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Studies on the oxygen uptake of leucocytes from the chacma baboon (Papio ursinus) and on a new medium for the in vitro study of intact leucocytes
Comp. Biochera. Physiol., 1969, Vol. 29, pp. 235 to 242. Pergamon Press. Printed in Great Britain
STUDIES ON T H E OXYGEN UPTAKE OF LEUCOCYTES FROM T H E CHACMA BABOON (PAPIO URSINUS) AND ON A NEW M E D I U M FOR T H E IN VITRO STUDY OF I N T A C T LEUCOCYTES* M I C H A E L J. W E I G H T and N E R I N A SAVAGE Department of Physiology, University of the Witwatersrand Medical School, Johannesburg, South Africa (Received 26 August 1968) A b s t r a c t - - 1 . The oxygen uptake of leucocytes isolated from the blood of the
Chacma baboon (Papio ursinus) has been determined in different media. 2. A new medium containing deionized pork skin gelatine and "HEPES" buffer for the study of intact leucocytes in vitro has been proposed. 3. The oxygen consumption in this medium is 126/~moles/101° leucocytes per hr.
INTRODUCTION MANYrespiratorystudies havebeen carriedout on humanleucocytes. In this study the respiratoryactivityof baboon leucocytesis described. Studies on the metabolism of intact leucocytesin vitro have been hinderedby the lackof a suitable medium. Leucocytesin wholeblood havebeenused in tracer studies, and then separated afterwards (Rowe et al., 1960). Alternatively, the leucocytes have been pre-separated and suspended in homologous serum or plasma, or in serum or plasma diluted with buffer (Bird et al., 1951; Warburg et al., 1958; Pastore, 1959; Miras et al., 1965). The latter media are not readily reproducible; the reasons why so many studies are carried out in these irreproducible media are twofold. Firstly, leucocytes agglutinate when suspended in buffers, and secondly, it is difficult to find a buffer system with sufficient buffer capacity to control the large quantities of lactic acid produced by the vigorous glycolysis of the leucocytes. Skoog & Beck (1956), after observing hundreds of leucocyte isolations, concluded that clumping always occurs to a greater or lesser extent in leucocyte preparations. Tullis (1953), Ruthberg & Terent'eva (1959), Leontovich&Abezgauz (1960) and Esmann (1962) found that 1-2% gelatine was capable of counteracting this agglutination, but these authors did not specify the kind of gelatine they had used. 200 mg% of sodium acetate was found by Tullis to have a similar restraining effect. * Supported by a grant from the Council for Scientific and Industrial Research, South Africa. 235
236
MICHAELJ. WEIGHTANDNERINASAVAGE
Esmann (1962) has described two buffers containing gelatine and sodium acetate for the study of leucocyte metabolism (Table 1). KRPAGA is a Krebs-RingerPhosphate-Acetate-Gelatine-Ascorbic acid buffer and KRBAGA is a KrebsRinger-Bicarbonate-Acetate-Gelatine-Ascorbic acid buffer. Esmann used the KRBAGA buffer throughout his studies as it maintained the pH during the TABLEI--BuFFERCOMPOSITIONS Esmarm (1962)
NaCI KCI CaC12 KH2PO, MgSO4 Na-acetate NaHCO3 NaH2PO4 Na2HPO4 Glucose Gelatine
Present authors
KRBAGA (raM)
KRPAGA (mM)
KRPAGA (mM)
91.8 4"7 2"5 1"2 1"2 24"4 25"0
95"9 4.7 2"5 1'2 1"2 24"4 --
95"9 4.7 2"5 1"2 1"2 24"4 --
95"9 4.7 2"5 1'2 1"2 24"4 --
1"9
1"9
--
12"5 16-7 1% (unspecified) 0"06 7.10
12'5 16"7 1% CSA 0"06 7"40
-16"7 1% PSH 0"06 7"40
--
-16"7 1% (unspecified) Ascorbic acid 0"06 pH 7"43
KRHAGA (mM)
incubations. However, this buffer is troublesome to use, and only allows the indirect Warburg technique to be used. In the latter technique the continuous uptake of oxygen cannot be followed, and in radioactive tracer studies the continuous collection of carbon dioxide is not possible. The KRPAGA, on the other hand, has insufficient buffer capacity and the phosphate concentration cannot be increased as it precipitates the calcium and magnesium ions which are present. Therefore the aim of the present study has been to modify the KRPAGA buffer described by Esmann using a specially prepared gelatine and a new buffer described by Good et al. (1966). This buffer has been investigated as regards its ability to keep the pH constant and to prevent the agglutination of baboon leucocytes. In accordance with the nomenclature given above it has been called KRHAGA or Krebs-Ringer-HEPES-Acetate-Gelatine-Ascorbic acid buffer. The suitability of this medium was investigated by studying the respiration of the leucocytes, since Tullis (1953) has demonstrated that leucocyte respiration decreases rapidly when they are damaged. This is the first function to be affected, even before those of migration and phagocytosis. MATERIALS AND METHODS Jlnimals
Studies were carried out on the Chacma baboon (Papio ursinus) which were bled after an overnight fast (about 16 hr).
OXYGEN UPTAKE OF LEUCOCYTES FROM THE CHACMA BABOON
237
"Method of isolation The following reagents were used: 1. Dextran. (A) Phosphate buffer 0.05 M, p H = 7.4: (i) 6"8 g KH~PO4 is dissolved in 1000 ml of distilled water; (ii) 8.7 g K2HPO4 is dissolved in 1000 rnl of distilled water. Mix (i) and (ii) in the ratio of 19 : 81 parts by volume. (B) Five g of Dextran 250 (Dextran fraction TDR-205-11-B-1 Pharmacia Uppsala), 0"9 g NaC1 0.3 g glucose were dissolved in 100 ml of the phosphate buffer 0-05 M, pH = 7"4 in a volumetric flask. Aliquots (12"5 ml) of the dextran saline solution were dispensed into glass vials, sealed and autoclaved at 15 lb/15 rain and stored at 4°C. 2. E D T A . A 4% solution of E D T A * (British Drug Houses, Poole) in 0"9% NaC1 was similarly dispensed into vials and autoclaved in the same manner. 3. P S H gelatine. (A) one M " H E P E S " t (Calbiochem, Lucerne): 23"6 g was dissolved in about 30 ml of distilled water and the pH adjusted to 7-4 with 2 N NaOH. This was then transferred to a 100-ml volumetric flask and made up to the mark. (B) Twenty g of American pork skin gelatine with an isoelectric point I.E.P. = 8.0 was dissolved in 150 ml of distilled water by heating to 40°C in a water-bath. This gelatine was then transferred to the funnel of a completely jacketed column which was circulated with water at about 50°C. The gelatine was allowed to flow through the column packed with Amberlite MB3 Monobed resin (Rohm & Haas Co., Philadelphia) at the rate of ~t ml/ml resin per min and collected in a 500-ml volumetric flask. After all the gelatine had passed through the column, the latter was washed through with warm distilled water. T h e gelatine solution was finally made up to the mark. The column should contain at least 100 ml of resin, which is self-indicating. Should the gelatine come off the column at a pH other than 8"0 it must be deionized again. (C) Twenty-five ml of the gelatine solution and 5 ml of 1 M " H E P E S " were transferred to 50-ml McCartney bottles and autoclaved at 15 lb for 15 rain and finally stored at 4°C. This amount of the deionized gelatine and " H E P E S " was sufficient to make up 100 ml K R H A G A , i.e. it is equivalent to 1 o/o gelatine. The final pH of K R H A G A was always 7"4. 4. C S A gelatine. A calf skin gelatine (Oxoid-Oxo, London) was deionized as described above and the I.E.P. was found to be 4"9 after deionization. The p H was raised to 7.4 with KOH. The medium with this gelatine as a base was buffered with either phosphate, T r i s HC1, T r i s - m a l e a t e - N a O H or imidazole.
Collection Twelve and a half ml of 5~o dextran and 1"6 ml E D T A were transferred to a suitably graduated Erlenmeyer flask. T h e flask was stoppered and transported in a thermos jar containing water at 37°C. While taking the blood the precaution of Martin & Green (1958) was taken by making the venipuncture with a large needle (17 gauge) inserted rapidly and cleanly. Fifty ml of blood were collected by allowing it to drip from the syringe needle into the mixture in the flask. The blood was normally taken from the ante-cubital vein.
Isolation As soon as possible after the collection of the blood it was distributed in Pyrex test tubes (14 x 125 ram) previously placed in a constant-temperature bath at 37°C. The red blood cells were allowed to sediment for exactly 50 rain and the supematant transferred to a 50-ml constricted neck centrifuge tube with a Pasteur pipette, and centrifuged at 200 g for exactly 4 re.in. T h e supematant was carefully decanted and the leucocyte pellet taken up in a little of the medium described (1-2 ml) and resuspended by aspirating slowly up and down with a Pasteur pipette. At this stage about 40 ml of the medium was added and the contents Ethylene-diamine-tetra-acetieacid disodium salt. t N-2-hydroxyethylpiperazine-N'-2"-ethanesulphonic acid. *
238
MICHAEL J. WEIGHT AND NERINA SAVAGE
carefully mixed with the pipette and centrifuged at 200 g for exactly 2 min. The supernatant was again carefully decanted and the leucocyte pellet made up in an appropriate volume of the medium. At this stage samples were taken for leucocyte counting.
Leucocyte counting The leucocytes were counted in a Neubauer counting chamber, after dilution (50 : 950) with gentian violet in 1% acetic acid. Respiration studies
Standard direct Warburg technique was used. Two ml of the leucocyte suspension (made up in one or other of the media) was pipet-ted into each flask prewarmed in an incubator at 37°C. The centre well was fitted with a roll of Whatman No. 40 filter paper soaked with 0.1 ml 40% KOH. The paper should project about 3-4 mm above the rim of the centre well. After a 10-min equilibration period the taps were closed and readings taken every 30 min during the 2-hr incubation period. RESULTS A comparison of the oxygen consumption of baboon leucocytes suspended in the medium KRPAGA containing both the CSA gelatine and phosphate buffer revealed results which were similar to those obtained by authors using phosphate buffers and human leucocytes (Table 2). The use of imidazole, Tris-HC1, or Tris-maleate-NaOH did not increase the oxygen consumption. In fact in higher concentrations these buffers had a severely inhibiting effect on leucocyte respiration, an observation also noted by Estes (1960) in the case of Tris. Imidazole at the level of 30 mM caused complete inhibition of respiration. Adjustment of the pH of the CSA gelatine solution to 7.4 with either K O H or Na2CO3 did not appear to affect the leucocyte respiration. Esmann found that the pH of his KRPAGA medium dropped to 6.0-6.3 when incubating leucocytes in the presence of glucose. The KRPAGA medium made up with the CSA gelatine used in this study showed a similar drop in pH during a 2-hr incubation, i.e. 6.0-6.2. The combination of the PSH gelatine and HEPES in the KRHAGA made an appreciable difference to the leucocyte respiration (Fig. 1). In nine experiments the oxygen consumption of baboon leucocytes was found to be 73 _+1.58/~moles* in the case of KRPAGA containing CSA gelatine. In eleven experiments the oxygen consumption was found to be 126 _+4.07 #moles in the case of the KRHAGA containing the PSH gelatine. The standard error of the difference between means was 12.24, P < 0.0005. Furthermore, the pH remained constant during the 2 hr incubation period and only dropped to pH = 7.2 over a 3-hr period. This could no doubt be avoided by increasing the concentration of " H E P E S " buffer. DISCUSSION Many studies have been published on the glycolytic and respiratory metabolism of the human leucocyte. Much of this work is difficult to interpret because of variations in experimental procedure, e.g. different incubation media or incubation periods, different leucocyte concentrations were used or no leucocyte counts were recorded and results expressed in terms of leucocyte dry weight. In the latter case * All oxygen uptakes expressed as/Lmoles/101°leucocytes per hr.
239
OXYGEN UPTAKE OF LEUCOCYTES FROM THE CHACMA BABOON
conversion factors have been published for the conversion of dry weight tO n u m b e r s of leucocytes; however, the values quoted are not in agreement. M a n y of the studies have been d o n e in plasma or s e r u m which gives a fairly high oxygen consumption, or in various buffers containing s e r u m or plasma T A B L E 2 - - C O M P A R I S O N OF RESULTS OBTAINED FOR HUMAN LEUCOCYTES BY VARIOUS AUTHORS
pmoles 02/101° Glu- leucocytes per hr cose + S.E.M.
Authors using human leucocytes
n
Warburg et al. (1958)
5 Plasma + saline
+ "
245 + 25
Bird et al. (1951)
5 Plasma + bicarbonate buffer
+
230 + 38
Pastore (1959)
3
Serum + phosphate buffer
+
178
Seelich et al. (1957)
9 9
Bicarbonate buffer-Krebs Ringer with air as the gas phase
+ -
402 + 22 473 __.27
Remmele (1955)
3 Bicarbonate buffer-Krebs Ringer
-
213
Bicarbonate buffer (KRBAGA)
+
172
Bicz (1960)
5 Bicarbonate buffer-Krebs Ringer + Tris
-
103
Seelich et al. (1957)
5 Phosphate buffer-Krebs Ringer 7 Phosphate buffer-Krebbs Ringer
+ -
219 -+14 228 -+ 17
? Phosphate buffer-Hanks McKinney & Rundles (1956) ? + resin plasma
+ -
92_+6 150 _+7
Athens et al. (1959)
3 Phosphate buffer-Hanks + resin plasma
-
173
Bisset & Alexander (1958) Bisset & Alexander (1960)
8 Phosphate buffer (Krebs No. 2) 6 containing 1"4 mM HC~3
+ ?
110 _+6 113 +_11
+ +
73 +_1"58 126 _+4"05
Esmann (1962)
77
Medium
McKinneyetaL(1952)
Present authors using baboon leucocytes
9 11
KRPAGA KRHAGA
(Table 2). T h i s was the case in the studies of Bird et al. (1951) and W a r b u r g et al. (1958) where citrated plasma was used. T h e results they obtained were 230 and 245/zmoles respectively. Pastore (1959) incubated leucocytes in phosphate buffer + 30~/o homologous s e r u m and found an oxygen consumption of 178/zmoles. Results obtained in bicarbonate buffer are also rather higher than in phosphate, and R e m m e l e (1955) reported an oxygen consumption of 213 tzmoles (although only three experiments were done and each value differed f r o m the average b y about
240
MICHAEL J.
W E I G H T A N D N E R I N A SAVAGE
50 per cent). Bicz (1960) found the oxygen uptake of leucocytes to be 103/~moles but he did have Tris present in the medium which probably inhibited the respiration. The values obtained by Seelich et al. (1957) of 402-473/Lmoles are very high.
250"
oq ua b- 200. U 0 U L.u _J _o o
- ~ 150' Z 0 >x 0 if) u,i ~ 100 E
0
/.// 3~0
60
;0
1 t2 0
MINUTES,
FIG. 1. Oxygen uptake of leucocytes: Q--O, in KRPAGA medium (each point mean of twenty-six observations); ©--©, in KRHAGA medium (each point mean of thirty-three observations). These workers used bicarbonate buffer with air as the gas phase and presumably the pH of this system must also have been very high. Esmann (1962) in a very thorough study of leucocyte respiration in bicarbonate-CO~, buffer (KRBAGA) found the oxygen consumption of leucocytes to be 172/zmoles. The lowest figures for oxygen uptakes for human leucocytes where phosphate buffer was used were those of McKinney et al. (1952) and Bisset & Alexander (1958, 1960) with buffer containing glucose, i.e. 92 gmoles and 111/~moles respectively. In the absence of glucose the consumption appears to be somewhat
OXYGEN U P T A K E OF LEUCOCYTES F R O M T H E C H A C M A BABOON
241
higher, and McKinney & Rundles (1956) reported a value of 150 #moles, and Athens et al. (1959) one of 170 #moles. Even these results in phosphate buffer are not really comparable as McKinney & Rundles and Athens et al. used resin plasma in their media, and that of Bisset & Alexander contained bicarbonate. In the present study the KRPAGA medium containing the CSA gelatine was compared with the K R H A G A medium containing the PSH gelatine. As in the case of phosphate buffers containing glucose the oxygen consumption is low in the KRPAGA medium. The only authors to get the higher results in human leucocytes are Seelich et al. (1957), who found roughly twice the values for leucocyte oxygen consumption in the case of both bicarbonate and phosphate buffers. The values obtained in phosphate could be due to the fairly rapid decrease in pH. In the case of K R H A G A buffer described in this paper the pH remained constant throughout the period of incubation and it is very likely that " H E P E S " has none of the toxic properties of Tris or imidazole. This medium could provide a standardized method for investigating the metabolism of intact leucocytes with little or no change in pH and no chance of agglutination. SUMMARY The oxygen consumption of leucocytes from the Chacma baboon (Papio ursinus) was measured in different media. A new buffer medium has been described for the study of intact leucocytes. The medium K R H A G A contains a deionized pork skin gelatine together with " H E P E S " buffer. In this medium the oxygen consumption of baboon leucocytes was greatly increased compared with the standard phosphate buffer (KRPAGA) and found to be 126 #moles/101° leucocytes per hr. The pH of the medium remains constant during the period of incubation which is not the case with phosphate buffer. The leucocytes do not agglutinate in the new medium. The new medium offers the opportunity to standardize studies on leucocyte metabolism. Acknowledgements--The authors express their gratitude to Professor C. P. Luck for constructive advice and criticism in the preparation of this manuscript. Also to Mr. H. V. Williams of Davis Gelatine Industries for a gift of American Pork-Skin Gelatine and much helpful advice, and to Mr. E. F. Allen for technical assistance with the animals. REFERENCES
ATHENSJ. W., MAUERA. M., ASHENBRUCKERH., CARTWRIGHTG. E. & WINTROBE(1959) Leucokinetic studies--I. A method for labelling leucocytes with diisopropylfluorophosphate (DFP32). Blood 14, 303-333. Blcz W. (1960) The influence of CO~ tension on the respiration of normal and leukaemic human leukocytes--I. Influence of endogenous respiration. Cancer Res. 20, 184-190. BIRD R. M., CLEMENSJ. A. & BECKERL. M. (1951) The metabolism of leucocytes taken from peripheral blood from leukaemic patients. Cancer 4, 1009-1014. BlSSET S. K. & ALEXANDERW. n. (1958) Effect of muscular activity prior to venepuncture on the respiration of leucocytes in vitro. Nature, Load. 181,909-910.
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MICHAELJ. WEIGHT AND NERINA SAVAGE
BISSET S. K. ~ ALEXANDERW. D. (1960) The effect of metabolic stimulants on the oxygen uptake of normal and leukaemic human leucocytes in vitro. Quart. j~. exp. Physiol. 45, 18-24.
ESMANN V. (1962) Carbohydrate metabolism and respiration in leucocytes from normal and diabetic subjects. Ph.D. Thesis, University of Aarus, Denmark. ESTES F. L., AUSTIN N. S. & GAST J. H. (1960) Metabolic characterisation of polymorphonuclear leucocytes. Clin. Chem. 6, 501-503. GOOD N. E., WINGEr G. D., WINTER W., CONOLLY T. N., IZAWAS. & SINGH R. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467-475. LEONTOVICHV. A. & ABEZGAUZN. N. (1960) Conditions prolonging the storage period of viable white cells. Probl. Gemat. 5, a A 50. McKINNEY G. R., MARTIN S. P., RUNDLES R. W. & GREEN R. (1952) Respiratory and glycolytic activities of human leucocytes in vitro, j~. appl. Physiol. 5, 335-340. McKINNEY G. R. & RUNDLES R. W. (1956) Lactate formation and glyoxylase activity in normal and leukaemic human leucocytes in vitro. Cancer Research 16, 67-69. MARTIN S. P. & GREEN T. (1958) Methods for the study of surviving leucocytes. In Methods in Medical Research, Vol. 7, pp. 136-137. Year Book Publishers, Chicago. MIRAS C. J., MANTOSJ. D. & LEVIS G. M. (1965) Fatty acid synthesis in human leucocytes. Biochem. biophys. Res. Commun. 19, 79-83. PASTORE E. J. (1959) Metabolism of acetate by human leucocytes. Ph.D. Thesis, Boston University Graduate School. REMMELE W. (1955) Atmung und Glykolyse der Leukozyten..4cta haemat. 13, 103-123. RowE C. E., ALLISON A. C. & LOVELOCKJ. D. (1960) Lipid biosynthesis in human blood; the incorporation of acetate into lipids by different types of human cells. Biochim. biophys. Acta 41,310-314. RUTHBERGR. A. & TERENT'EVAE. I. (1959) Preservation of viable packed white cells. Probl. Gemat. 4, 50-54. SEELICH F., LETNANSKYW., FRISCH W. & SCHNECKO. (1957) Zur Frage der Stoffwechsels normaler und pathologischer menschlicher Leukozyten. Z. Krebsforsch 62, 1-8. SKOOG W. A. & BECK W. S. (1956) Studies on the fibrinogen, dextran and phytohaemagglutinin methods of isolating leucocytes. Blood11,436-454. TULLIS J. L. (1953) Preservation of leucocytes. Blood 8, 563-575. TULLIS J. L. (1953) The Origin, Properties, Function and Preservation of White Blood Cells in Blood Cells and Plasma Proteins, pp. 276-279. Academic Press, New York. WARBURG O., GAwEHN K. & GEISSLERA. W. (1958) Stoffwechsel der weissen Blutzellen. Z. Naturforsch. 13, 515-516. Key Word Index---Oxygen consumption; leucocytes; leucocyte medium; baboon; human leucocytes; Papio ursinus.
STUDIES ON T H E OXYGEN UPTAKE OF LEUCOCYTES FROM T H E CHACMA BABOON (PAPIO URSINUS) AND ON A NEW M E D I U M FOR T H E IN VITRO STUDY OF I N T A C T LEUCOCYTES* M I C H A E L J. W E I G H T and N E R I N A SAVAGE Department of Physiology, University of the Witwatersrand Medical School, Johannesburg, South Africa (Received 26 August 1968) A b s t r a c t - - 1 . The oxygen uptake of leucocytes isolated from the blood of the
Chacma baboon (Papio ursinus) has been determined in different media. 2. A new medium containing deionized pork skin gelatine and "HEPES" buffer for the study of intact leucocytes in vitro has been proposed. 3. The oxygen consumption in this medium is 126/~moles/101° leucocytes per hr.
INTRODUCTION MANYrespiratorystudies havebeen carriedout on humanleucocytes. In this study the respiratoryactivityof baboon leucocytesis described. Studies on the metabolism of intact leucocytesin vitro have been hinderedby the lackof a suitable medium. Leucocytesin wholeblood havebeenused in tracer studies, and then separated afterwards (Rowe et al., 1960). Alternatively, the leucocytes have been pre-separated and suspended in homologous serum or plasma, or in serum or plasma diluted with buffer (Bird et al., 1951; Warburg et al., 1958; Pastore, 1959; Miras et al., 1965). The latter media are not readily reproducible; the reasons why so many studies are carried out in these irreproducible media are twofold. Firstly, leucocytes agglutinate when suspended in buffers, and secondly, it is difficult to find a buffer system with sufficient buffer capacity to control the large quantities of lactic acid produced by the vigorous glycolysis of the leucocytes. Skoog & Beck (1956), after observing hundreds of leucocyte isolations, concluded that clumping always occurs to a greater or lesser extent in leucocyte preparations. Tullis (1953), Ruthberg & Terent'eva (1959), Leontovich&Abezgauz (1960) and Esmann (1962) found that 1-2% gelatine was capable of counteracting this agglutination, but these authors did not specify the kind of gelatine they had used. 200 mg% of sodium acetate was found by Tullis to have a similar restraining effect. * Supported by a grant from the Council for Scientific and Industrial Research, South Africa. 235
236
MICHAELJ. WEIGHTANDNERINASAVAGE
Esmann (1962) has described two buffers containing gelatine and sodium acetate for the study of leucocyte metabolism (Table 1). KRPAGA is a Krebs-RingerPhosphate-Acetate-Gelatine-Ascorbic acid buffer and KRBAGA is a KrebsRinger-Bicarbonate-Acetate-Gelatine-Ascorbic acid buffer. Esmann used the KRBAGA buffer throughout his studies as it maintained the pH during the TABLEI--BuFFERCOMPOSITIONS Esmarm (1962)
NaCI KCI CaC12 KH2PO, MgSO4 Na-acetate NaHCO3 NaH2PO4 Na2HPO4 Glucose Gelatine
Present authors
KRBAGA (raM)
KRPAGA (mM)
KRPAGA (mM)
91.8 4"7 2"5 1"2 1"2 24"4 25"0
95"9 4.7 2"5 1'2 1"2 24"4 --
95"9 4.7 2"5 1"2 1"2 24"4 --
95"9 4.7 2"5 1'2 1"2 24"4 --
1"9
1"9
--
12"5 16-7 1% (unspecified) 0"06 7.10
12'5 16"7 1% CSA 0"06 7"40
-16"7 1% PSH 0"06 7"40
--
-16"7 1% (unspecified) Ascorbic acid 0"06 pH 7"43
KRHAGA (mM)
incubations. However, this buffer is troublesome to use, and only allows the indirect Warburg technique to be used. In the latter technique the continuous uptake of oxygen cannot be followed, and in radioactive tracer studies the continuous collection of carbon dioxide is not possible. The KRPAGA, on the other hand, has insufficient buffer capacity and the phosphate concentration cannot be increased as it precipitates the calcium and magnesium ions which are present. Therefore the aim of the present study has been to modify the KRPAGA buffer described by Esmann using a specially prepared gelatine and a new buffer described by Good et al. (1966). This buffer has been investigated as regards its ability to keep the pH constant and to prevent the agglutination of baboon leucocytes. In accordance with the nomenclature given above it has been called KRHAGA or Krebs-Ringer-HEPES-Acetate-Gelatine-Ascorbic acid buffer. The suitability of this medium was investigated by studying the respiration of the leucocytes, since Tullis (1953) has demonstrated that leucocyte respiration decreases rapidly when they are damaged. This is the first function to be affected, even before those of migration and phagocytosis. MATERIALS AND METHODS Jlnimals
Studies were carried out on the Chacma baboon (Papio ursinus) which were bled after an overnight fast (about 16 hr).
OXYGEN UPTAKE OF LEUCOCYTES FROM THE CHACMA BABOON
237
"Method of isolation The following reagents were used: 1. Dextran. (A) Phosphate buffer 0.05 M, p H = 7.4: (i) 6"8 g KH~PO4 is dissolved in 1000 ml of distilled water; (ii) 8.7 g K2HPO4 is dissolved in 1000 rnl of distilled water. Mix (i) and (ii) in the ratio of 19 : 81 parts by volume. (B) Five g of Dextran 250 (Dextran fraction TDR-205-11-B-1 Pharmacia Uppsala), 0"9 g NaC1 0.3 g glucose were dissolved in 100 ml of the phosphate buffer 0-05 M, pH = 7"4 in a volumetric flask. Aliquots (12"5 ml) of the dextran saline solution were dispensed into glass vials, sealed and autoclaved at 15 lb/15 rain and stored at 4°C. 2. E D T A . A 4% solution of E D T A * (British Drug Houses, Poole) in 0"9% NaC1 was similarly dispensed into vials and autoclaved in the same manner. 3. P S H gelatine. (A) one M " H E P E S " t (Calbiochem, Lucerne): 23"6 g was dissolved in about 30 ml of distilled water and the pH adjusted to 7-4 with 2 N NaOH. This was then transferred to a 100-ml volumetric flask and made up to the mark. (B) Twenty g of American pork skin gelatine with an isoelectric point I.E.P. = 8.0 was dissolved in 150 ml of distilled water by heating to 40°C in a water-bath. This gelatine was then transferred to the funnel of a completely jacketed column which was circulated with water at about 50°C. The gelatine was allowed to flow through the column packed with Amberlite MB3 Monobed resin (Rohm & Haas Co., Philadelphia) at the rate of ~t ml/ml resin per min and collected in a 500-ml volumetric flask. After all the gelatine had passed through the column, the latter was washed through with warm distilled water. T h e gelatine solution was finally made up to the mark. The column should contain at least 100 ml of resin, which is self-indicating. Should the gelatine come off the column at a pH other than 8"0 it must be deionized again. (C) Twenty-five ml of the gelatine solution and 5 ml of 1 M " H E P E S " were transferred to 50-ml McCartney bottles and autoclaved at 15 lb for 15 rain and finally stored at 4°C. This amount of the deionized gelatine and " H E P E S " was sufficient to make up 100 ml K R H A G A , i.e. it is equivalent to 1 o/o gelatine. The final pH of K R H A G A was always 7"4. 4. C S A gelatine. A calf skin gelatine (Oxoid-Oxo, London) was deionized as described above and the I.E.P. was found to be 4"9 after deionization. The p H was raised to 7.4 with KOH. The medium with this gelatine as a base was buffered with either phosphate, T r i s HC1, T r i s - m a l e a t e - N a O H or imidazole.
Collection Twelve and a half ml of 5~o dextran and 1"6 ml E D T A were transferred to a suitably graduated Erlenmeyer flask. T h e flask was stoppered and transported in a thermos jar containing water at 37°C. While taking the blood the precaution of Martin & Green (1958) was taken by making the venipuncture with a large needle (17 gauge) inserted rapidly and cleanly. Fifty ml of blood were collected by allowing it to drip from the syringe needle into the mixture in the flask. The blood was normally taken from the ante-cubital vein.
Isolation As soon as possible after the collection of the blood it was distributed in Pyrex test tubes (14 x 125 ram) previously placed in a constant-temperature bath at 37°C. The red blood cells were allowed to sediment for exactly 50 rain and the supematant transferred to a 50-ml constricted neck centrifuge tube with a Pasteur pipette, and centrifuged at 200 g for exactly 4 re.in. T h e supematant was carefully decanted and the leucocyte pellet taken up in a little of the medium described (1-2 ml) and resuspended by aspirating slowly up and down with a Pasteur pipette. At this stage about 40 ml of the medium was added and the contents Ethylene-diamine-tetra-acetieacid disodium salt. t N-2-hydroxyethylpiperazine-N'-2"-ethanesulphonic acid. *
238
MICHAEL J. WEIGHT AND NERINA SAVAGE
carefully mixed with the pipette and centrifuged at 200 g for exactly 2 min. The supernatant was again carefully decanted and the leucocyte pellet made up in an appropriate volume of the medium. At this stage samples were taken for leucocyte counting.
Leucocyte counting The leucocytes were counted in a Neubauer counting chamber, after dilution (50 : 950) with gentian violet in 1% acetic acid. Respiration studies
Standard direct Warburg technique was used. Two ml of the leucocyte suspension (made up in one or other of the media) was pipet-ted into each flask prewarmed in an incubator at 37°C. The centre well was fitted with a roll of Whatman No. 40 filter paper soaked with 0.1 ml 40% KOH. The paper should project about 3-4 mm above the rim of the centre well. After a 10-min equilibration period the taps were closed and readings taken every 30 min during the 2-hr incubation period. RESULTS A comparison of the oxygen consumption of baboon leucocytes suspended in the medium KRPAGA containing both the CSA gelatine and phosphate buffer revealed results which were similar to those obtained by authors using phosphate buffers and human leucocytes (Table 2). The use of imidazole, Tris-HC1, or Tris-maleate-NaOH did not increase the oxygen consumption. In fact in higher concentrations these buffers had a severely inhibiting effect on leucocyte respiration, an observation also noted by Estes (1960) in the case of Tris. Imidazole at the level of 30 mM caused complete inhibition of respiration. Adjustment of the pH of the CSA gelatine solution to 7.4 with either K O H or Na2CO3 did not appear to affect the leucocyte respiration. Esmann found that the pH of his KRPAGA medium dropped to 6.0-6.3 when incubating leucocytes in the presence of glucose. The KRPAGA medium made up with the CSA gelatine used in this study showed a similar drop in pH during a 2-hr incubation, i.e. 6.0-6.2. The combination of the PSH gelatine and HEPES in the KRHAGA made an appreciable difference to the leucocyte respiration (Fig. 1). In nine experiments the oxygen consumption of baboon leucocytes was found to be 73 _+1.58/~moles* in the case of KRPAGA containing CSA gelatine. In eleven experiments the oxygen consumption was found to be 126 _+4.07 #moles in the case of the KRHAGA containing the PSH gelatine. The standard error of the difference between means was 12.24, P < 0.0005. Furthermore, the pH remained constant during the 2 hr incubation period and only dropped to pH = 7.2 over a 3-hr period. This could no doubt be avoided by increasing the concentration of " H E P E S " buffer. DISCUSSION Many studies have been published on the glycolytic and respiratory metabolism of the human leucocyte. Much of this work is difficult to interpret because of variations in experimental procedure, e.g. different incubation media or incubation periods, different leucocyte concentrations were used or no leucocyte counts were recorded and results expressed in terms of leucocyte dry weight. In the latter case * All oxygen uptakes expressed as/Lmoles/101°leucocytes per hr.
239
OXYGEN UPTAKE OF LEUCOCYTES FROM THE CHACMA BABOON
conversion factors have been published for the conversion of dry weight tO n u m b e r s of leucocytes; however, the values quoted are not in agreement. M a n y of the studies have been d o n e in plasma or s e r u m which gives a fairly high oxygen consumption, or in various buffers containing s e r u m or plasma T A B L E 2 - - C O M P A R I S O N OF RESULTS OBTAINED FOR HUMAN LEUCOCYTES BY VARIOUS AUTHORS
pmoles 02/101° Glu- leucocytes per hr cose + S.E.M.
Authors using human leucocytes
n
Warburg et al. (1958)
5 Plasma + saline
+ "
245 + 25
Bird et al. (1951)
5 Plasma + bicarbonate buffer
+
230 + 38
Pastore (1959)
3
Serum + phosphate buffer
+
178
Seelich et al. (1957)
9 9
Bicarbonate buffer-Krebs Ringer with air as the gas phase
+ -
402 + 22 473 __.27
Remmele (1955)
3 Bicarbonate buffer-Krebs Ringer
-
213
Bicarbonate buffer (KRBAGA)
+
172
Bicz (1960)
5 Bicarbonate buffer-Krebs Ringer + Tris
-
103
Seelich et al. (1957)
5 Phosphate buffer-Krebs Ringer 7 Phosphate buffer-Krebbs Ringer
+ -
219 -+14 228 -+ 17
? Phosphate buffer-Hanks McKinney & Rundles (1956) ? + resin plasma
+ -
92_+6 150 _+7
Athens et al. (1959)
3 Phosphate buffer-Hanks + resin plasma
-
173
Bisset & Alexander (1958) Bisset & Alexander (1960)
8 Phosphate buffer (Krebs No. 2) 6 containing 1"4 mM HC~3
+ ?
110 _+6 113 +_11
+ +
73 +_1"58 126 _+4"05
Esmann (1962)
77
Medium
McKinneyetaL(1952)
Present authors using baboon leucocytes
9 11
KRPAGA KRHAGA
(Table 2). T h i s was the case in the studies of Bird et al. (1951) and W a r b u r g et al. (1958) where citrated plasma was used. T h e results they obtained were 230 and 245/zmoles respectively. Pastore (1959) incubated leucocytes in phosphate buffer + 30~/o homologous s e r u m and found an oxygen consumption of 178/zmoles. Results obtained in bicarbonate buffer are also rather higher than in phosphate, and R e m m e l e (1955) reported an oxygen consumption of 213 tzmoles (although only three experiments were done and each value differed f r o m the average b y about
240
MICHAEL J.
W E I G H T A N D N E R I N A SAVAGE
50 per cent). Bicz (1960) found the oxygen uptake of leucocytes to be 103/~moles but he did have Tris present in the medium which probably inhibited the respiration. The values obtained by Seelich et al. (1957) of 402-473/Lmoles are very high.
250"
oq ua b- 200. U 0 U L.u _J _o o
- ~ 150' Z 0 >x 0 if) u,i ~ 100 E
0
/.// 3~0
60
;0
1 t2 0
MINUTES,
FIG. 1. Oxygen uptake of leucocytes: Q--O, in KRPAGA medium (each point mean of twenty-six observations); ©--©, in KRHAGA medium (each point mean of thirty-three observations). These workers used bicarbonate buffer with air as the gas phase and presumably the pH of this system must also have been very high. Esmann (1962) in a very thorough study of leucocyte respiration in bicarbonate-CO~, buffer (KRBAGA) found the oxygen consumption of leucocytes to be 172/zmoles. The lowest figures for oxygen uptakes for human leucocytes where phosphate buffer was used were those of McKinney et al. (1952) and Bisset & Alexander (1958, 1960) with buffer containing glucose, i.e. 92 gmoles and 111/~moles respectively. In the absence of glucose the consumption appears to be somewhat
OXYGEN U P T A K E OF LEUCOCYTES F R O M T H E C H A C M A BABOON
241
higher, and McKinney & Rundles (1956) reported a value of 150 #moles, and Athens et al. (1959) one of 170 #moles. Even these results in phosphate buffer are not really comparable as McKinney & Rundles and Athens et al. used resin plasma in their media, and that of Bisset & Alexander contained bicarbonate. In the present study the KRPAGA medium containing the CSA gelatine was compared with the K R H A G A medium containing the PSH gelatine. As in the case of phosphate buffers containing glucose the oxygen consumption is low in the KRPAGA medium. The only authors to get the higher results in human leucocytes are Seelich et al. (1957), who found roughly twice the values for leucocyte oxygen consumption in the case of both bicarbonate and phosphate buffers. The values obtained in phosphate could be due to the fairly rapid decrease in pH. In the case of K R H A G A buffer described in this paper the pH remained constant throughout the period of incubation and it is very likely that " H E P E S " has none of the toxic properties of Tris or imidazole. This medium could provide a standardized method for investigating the metabolism of intact leucocytes with little or no change in pH and no chance of agglutination. SUMMARY The oxygen consumption of leucocytes from the Chacma baboon (Papio ursinus) was measured in different media. A new buffer medium has been described for the study of intact leucocytes. The medium K R H A G A contains a deionized pork skin gelatine together with " H E P E S " buffer. In this medium the oxygen consumption of baboon leucocytes was greatly increased compared with the standard phosphate buffer (KRPAGA) and found to be 126 #moles/101° leucocytes per hr. The pH of the medium remains constant during the period of incubation which is not the case with phosphate buffer. The leucocytes do not agglutinate in the new medium. The new medium offers the opportunity to standardize studies on leucocyte metabolism. Acknowledgements--The authors express their gratitude to Professor C. P. Luck for constructive advice and criticism in the preparation of this manuscript. Also to Mr. H. V. Williams of Davis Gelatine Industries for a gift of American Pork-Skin Gelatine and much helpful advice, and to Mr. E. F. Allen for technical assistance with the animals. REFERENCES
ATHENSJ. W., MAUERA. M., ASHENBRUCKERH., CARTWRIGHTG. E. & WINTROBE(1959) Leucokinetic studies--I. A method for labelling leucocytes with diisopropylfluorophosphate (DFP32). Blood 14, 303-333. Blcz W. (1960) The influence of CO~ tension on the respiration of normal and leukaemic human leukocytes--I. Influence of endogenous respiration. Cancer Res. 20, 184-190. BIRD R. M., CLEMENSJ. A. & BECKERL. M. (1951) The metabolism of leucocytes taken from peripheral blood from leukaemic patients. Cancer 4, 1009-1014. BlSSET S. K. & ALEXANDERW. n. (1958) Effect of muscular activity prior to venepuncture on the respiration of leucocytes in vitro. Nature, Load. 181,909-910.
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MICHAELJ. WEIGHT AND NERINA SAVAGE
BISSET S. K. ~ ALEXANDERW. D. (1960) The effect of metabolic stimulants on the oxygen uptake of normal and leukaemic human leucocytes in vitro. Quart. j~. exp. Physiol. 45, 18-24.
ESMANN V. (1962) Carbohydrate metabolism and respiration in leucocytes from normal and diabetic subjects. Ph.D. Thesis, University of Aarus, Denmark. ESTES F. L., AUSTIN N. S. & GAST J. H. (1960) Metabolic characterisation of polymorphonuclear leucocytes. Clin. Chem. 6, 501-503. GOOD N. E., WINGEr G. D., WINTER W., CONOLLY T. N., IZAWAS. & SINGH R. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467-475. LEONTOVICHV. A. & ABEZGAUZN. N. (1960) Conditions prolonging the storage period of viable white cells. Probl. Gemat. 5, a A 50. McKINNEY G. R., MARTIN S. P., RUNDLES R. W. & GREEN R. (1952) Respiratory and glycolytic activities of human leucocytes in vitro, j~. appl. Physiol. 5, 335-340. McKINNEY G. R. & RUNDLES R. W. (1956) Lactate formation and glyoxylase activity in normal and leukaemic human leucocytes in vitro. Cancer Research 16, 67-69. MARTIN S. P. & GREEN T. (1958) Methods for the study of surviving leucocytes. In Methods in Medical Research, Vol. 7, pp. 136-137. Year Book Publishers, Chicago. MIRAS C. J., MANTOSJ. D. & LEVIS G. M. (1965) Fatty acid synthesis in human leucocytes. Biochem. biophys. Res. Commun. 19, 79-83. PASTORE E. J. (1959) Metabolism of acetate by human leucocytes. Ph.D. Thesis, Boston University Graduate School. REMMELE W. (1955) Atmung und Glykolyse der Leukozyten..4cta haemat. 13, 103-123. RowE C. E., ALLISON A. C. & LOVELOCKJ. D. (1960) Lipid biosynthesis in human blood; the incorporation of acetate into lipids by different types of human cells. Biochim. biophys. Acta 41,310-314. RUTHBERGR. A. & TERENT'EVAE. I. (1959) Preservation of viable packed white cells. Probl. Gemat. 4, 50-54. SEELICH F., LETNANSKYW., FRISCH W. & SCHNECKO. (1957) Zur Frage der Stoffwechsels normaler und pathologischer menschlicher Leukozyten. Z. Krebsforsch 62, 1-8. SKOOG W. A. & BECK W. S. (1956) Studies on the fibrinogen, dextran and phytohaemagglutinin methods of isolating leucocytes. Blood11,436-454. TULLIS J. L. (1953) Preservation of leucocytes. Blood 8, 563-575. TULLIS J. L. (1953) The Origin, Properties, Function and Preservation of White Blood Cells in Blood Cells and Plasma Proteins, pp. 276-279. Academic Press, New York. WARBURG O., GAwEHN K. & GEISSLERA. W. (1958) Stoffwechsel der weissen Blutzellen. Z. Naturforsch. 13, 515-516. Key Word Index---Oxygen consumption; leucocytes; leucocyte medium; baboon; human leucocytes; Papio ursinus.