ENZYRIE STUDIES ON WHITE BLOOD CELLS*

By Richard Wagner and Rose Sparaco Research Laboratory of the Boston Floating Hospital and the Department of Pediatrics, Ti&

Unii errity Scbool of Medicine, Boslon, &f ass.

Introduction Enzyme studies on white blood cells are difficult to evaluate for many

reasons, one of which is the problem of obtaining material free from con- taminants, particularly blood platelets. In the case of the respiratory enzymes this problem is complicated further by the fact that the reaction occurs through the mediation of a series of enzymes, each of which is necessary to obtain oxygen uptake.

Tlae Subslrales of Endogenous Oxygen Uplake Otto Heinrich Warburgl attributed most of the oxidations in the blood to

the blood platelets because of their great number as compared to the relatively small number of leukocytes. However, this view is no longer entirely ac- cepted. In previous studies2 we have shown that the endogenous oxygen uptake of the individual horse blood leukocyte is a t least 45 times that of a blood platelet. The QU,t of the leukocyte was found to be -32.6, in contrast to - 11.6 for rat liver slices. Also, there is another essential difference between leukocytes and liver, which have many enzymatic peculiarities in common: namely, the fact that the principal substrate for oxygen uptake in the liver is succinate, while for the leukocyte it is a-glycerophosphate.

The experiments on the identification of the substrates responsible for the endogenous uptake were performed on horse blood leukocytes; we have de- scribed the isolation of these cells and the enzymatic technique elsewhere.? A good test for the study of oxygen uptake is leukocyte dialysis against distilled water. After such dialysis the endogenous oxygen consumption in leukocyte suspensions is reduced almost to zero, but it can be restored by adding some substrates, such as succinate or a-glycerophosphate. There is good experi- mental evidence that both of these substrates are removed by dialysis. We examined the cell suspensions for the presence of the two substrates in un- dialyzed and dialyzed conditions, by paper chromatography according to the method of Hanes and I s h e r ~ o o d , ~ and by column chromatography for a- glycerophosphate as described by Khym and Cohn4; that is, by ion exchange with the use of the different borate fraction4. For the fermentation acids, paper chromatography was done by the technique of van D u ~ r e n , ~ and column

* The norb reported in this paper was supported by Research Grant No. PHS H-1652 (C9) from the Division of Research Grants and Fellowships of the National Institutes of Health, Public Health Service, Bethesda, Md.

This is the sixth of a series of articles on enzyme studies on white blood cells and blood platelets.

t The symbol (70% represents oxygen consumption in terms of the number of microliters consumed in 1 hour by 1 mg. (dry weight) of tissue. By convention the consumption of oxy- gen is given a negative value.

16

Wagner & Sparaco: Enzymes in White Blood Cells 17

I I I I I

141 1 ORIGINAL 24 H R . 48 H R . 72 H R -- GLYCERO- CELL 0 I ALY ZE D

PHOSPHATE SUSPENSION AGAINST DISTILLED WATER

FIGURE 1. Leukocyte-platelet suspension. The material is dispersed, partially crushed, and deproteinized with perchloric acid. This chromatogram was performed by the method of Hanes and Isherwood.3 The amount spotted is equivalent to 30 X lo6 leukocytes.

. 5 9 @ .57#

I I I I I I

OIALVSATE 1 901 WT. 78 a 104 olALYSATE2 a71 AGIO PHOSPH. GLUCOSE I- GLUCOSE 6- Pnosmo- + 540 PHOSPHATE PHOSPHATE GLYCERIC a-GLICERO- ACID PHOSPHATE

FIGURE 2. Dialysate of horse leukocyte-platelet suspension. This chromatogram was The amount spotted is equivalent to preformed by the method of Hanes and I s h e r ~ o o d . ~

30 X lo6 leukocytes.

18

chromatography by the melhod dcscribed by Neiih.fi The rlialysate5 were also investigated for the two substrates.

FICXJRL 1 shows i n

the undialyzed suspension a spot of a-glycerophosphate and spots of sonic other phosphorylated intermediates. Their K , s are near those of glucose- 1- and glu~ose-6-phospliate. I n the dialyzed suspensions a-glycerophosphate is no longer detectable, and only faint spots of another intermediate can be seen, the R, being ncar hexose diphosphate. On column chromatography a-glycerophosphste can be eluted together with glucose-6-phosphate.

Aiinals New Yo& Academy of Sciences

The results are presented in the following illustrations.

.43 a .43 45 t8

1 I i I I

90 E 300 r 300 r PERCHURIC ACID TUNGSTATE SUCCl N IC LACTIC LACTIC a ACID ACID ACID 90 d DEPROTEINIZATION

SUCCINIC ACID

FIG~JKE 3 . Horse leukocyte-platelet suspension. 'This chromatogram was performed The amount spotted is equivalent to 280 X lo6 according to the method of van Duuren.5

leukocytes.

PERCHWRIC ACID 300 8 LACTIC ACID 8

DEPROTEINIZATION 120 X SUCCINIC ACID

F I C ~ ~ K E 4. Horse leuhucy te-platelet suspension. This chromatogram was performed ac cording to the method of van D u ~ r e n . ~ 'I'he amount spotted is equivalent to 280 X 10'' leukocytes.

Wagner & Sparaco: Enzymes in White Blood Cells 19

FIGURE 2 is the chromatogram of two different dialysates. Glucosed-phos- phate and a-glycerophosphate were identified in the first, and phosphoglyceric acid was identified in the second. FIGURE 3 is a chromalogram made by the method of van D ~ u r e n . ~ Lactic acid is detectable with perchloric acid de- proteinization, and succinic acid by acid-tungstate deproteinization. How- ever, these reactions are not always reproducible; in most instances another acid was found, as shown in FIGURE 4. In addition to lactic acid, two spots appear that have an R, lower than that of succinic acid. We have been unable to identify this acid to date. These spots occurred in many of our chromatograms and require further study.

FIGURE 5 is a chromatogram of a dialysate showing phosphoglyceric acid,

1 1 I

ma LACTICB DIALYSATE 076 PHOSRIO- 1206 SUCClNlC GLYCERIC .ACID

ACID

FIGURE 5. Dialysate of horse leukocyte-platelet suspension. This chroniatogram was 'l'he amount spotted is equivalent to performed according to the method of van Duuren.$

30 X 10' leukocytes.

20 Annals New York Academy of Sciences in analogy with the results shown in FIGURE 2, in which the acid was identified by the method of Hanes and Isherwood.3

These chromatographic studies may be considered proof that the loss of endogenous oxygen uptake following dialysis against distilled water is the result of a loss of substrates. The addition of the dialysate, sterilized with penicillin and streptomycin, to a dialyzed cell suspension did not restore the dehydrogenase activity. Obviously the concentration of the substrates is too low.

PL.

"""1 ENDOG. OXYGEN UPTAKE - a -GLYCEROPHOSPHAl€ (0.083 W) AHD CYTOCHROWE c (1.33 mg. par ml. REACTION MIXTURE) ADOED

691s a -GLYGEROPHOSPHATE ( 0 . 0 ~ 1 3 ~ ) ADDED 7001 6001 500

NOdAL- NORMAL- LEUK. LEUK.

P DIALYZED

L

DIM

DIA UZED -4L m a u M D - FIGURE 6 . Comparison between normal and leukemic leukocytes: microliters of oxygen

uptake per 1000 X lofi leukocytes in 90 min.

Wagner & Sparaco: Enzymes in White Blood Cells 21

a-Glycerophosphate Dehydrogenase Aclivicy in Leukemic Leukocytes The technique of studying oxygen uptake on dialyzed cell material with

added substrates has proved to be particularly informative in the study of leukocytes obtained from patients with chronic myelogenous leukemia. FIG- URE 6 shows that the increase of oxygen uptake above the level of the endog- enous respiration when a-glycerophosphate is used as substrate does not occur in leukemia when the cell suspension is undialyzed. When the cell material is dialyzed the oxygen uptake of leukemic cells is only approximately 25 per cent that of normal human white blood cells. In the third group of bars one can see that addition of cytochrome c in excess considerably increases a-glycerophosphate dehydrogenase activity in both undialyzed and dialyzed normal human leukocytes. In undialyzed horse blood leukocytes this increase is missing? However, in horse blood leukocytes there is a considerable in- crease in activity if the cells are dialyzed. I n dialyzed leukemic leukocytes, there is no increase of a-glycerophosphate dehydrogenase activity following addition of cytochrome c to the reaction mixture. Addition of cytochrome c without a- glycerophosphate to dialyzed human or horse leukocytes does not increase oxygen uptake.

We cannot conclude from our studies that it is the a-glycerophosphate dehydrogenase activity which is a t a lower level in leukemic leukocytes. The defect can be in any of the series of enzymes necessary to obtain oxygen up- take. The behavior of leukemic leukocytes may be due to an actual difference in the cytochrome concentration in the cells or it may be the result of differ- ences in the extractibility of cytochrome c. Quantitative cytochrome deter- minations are in progress.

References 1. WARBURG, 0.

2. WAGNER, R., N. MEYERRIECKS & R. SPARACO.

1911. Untersuchungen uber die Oxydationsprozesse in Zellen. Munch. med. Wochschr. 68: 289.

Enzyme studies on white blood cells and blood platelets. v. Dehydrogenase activity. Arch. Biochem. Biophys. 61: 278.

3. HANES, C. S. & F. A. ISHERWOOD. Separation of the phosphoric esters on the fil- ter paper chromatogram.

4. KHYM, J. X. & W. E. COHN. The separation oi sugar phosphates by ion exchange with the use of the borate complex.

5. VAN DUUREN, A. J. 1953. Determination of organic acids in plant material by paper chromatography. Rec. trav. chim. 72: 889.

6. NEISR, A. C. 1949. Production and properties of 2,d-butanediol. XXX. Determina- tion of the fermentation acids by partition chromatography Can. J.Research. B27: 6.

1956.

1949. Nature. 164: 1107. 1953.

J. Am. Chem. SOC. 76: 1153.