowers and even inhibits respiration of leucocytes in vitro. Ether does not affect glycolysis when administered for short periods, but when the anesthetic acts for some hours even glycolysis is lowered.

,X

CHART 2. Variation in the glucose consumption of leucocytes at differ- ent concentrations. The solid line represents glycolysis of granulocytes; the broken line, glycolysis of lymphocytes. The material was obtained from the same sources as in Chart 1.

Amytal and urethane also inhibit both processes. A detailed re- port of these effects on blood cell metabolism will be made later.

It was only after much data had been obtained from the study of blood from patients with leucocytosis and leucemia, as well as

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Studies on Blood Cell Metabolism. V

from experiments with dog lymphocytes, that a fact of importance emerged whose significance we believe has been neglected in many previous studies; this is, that cell concentration has a very marked influence on metabolic activity. When the oxygen consumption is calculated per million of cells per cmm. and per unit of time,

K= mm.3 O2

cells (millions) X time (hours)’ it tends to be in inverse ratio

to the cell concentration present. This depressing effect of con- centration upon respiratory activity was found to affect the process of glycolysis as well. An example of experiments upon blood cells obtained from a common source, at different serum dilutions, is shown in Charts 1 and 2. It is indicated clearly that the optimum concent.ration for maximal respiration and maximal glycolysis of both granulocytes and lymphocytes is approximately 10,000 cells per cmm.

Since normal blood generally contains from 65 to 70 per cent of polynuclear leucocytes and from 25 to 35 per cent of mononuclear cells, it was decided to use only those specimens of blood which contained an excess of one or the other type of cell which it was desired to study. Aside from the blood of patients with lym- phatic leucemia, lymph from the thoracic duct of dogs furnishes the best material for the study of the metabolism of lymphocytes. This can be obtained by cannulation of the dog’s thoracic duct, provided the anesthetic employed for the operation is not harmful to the cells2

Methods.

The methods used were as follows: Blood was collected in purified heparin3 and centrifuged at once for 2 to 3 minutes. The supernatant fluid was pipetted off, a portion taken for cell counts and smears, and t.he rest used immediately without being permitted to cool. Oxygen absorption was measured in the Barcroft-Warburg manometers at 37.5” and glycolysis and lactic

2 That dog lymph contains practically only lymphocytes has been shown by ROUS, Davis, and Carlson, and others (Bloom, W., A&. ezp. Zdlfomch., 5,269 (1928)).

3 This heparin is of a high degree of purity and contains no inorganic salts. It was furnished to us through the kindness of Professor W. II. Howell.

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. S. G. Barron and G. A. Harrop, Jr. 95

acid formation in flasks kept at the same temperature in a con- stant shaking device. For anaerobic glycolysis either KCN was added to one flask or purified nitrogen was passed through the flask during the experiment, as indicated in Table I. The sugar was determined by Benedict’s modification of the Folin-Wu method and the lactic acid by the Friedemann-Shaffer method,

TABLE II.

Oxygen Consumption of Leucocytes, with E

Source of material.

1. Chronic myelogenous leucemia.. . 2. “ ‘I “

3. ‘( “ ‘I . 4. “ “ “

5. “ “ “

6. Empyema... . . 7. Pneumonia.. ., 8. Empyema.. ., 9. Hodgkin’s disease (?).. . .

1. Chronic lymphatic leucemia.. ., 2. CL ‘( ‘L . . 3. (‘ “ “ . . . 1. Dog lymph lymphocytes . . 2. “ “ “ 3. “ “ ‘L y:::::::::

No. of ce!ls per

c.mm.

-

_ .

02 con- rumption

per hr. K’ I

-

Differen- ,ial count. 1

POlynU- clears.

48,000 72,000

162,000 123,000 448 ) 000

30 ) 000 30,000 25,400 75,000

c.mm.

46 60

127 100.6 268

39 41.4 43.0 55

0.96 0.84 0.78 0.82 0.60 1.30 1.38 1.52 0.73

per cent

90 92 90 90 91 95 84 86 93

-

- Lympho-

cytes.

150,000 60.0 0.40 95 52,800 56.2 1.06 90

320,000 54.0 0.017 97 10,000 25 2.5 98

8,000 23 2.87 92 6,000 12.5 2.08 93

:cts of OvercrowGng.

*K= c.mm. 02 consumed

cell concentration (millions) X time (hours)

before and after incubation. This seemed a more direct, accurate, and specific technique than that used in earlier studies (Fleisch- mann and Kubowitz (S), Fujita (9)), with the Warburg mano- meters, and we believe more accurately determines the lactic acid production as a result of glycolysis. From comparative studies with whole blood (erythrocytes do not respire appreciably) we were able to convince ourselves that these manipulative pro-

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

96 Studies on Blood Cell Metabolism. V

cedures, including this brief period of centrifuging, do not affect the oxidative metabolism of leucocytes.

In human blood, the oxygen consumption of polynuclear and mononuclear leucocytes seems to be roughly of the same magni- tude, with the single difference that polynuclear leucocytes resist overcrowding better than do mononuclear leucocytes (Table II and Chart 1). Doubtless the greater resistance of the glycolytic function to overcrowding is of value in the metabolism of pus cells, for in pus pockets the oxygen supply must be deficient and the cells must depend almost entirely on anaerobic processes of

TABLE III.

Aerobic Glycolysis Calculated per Hour and per Million Cells, Showing E$ects of Overcroruding upon Sugar Utilization.

Experi- ment

NO.

I

2 3 4 5 6 7 8 9

10 11

Source of material. No. of cells per c.mm.

/ Glucose d&roved per million cells

per hr.

Chronic myelogenous leucemia. “ “ “ “ “ “ ‘I “ “ “ “ “

Empyema. “

Hodgkins disease (?). Chronic lymphatic leucemia.

“ “ “ ‘I “ “

48,000 72,000

162,000 123,000 448 ) 000

30,000 28,400 75 ) 000

150,000 25,000

320,000

w7.

6.25 X 1O-3 3.45 x 10-a 2.95 x IO-3 4.76 X 1OF 1.32 X 1O-3 7.68 x 1O-3 1.05 x 10-z 8.24 X 1O-3 5.84 x 10-d 2.0 x 10-a 2.19 x 10-d

metabolism for their source of energy. Dog lymphocytes showed a decidedly higher oxygen consumption than lymphocytes ob- tained from cases of leucemia, but the concentrations used were optimal. The figures in Table II are taken only from those ex- periments where very slight ether anesthesia was employed.

In their behavior towards glycolysis there is an undoubted difference between polynuclear leucocytes and mononuclear leu- cocytes. Polynuclear leucocytes possess a higher aerobic and anaerobic glycolysis than mononuclear cells. This is clearly shown in Table III, where the aerobic glycolysis of each variety of cell had been calculated per million cells and per hour of incuba-

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. S. G. Barron and G. A. Harrop, Jr. 97

tion. In this table it is seen that polynuclear leucocytes appear to have a glycolytic power at least 5 times as great as that of mononuclear cells if we compare experiments in which the con- centration is approximately the same (for example, Experiments 3 and 9, and 7 and 10). We have found that polynuclear leu- cocytes have a higher anaerobic metabolism than was observed by Fujita. It is possible that the explanation of this discrepancy lies in the fact that Fujita was working with a mixture of both poly- nuclear leucocytes and mononuclear leucocytes. Since lympho- cytes have the higher aerobic metabolism, a mean value would thus be obtained under such conditions. Most of our cell prepa- rations have been studied for motility with the dark-field tech- nique and it seems clear that oxygen consumption is greatest in actively motile cells. On the other hand glycolysis and lactic acid ,formation, and the power of the cell to reduce methylene blue are unrelated to the motility of the cell, for these properties re- mained unaltered in mature granulocytes in which motility had entirely ceased.

The fundamental researches of Pasteur on “la vie sans oxygen” established the fact that fermentative and oxidative processes are not independent, but closely related. If a cell which ferments sugar in anaerobiosis is put in oxygen, the respiration thus estab- lished has a tendency to lower or replace completely the fermenta- tion. Warburg (10) has recently applied Pasteur’s fundamental ideas to his classification of the metabolic activity of cells. He properly names as the “Pasteur reaction” this relationship be- tween respiration and fermentation. Taking the maximum value obtained when the cells are put in a condition of optimum oxidative activity and comparing them with those obtained under anaerobic conditions, he calculates the ‘(excess of fermentation” by the following equation.

U (excess of fermentation) = QE; - (2 C&J*

* Q”,z is the lactic acid produced in anaerobic conditions; QO, is the

oxygen consumption.

Since U is the excess of fermentation which remains when the Pasteur reaction reaches its maximum effect, it will be nil when the anaerobic fermentation is twice the respiration, and it will be

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Calcu

lation

?f

Valu

e SO

T Pa

stew

Reac

tion

(Wn~

burg

) jar

Gr

anulo

cyte

s an

d Ly

mph

ocyte

s.

Diag

nosis

.

Chro

nic

mye

loid

leuc

emia

.. “

“ “

“ “

“

“ “

“

Empy

ema.

. Po

lycyth

emia

Vera

.. (‘h

ronic

m

yeloi

d le

ucem

ia.

Pneu

mon

ia..

Empy

ema.

. Ho

dgkin

’s di

seas

e..

Chro

nic

lymph

atic

leuc

emia

“

“ “

“ “

“

Gluc

ose,

mg.

pe

r cc

. se

rum

. 02

co

n-

2

, su

mpt

ion

2 .F d

.L

j d

2 C.

Inlll.

ca

lculat

ed

e.2

$E

for

time

,4

OE

$2

$2

of

incu-

2

2 ba

tion.

* m

-4

0

Paste

ur re

acw-

tio

n.

Perc

enta

ge

of

cells

.

3 1.

100.

250.

85

138

24

2.42

1.62

0.80

15

0 2

2.45

0.90

1.55

25

4 2;

1.

590.

191.

40

228

2 1.

260

740.

52

78

2 2.

281.

101.

18

140

l+

1.84

0.25

1.59

40

2 2

1.04

0.25

0.79

82

.8

14

0.91

0.37

0.54

65

.0

1:

0.97

0.17

0.80

11

1.0

3 0.

830.

580.

25

180.

0 2

1.05

0.78

0.27

11

2.4

l$

0.75

0.42

0.33

15

0.0

+0.4

8 90

gr

anulo

cyte

s (3

0Y0

mat

ure

polyn

uclea

rs).

+0.4

0 92

(‘

(50%

“

“ &

f0.8

7 90

“

(40%

“

(‘ +0

.79

90

“ i:

g (2

0%

“ “

). f0

.31

95

(‘ (a

ll “

cells

). +0

.805

88

“

(86%

“

“ ).

z

+0.5

13

91

“ (a

ll “

“ ).

s

1-0.

566

84

“ *‘

C‘

1.

$0.3

66

86

“ ;::

“

“).

5

to.5

0 93

‘(

(90%

“

“ ).

g

-0.2

35

95

lymph

ocyte

s. ?

-0.0

3 90

‘(

-0.0

7 97

“

-4

* 1

c.mm

. O2

=

1.34

X

lo-”

mg.

of

gl

ucos

e.

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

E. S. G. Barron and G. A. Harrop, Jr.

negative or positive when twice the respiration is greater or smaller than the anaerobic fermentation. Normal organs, the retina excepted, give negative values under such conditions, while tissues from malignant tumors give positive values for U. Ac- cording to Fleischmann and Kubowitz, blood cells give positive U values, thus being similar to the cells of the retina. As seen from Table IV, the present study shows that the values for granu- locytic cells are positive and thus indicate that their metabolism resembles that of tumor cells and the cells of the retina. On the other hand, the lymphocytes from cases of chronic lymphatic leucemia all gave negative values, low it is true, but corresponding to the behavior of the cells from normal tissues.

DISCUSSION.

The difference in the anaerobic metabolism (glycolysis) of granulocytic and of lymphocytic leucocytes seems to afford some substantiation to the view of Sabin (11) and her coworkers, based on anatomical grounds that the two types of cells have different origins as well as functions. Glycolytic activity seems to be a more stable type of activity and its greater magnitude in the polynuclear leucocyte must be vital to the activities of this cell when it is placed, as must often be the case, in conditions of very low oxygen tension.

Our results give no support to the suggestion, particularly made by Daland and Isaacs (12), that the respiratory activity of mature polynuclear cells is higher than that of the immature cells. We are unable with our present methods to find any distinct differ- ences in the metabolic activities of immature and mature leuco- cytes. This fact, in addition to the further observation that the leucocytes from the blood of patients with leucemia do not differ in their metabolism from the corresponding cells of blood from non-leucemic patients, seems to us to indicate that in leucemia we are not dealing with cells with pathologically altered metabolism, such as are found in cancer tissue. Leucemia is not analogous to cancer, at least as regards cell metabolism. It rather seems to be a condition in which large numbers of cells, normal as regards their fermentative and respfratory activities, are thrown out into the circulation as a result of a disturbance in the mecha- nism which regulates the rate of blood cell formation.

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

100 Studies on Blood Cell Metabolism. V

SUMMARY.

1. The basal optimal conditions required for measurement of the metabolism of the white cells of the blood in vitro have been defined. These consist primarily in measurement at low concen- tration, measurement over short intervals of time, use of anti- coagulants which do not damage respiration, very short centri- fugation, and avoidance of any cooling.

2. No differences have been found in the metabolism of mature and immature granulocytes, or of granulocytes from cases of leucocytosis and of myeloid leucemia.

3. Measured under comparable conditions of concentration, lymphocytes differ in their metabolism from granulocytes in the following particulars: (a) The glycolysis of polynuclear leucocytes is about 5 times greater than the glycolysis of lymphocytes. (6) There is no marked difference in the oxygen consumption of these two kinds of leucocytes (human) when measured at low concen- tration, but lymphocytes show a diminution of respiratory power as a consequence of overcrowding more easily than do polynuclear cells. (c) The Pasteur reaction in lymphocytes resembles that in normal tissues; the Pasteur reaction in granulocytes resembles that in tumor cells.

BIBLIOGRAPHY.

1. Harrop, G. A., Jr., and Barron, E. S. G., J. Exp. Med., 48, 207 (1928); J. Biol. Chem., 79, 65 (1928). Barron, E. S. G., J. Biol. Chem., 81, 445 (1929).

2. Levene, P. A., andMeyer, G. M., J. Biol. Chem., 11,361 (1912). 3. Slosse, A., Arch. internat. physiol., 11,154 (1912). 4. Maclean, H., and Weir, H. B., Biochem. J., 9,412 (1915). 5. Fleischmann, W., Ergebn. Physiol., 27,1 (1928). 6. Bakker, A., Dissertation, Groningen (1926); Klin. Woch., 8,252 (1927). 7. Hamburger, H. J., in Abderhalden, E., Handbuch der biologischen

Arbeitsmethoden, IV. Abt., Berlin and Vienna, pt. 4, 970 (1927). 8. Fleischmann, W., and Kubowitz, F., Biochem. Z., 181, 395 (1927). 9. Fujita, KZin. Woch., 7, 897 (1928).

10. Warburg, O., Biochem. Z., 184,484 (1927). 11. Sabin, F. R., Physiol. Rev., 11,38 (1922). 12. Daland, G. A., and Isaacs, R., J. Exp. Med., 46,53 (1927).

by guest on October 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Jr.E. S. Guzman Barron and George A. Harrop,

OF LEUCOCYTESMETABOLISM: V. THE METABOLISM

STUDIES ON BLOOD CELL

1929, 84:89-100.J. Biol. Chem. 

  http://www.jbc.org/content/84/1/89.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  #ref-list-1

http://www.jbc.org/content/84/1/89.citation.full.htmlaccessed free atThis article cites 0 references, 0 of which can be by guest on O

ctober 29, 2018http://w

ww

.jbc.org/D

ownloaded from