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The effect of primaquine on lecithin metabolism in human erythrocytes
594 BBA
SHORTCOMMUNICATIONS 53102
The effect of primaquine on lecithin metabolism in human erythrocytes Human erythrocytes exposed to prelytic concentrations of primaquine in vitro manifest increased leakage of K+ and hypersensitivity to osmotic lysisl. Both of these responses
are considered
to indicate
The recent demonstration
an impaired
integrity
of an active phospholipid
of the red cell membrane.
metabolism
the mature human red ce112m4suggested that these functional brane effected metabolism.
by primaquine
might
be accompanied
in the membrane
alterations
by changes
of
of the mem-
in phospholipid
Venous blood was obtained from clinically normal adult males. The red cells were isolated by diluting the freshly drawn blood with an equal volume of a 3% solution
of bovine
fibrinogen-citrate
in isotonic
NaCl. After
complete
mixing,
the
blood cells were allowed to sediment at room temperature and the buffy coat and top 10% of the red cell layer were removed. The red cell mass was then washed 3 times with phosphate-buffered,
isotonic
NaCl (pH 7.4), at o-4”, the top 3-5 o/0of the
red cell layer being discarded each time. The red cells were finally suspended in ice cold calcium-free Krebs-Ringer phosphate solution (pH 7.4), to a hematocrit of 75 o/o and used immediately.
As determined
by cell counts,
white cell contamination
of the
red cell mass prepared by this method ranged from o-2 white cells per IOOOOO red cells. This was confirmed by the rarity of white cells as well as the virtual absence of platelets
identified
in stained blood films.
Red cell membranes were prepared by a modification of the method of DODGE, MITCHELL AND HANAHAN~. Hemolysis was accomplished by placing the red cells in IO vol. of an aqueous
solution
of IO-* M EDTA
and 1.7 . IO-~ M Tris (pH 7.4), at
o-4” for IO min. The membranes were sedimented by centrifuging at 54 500 x g for 15 min and then washed 3 times with the EDTA-Tris solution and once with calciumfree Krebs-Ringer
phosphate
(pH 7.4).
Metabolic reactions were carried out in a shaking water bath at 37’ using 25 ml erlenmeyer flasks with air as the gaseous phase. The reactions were terminated by initiating the preparation of membranes when intact red cells were used or starting the lipid extraction when membranes were used. Extraction, chromatographic separation, and preparation of phospholipids for liquid scintillation spectroscopy were done as previously
describeds.
[I-l%]Palmitic acid, [I-%]linoleic acid, [r-14C]palmityl-CoA and palmityl-CoA were prepared or obtained as described previously 8. Lecithin containing [I-X]palmitic acid was prepared by incubating red cell membranes withlysolecithinand[r-14C]palmityl-CoA followed by extraction and chromatographic separation of the lecithin. Lysolecithin was prepared from egg lecithin by the method of LANG AND PENNY'. Bovine fibrinogen-citrate was obtained from Armour Pharmaceutical Co., Kankakee, Ill. Primaquine phosphate was a gift from Dr. R. 0. CLINTON, Sterling-Winthrop Research Institute, Rensselaer, New York. The rate of [I-%]linoleic acid incorporation into lecithin of intact red cells was markedly enhanced in the presence of primaquine. This stimulation was observed for periods as long as 90 min and was proportional to the primaquine concentration (Figs, I and 2). Incorporation of [I-‘4C]palmitic acid into lecithin was less rapid than Biochim.
Biophys.
Acta,
125 (1966)
594-597
505
SHORT COMMUNICATIONS
that of linoleic acid, but the degree of stimulation effected by the primaquine was similar. The accelerated formation of [Xllecithin in the red cells in the presence of primaquine predicated a concomitant increase in the availability of phosphoglyceride
/
Fig. I. The effect in the presence of min per mpmole), Ringer phosphate incubation was at
d
I
1
I
2
Primaqui ne
of duration of incubation on [W]lecithin formation by intact human red cells primaquine. The system contained 80 mpmoles of linoleic acid (1600 counts/ 1.35 ml of red cells suspended to a 75 yO hematocrit in calcium-free Krebsbuffer (pH 7.4). and IO pmolcs of primaquine. Final volume was 2.0 ml and 37”.
I
I
6 4 Primoauine fpmoles)
I
I
8
to’
Fig. a. The effect of primaquine on [f*C]lecithin formation by intact human red cells. The system contained 80 mpmoles of linoleic acid (rdoo counts/min per mpmole), 1.35 ml of red cells suspended to a 75 yO hematocrit in calcium-free Krebs-Ringer phosphate buffer (pH 7.4), and primaquine as indicated. Final volume was 2.0 ml and incubation was at 37O for go min.
acceptor. Several investigators, however, have been unable to demonstrate phospholipase A activity in the red cell 2-4, and an attempt to detect a primaquine-activated lecithinase in the red cell was likewise unsuccessful (Table I). Since de BOVOsynthesis of lecithin in the red cell appears to be insignificant s, and, in these experiments, no exogenous acceptor was added to the reaction system and the red cells were thoroughly washed free of serum, endogenous lysophosphoglyceride appears to have served as Biochina.Biofihys.
Acta,
125 (1966) 594-597
SHORT COMMUNICATIONS
596 TABLE
I
PHOSPHOLIPASEA ACTIVITY IN THE RED CELL The system contained 4 m,mnoles of [%jlecithin (2250 counts/min per mpmole), and 1.35 ml of intact red cells suspended in calcium-free Krebs-Ringer phosphate buffer (pH 7.4), (75 o/0 hematocrit). In addition, tubes z and 4 contained IO Llmoles of primaquine and tubes 3 and 4, IOO mpmoles of non-radioactive palmitic acid. Final volume was 2.0 ml and incubation was at 37’ for 60 min. Pgimaquine
Tube
I
[W]Lecithin (counts~min)
14C-labeled fatty acid
(c0u&/min,l
4279
-
2
+
3 4
+
0
4558 4699 4458
0 0 0
the major substrate
for acylation.
mation
an otherwise
by exposing
to the activated
fatty
Thus, primaquine inaccessable
of lysolecithin
[14C]lecithin forin the red cell
acids.
Phospholipids and phospholipid metabolism Although primaquine consistently
membrane2y6. incorporation
may accelerate
reservoir
into lecithin
of intact
in the red cell are confined to the accelerated long chain fatty acid
red cells, incorporation
of long chain fatty
acid
into lecithin of membranes prepared from red cells by osmotic lysis was, paradoxically, inhibited (Table II). This inhibition did not appear to be due to removal of a required TABLE
II
INFLUENCEOF RED CELL INTEGRITY ON PRIMAQUINESTIMULATED[~X]LECITHIN
FORMATION
The system contained 25 mpmoles of [‘%]palmityl-CoA (2500 counts/min per mpmole), 10 /,&moles of primaquine as indicated, and 1.35 ml of intact red cells suspended in calcium-free Krebs-Ringer phosphate buffer, (pH 7.4), to a 750/u hematocrit, or the equivalent of red cells hemolyzed by repeated freezing and thawing, or red cell membranes (1.5 mg protein) suspended in the buffer to a 20 y0 hematocrit with or without I ml of the “supernatant” prepared by removing the membranes of hemolyzed red cells. Final volume was 2.0 ml and incubation was at 37’ for 30 min. Primaquine (counts/min)
Erythroc_vtes
y; Change
+ ~Intact Hemolyzed Membranes Membranes
+ “Supernatant”
5’6 7676 9900 8159
970 50’0 3152 3636
$88 -34 -68 -55
cofactor during membrane preparation, since lecithin formation was also by primaquine in hemolysates from which the membranes had not been These observations suggest that a molecular rearrangement occurs during so that primaquine has an inhibitory effect on the lysolecithin acylating In erythrocytes exposed to primaquine in vitro and in viva, hydrogen
depressed separated. hemolysis enzymes. peroxide is
generated at increased ratesO. The formation of the peroxide requires the presence of ferro-hemoglobin, and the peroxide generated is rapidly detoxified via the glutathione peroxidase pathway which requires the presence of glucose to maintain adequate levels of reduced glutathione. In order to determine whether the effect of primaquine on lecithin metabolism was a consequence of hydrogen peroxide accumulation, 3 types Biochim. Biophys.
Acta, 125 (1966) 594-597
597
SHORT COMMUNICATIONS
of experiments were carried out: (I) the potential formation of peroxide in red cells incubated with primaquine was blocked by prior conversion of the hemoglobin to methemoglobin; (2)the accumulation of peroxide was prevented by incubating the red cells with primaquine in the presence of glucose; and (3) hydrogen peroxide rather than primaquine was added directly to the red cells by a gaseous diffusion technique. In the first 2 types of experiments, the effect of primaquine on [Xllecithin formation was unchanged despite the fact that peroxide could not be generated in the presence of methemoglobin and despite the fact that if peroxide were formed it would be rapidly eliminated in the presence of glucose. Lastly, no evidence was found that peroxide itself mimics the effect of primaquine on phospholipid metabolism. Thus, the mechanism whereby primaquine affects lecithin metabolism in the red cell does not appear to be directly related to hydrogen peroxide and oxy-redox reactions of these cells. In recent years, a number of investigators have attempted to relate the flux of Na+ and K+ in the red cell to its phospholipid metabolismlO. The observation that primaquine, an agent known to cause an accelerated loss of K+ and gain of Na+ in the red cell, has an effect on erythrocyte phospholipid metabolism lends support to this postulated association. The mechanism whereby primaquine produces this dual effect on the red cell remains to be elucidated. This investigation was supported by Grant HE 10090 from the National Heart Institute, National Institutes of Health, United States Public Health Service, Grant 64-G-116 from the American Heart Association, and Grant GB 1416 from the National Science Foundation. Departments of Pathology, amd Physiology Duke University
and Pharmacology,
BENJAMIN
Medical Center,
PAUL
WITTELS
HOCHSTEIN
Durham, N. C. (U.S.A.) I R. WEED, J. EBER AND A. ROTHSTEIN, J. C&n. Invest., 40 (1961) 130. 2 M. M. OLIVEIRA AND M. VAUGHAN, J. Lipid Res., 5 (1964)156. 3 A. F. ROBERTSON AND W. E. M. LANDS, J. Lipid Res., 5 (1964) 88. 4 E. MULDER AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 106 (1965)
106. 5 J. T. DODGE, C. MITCHELL AND D. J. HANAHAN, Arch. Biochem. Biophys., IOO (1963) 119. 6 B. WITTELS AND R. BRESSLER, J. Clin. Invest., 44 (1965) 1639. 7 C. LONG AND I. F. PENNY, Biochem. J., 65 (1957) 382. 8 E. MULDER AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 106 (1965) 348. g G. COHEN AND P. HOCHSTEIN, Biochemistry, 3 (1964) 895. IO L. L. M. VAN DEENEN, in R. T. HOLMAN, Pryress in the Chemistry of Fats and other Lipids, Vol.8,Part I, Pergamon Press, New York, 1965.
Received June 23rd, 1966 Biochim. BBA
Biophys.
Acta,
125 (1966) 594-597
53106
On the positional specificity of pancreatic lipase A number of experiments performed in several laboratories have demonstrated that pancreatic lipase (EC 3.1.1.3) displays a very strong specificity towards the exB&him.
Biophys.
Acta,
125 (1966) 597-600
SHORTCOMMUNICATIONS 53102
The effect of primaquine on lecithin metabolism in human erythrocytes Human erythrocytes exposed to prelytic concentrations of primaquine in vitro manifest increased leakage of K+ and hypersensitivity to osmotic lysisl. Both of these responses
are considered
to indicate
The recent demonstration
an impaired
integrity
of an active phospholipid
of the red cell membrane.
metabolism
the mature human red ce112m4suggested that these functional brane effected metabolism.
by primaquine
might
be accompanied
in the membrane
alterations
by changes
of
of the mem-
in phospholipid
Venous blood was obtained from clinically normal adult males. The red cells were isolated by diluting the freshly drawn blood with an equal volume of a 3% solution
of bovine
fibrinogen-citrate
in isotonic
NaCl. After
complete
mixing,
the
blood cells were allowed to sediment at room temperature and the buffy coat and top 10% of the red cell layer were removed. The red cell mass was then washed 3 times with phosphate-buffered,
isotonic
NaCl (pH 7.4), at o-4”, the top 3-5 o/0of the
red cell layer being discarded each time. The red cells were finally suspended in ice cold calcium-free Krebs-Ringer phosphate solution (pH 7.4), to a hematocrit of 75 o/o and used immediately.
As determined
by cell counts,
white cell contamination
of the
red cell mass prepared by this method ranged from o-2 white cells per IOOOOO red cells. This was confirmed by the rarity of white cells as well as the virtual absence of platelets
identified
in stained blood films.
Red cell membranes were prepared by a modification of the method of DODGE, MITCHELL AND HANAHAN~. Hemolysis was accomplished by placing the red cells in IO vol. of an aqueous
solution
of IO-* M EDTA
and 1.7 . IO-~ M Tris (pH 7.4), at
o-4” for IO min. The membranes were sedimented by centrifuging at 54 500 x g for 15 min and then washed 3 times with the EDTA-Tris solution and once with calciumfree Krebs-Ringer
phosphate
(pH 7.4).
Metabolic reactions were carried out in a shaking water bath at 37’ using 25 ml erlenmeyer flasks with air as the gaseous phase. The reactions were terminated by initiating the preparation of membranes when intact red cells were used or starting the lipid extraction when membranes were used. Extraction, chromatographic separation, and preparation of phospholipids for liquid scintillation spectroscopy were done as previously
describeds.
[I-l%]Palmitic acid, [I-%]linoleic acid, [r-14C]palmityl-CoA and palmityl-CoA were prepared or obtained as described previously 8. Lecithin containing [I-X]palmitic acid was prepared by incubating red cell membranes withlysolecithinand[r-14C]palmityl-CoA followed by extraction and chromatographic separation of the lecithin. Lysolecithin was prepared from egg lecithin by the method of LANG AND PENNY'. Bovine fibrinogen-citrate was obtained from Armour Pharmaceutical Co., Kankakee, Ill. Primaquine phosphate was a gift from Dr. R. 0. CLINTON, Sterling-Winthrop Research Institute, Rensselaer, New York. The rate of [I-%]linoleic acid incorporation into lecithin of intact red cells was markedly enhanced in the presence of primaquine. This stimulation was observed for periods as long as 90 min and was proportional to the primaquine concentration (Figs, I and 2). Incorporation of [I-‘4C]palmitic acid into lecithin was less rapid than Biochim.
Biophys.
Acta,
125 (1966)
594-597
505
SHORT COMMUNICATIONS
that of linoleic acid, but the degree of stimulation effected by the primaquine was similar. The accelerated formation of [Xllecithin in the red cells in the presence of primaquine predicated a concomitant increase in the availability of phosphoglyceride
/
Fig. I. The effect in the presence of min per mpmole), Ringer phosphate incubation was at
d
I
1
I
2
Primaqui ne
of duration of incubation on [W]lecithin formation by intact human red cells primaquine. The system contained 80 mpmoles of linoleic acid (1600 counts/ 1.35 ml of red cells suspended to a 75 yO hematocrit in calcium-free Krebsbuffer (pH 7.4). and IO pmolcs of primaquine. Final volume was 2.0 ml and 37”.
I
I
6 4 Primoauine fpmoles)
I
I
8
to’
Fig. a. The effect of primaquine on [f*C]lecithin formation by intact human red cells. The system contained 80 mpmoles of linoleic acid (rdoo counts/min per mpmole), 1.35 ml of red cells suspended to a 75 yO hematocrit in calcium-free Krebs-Ringer phosphate buffer (pH 7.4), and primaquine as indicated. Final volume was 2.0 ml and incubation was at 37O for go min.
acceptor. Several investigators, however, have been unable to demonstrate phospholipase A activity in the red cell 2-4, and an attempt to detect a primaquine-activated lecithinase in the red cell was likewise unsuccessful (Table I). Since de BOVOsynthesis of lecithin in the red cell appears to be insignificant s, and, in these experiments, no exogenous acceptor was added to the reaction system and the red cells were thoroughly washed free of serum, endogenous lysophosphoglyceride appears to have served as Biochina.Biofihys.
Acta,
125 (1966) 594-597
SHORT COMMUNICATIONS
596 TABLE
I
PHOSPHOLIPASEA ACTIVITY IN THE RED CELL The system contained 4 m,mnoles of [%jlecithin (2250 counts/min per mpmole), and 1.35 ml of intact red cells suspended in calcium-free Krebs-Ringer phosphate buffer (pH 7.4), (75 o/0 hematocrit). In addition, tubes z and 4 contained IO Llmoles of primaquine and tubes 3 and 4, IOO mpmoles of non-radioactive palmitic acid. Final volume was 2.0 ml and incubation was at 37’ for 60 min. Pgimaquine
Tube
I
[W]Lecithin (counts~min)
14C-labeled fatty acid
(c0u&/min,l
4279
-
2
+
3 4
+
0
4558 4699 4458
0 0 0
the major substrate
for acylation.
mation
an otherwise
by exposing
to the activated
fatty
Thus, primaquine inaccessable
of lysolecithin
[14C]lecithin forin the red cell
acids.
Phospholipids and phospholipid metabolism Although primaquine consistently
membrane2y6. incorporation
may accelerate
reservoir
into lecithin
of intact
in the red cell are confined to the accelerated long chain fatty acid
red cells, incorporation
of long chain fatty
acid
into lecithin of membranes prepared from red cells by osmotic lysis was, paradoxically, inhibited (Table II). This inhibition did not appear to be due to removal of a required TABLE
II
INFLUENCEOF RED CELL INTEGRITY ON PRIMAQUINESTIMULATED[~X]LECITHIN
FORMATION
The system contained 25 mpmoles of [‘%]palmityl-CoA (2500 counts/min per mpmole), 10 /,&moles of primaquine as indicated, and 1.35 ml of intact red cells suspended in calcium-free Krebs-Ringer phosphate buffer, (pH 7.4), to a 750/u hematocrit, or the equivalent of red cells hemolyzed by repeated freezing and thawing, or red cell membranes (1.5 mg protein) suspended in the buffer to a 20 y0 hematocrit with or without I ml of the “supernatant” prepared by removing the membranes of hemolyzed red cells. Final volume was 2.0 ml and incubation was at 37’ for 30 min. Primaquine (counts/min)
Erythroc_vtes
y; Change
+ ~Intact Hemolyzed Membranes Membranes
+ “Supernatant”
5’6 7676 9900 8159
970 50’0 3152 3636
$88 -34 -68 -55
cofactor during membrane preparation, since lecithin formation was also by primaquine in hemolysates from which the membranes had not been These observations suggest that a molecular rearrangement occurs during so that primaquine has an inhibitory effect on the lysolecithin acylating In erythrocytes exposed to primaquine in vitro and in viva, hydrogen
depressed separated. hemolysis enzymes. peroxide is
generated at increased ratesO. The formation of the peroxide requires the presence of ferro-hemoglobin, and the peroxide generated is rapidly detoxified via the glutathione peroxidase pathway which requires the presence of glucose to maintain adequate levels of reduced glutathione. In order to determine whether the effect of primaquine on lecithin metabolism was a consequence of hydrogen peroxide accumulation, 3 types Biochim. Biophys.
Acta, 125 (1966) 594-597
597
SHORT COMMUNICATIONS
of experiments were carried out: (I) the potential formation of peroxide in red cells incubated with primaquine was blocked by prior conversion of the hemoglobin to methemoglobin; (2)the accumulation of peroxide was prevented by incubating the red cells with primaquine in the presence of glucose; and (3) hydrogen peroxide rather than primaquine was added directly to the red cells by a gaseous diffusion technique. In the first 2 types of experiments, the effect of primaquine on [Xllecithin formation was unchanged despite the fact that peroxide could not be generated in the presence of methemoglobin and despite the fact that if peroxide were formed it would be rapidly eliminated in the presence of glucose. Lastly, no evidence was found that peroxide itself mimics the effect of primaquine on phospholipid metabolism. Thus, the mechanism whereby primaquine affects lecithin metabolism in the red cell does not appear to be directly related to hydrogen peroxide and oxy-redox reactions of these cells. In recent years, a number of investigators have attempted to relate the flux of Na+ and K+ in the red cell to its phospholipid metabolismlO. The observation that primaquine, an agent known to cause an accelerated loss of K+ and gain of Na+ in the red cell, has an effect on erythrocyte phospholipid metabolism lends support to this postulated association. The mechanism whereby primaquine produces this dual effect on the red cell remains to be elucidated. This investigation was supported by Grant HE 10090 from the National Heart Institute, National Institutes of Health, United States Public Health Service, Grant 64-G-116 from the American Heart Association, and Grant GB 1416 from the National Science Foundation. Departments of Pathology, amd Physiology Duke University
and Pharmacology,
BENJAMIN
Medical Center,
PAUL
WITTELS
HOCHSTEIN
Durham, N. C. (U.S.A.) I R. WEED, J. EBER AND A. ROTHSTEIN, J. C&n. Invest., 40 (1961) 130. 2 M. M. OLIVEIRA AND M. VAUGHAN, J. Lipid Res., 5 (1964)156. 3 A. F. ROBERTSON AND W. E. M. LANDS, J. Lipid Res., 5 (1964) 88. 4 E. MULDER AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 106 (1965)
106. 5 J. T. DODGE, C. MITCHELL AND D. J. HANAHAN, Arch. Biochem. Biophys., IOO (1963) 119. 6 B. WITTELS AND R. BRESSLER, J. Clin. Invest., 44 (1965) 1639. 7 C. LONG AND I. F. PENNY, Biochem. J., 65 (1957) 382. 8 E. MULDER AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 106 (1965) 348. g G. COHEN AND P. HOCHSTEIN, Biochemistry, 3 (1964) 895. IO L. L. M. VAN DEENEN, in R. T. HOLMAN, Pryress in the Chemistry of Fats and other Lipids, Vol.8,Part I, Pergamon Press, New York, 1965.
Received June 23rd, 1966 Biochim. BBA
Biophys.
Acta,
125 (1966) 594-597
53106
On the positional specificity of pancreatic lipase A number of experiments performed in several laboratories have demonstrated that pancreatic lipase (EC 3.1.1.3) displays a very strong specificity towards the exB&him.
Biophys.
Acta,
125 (1966) 597-600