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Incorporation of fatty acids into phospholipids of erythrocytes from humans with muscular dystrophy
Neuroscience Letters, 20 (1980) 115-118 © Elsevier/North-Holland Scientific Publishers Ltd.
115
INCORPORATION OF FATTY ACIDS INTO P H O S P H O L I P I D S E R Y T H R O C Y T E S FROM H U M A N S W I T H M U S C U L A R D Y S T R O P H Y
OF
A.P. SHERBLOM, D.J. McALLISTER, M.V. MACUL and J.L. HOWLAND Committee on Biochemistry, Bowdoin College, Brunswick, ME 04011 (U.S.A.) (Received May 14th, 1980; Accepted July 15th, 1980)
Erythrocytes from patients with either Duchenne or myotonic muscular dystrophy exhibit a diminished ability to incorporate exogenous 14C-labeled oleate into membrane phospholipids. In control cells, the incorporation is stimulation by calcium (plus the bivalent cation ionophore A23187); in dystrophic cells the stimulation is considerably lessened. A calcium-induced uptake of unsaturated fatty acids into normal erythrocytes, together with its diminution in dystrophic cells, may account for observations of reduced amounts of palmitoleate in erythrocytes from dystrophic individuals. Defective capacity for acylation of lysophospholipids could account for some of the membrane abnormalities reported to be associated with these diseases.
Muscular dystrophies are associated with membrane abnormalities affecting various tissues and it has been argued that a membrane defect underlies the pathogenesis of such diseases [12]. For example, erythrocytes from dystrophic individuals exhibit a wide variety of alterations, including abnormal activities of a number of membrane-bound enzymes [6, 11, 14] as well as abnormal ionic transport [3, 8]. The diversity of alterations in metabolically unrelated functions suggests a common underlying source in an abnormal hydrophobic (lipid) phase of the erythrocyte plasma membrane and alterations in the lipid composition of erythrocyte membranes have been reported [2, 4, 13]. For instance, we reported that cells from Duchenne-dystrophic and myotonic-dystrophic individuals contained less palmitoleic acid than did normal controls [4], and this observation was subsequently confirmed in other laboratories [2, 13]. Similarly, dystrophic hamsters exhibit significantly diminished palmitoleate in their intracellular membranes [1]. In contrast, Plishker and Appel [10] and McLaughlin and Engel [7] failed to confirm the finding of diminished palmitoleate and pointed out that our values for that fatty acid in normal cells were higher than many reported in the literature. These authors confirmed, moreover, the generally accepted observation [5] that phospholipid composition was identical to control values. We have therefore been concerned to seek both an explanation for the apparent discrepancy in palmitoleate content in control versus dystrophic erythrocyte membranes and a mechanism by which the hydrophobic region of the membrane might become altered (even in the face of
116 normal phospholipid composition) and so exert an abnormal influence of the several membrane-bound enzymes. We report here incorporation of unsaturated fatty acids into erythrocyte phospholipids, a process that is substantially diminished in cells from dystrophic individuals. This incorporation, together with its diminution in the dystrophic case, can, in principle, account for the observed differences in apparent palmitoleic acid content. Table I shows incorporation of 14C-labeled oleate (18:1) into total phospholipids from control and Duchenne-dystrophic cells. Blood was drawn into citratecontaining tubes, the buffy coat removed, and cells washed three times in isotonic saline solution containing 10 mM Tris. HC1, pH 7.4. Cells were incubated with shaking at 37°C for 1 h in a medium containing 8 mM glucose, 8 mM Tris. HC1, pH 7.4, 9 mM KC1, 130 mM NaC1, and 13 nmol of [~4C]oleate with approximately 105 cpm. The oleate was suspended in the medium prior to addition of cells using an ultrasonic probe for 30 sec. Labeled fatty acids were obtained from New England Nuclear Corp. The reaction volume was 0.25 ml. The reaction was terminated by rapid addition of 0.6 ml methanol plus 0.3 ml chloroform. After 30 min, 0.3 ml methanol plus 0.3 ml 2 M KC1 were added to each tube and the mixture was centrifuged at 1000 × g for 5 min. The chloroform layer was retained and evaporated to dryness under nitrogen. In experiments, such as in Table l, where incorporation into total phospholipids was measured, lipids were separated on a Silica gel H thin layer plate developed with hexane:diethyl ether: formic acid (70 : 30 : 2). This system separated a single phospholipid spot from various classes of neutral lipids. Virtually all label ran with the phospholipid spot; uptake into triglycerides, diglycerides, monoglycerides and cholesterol esters were entirely due to contaminating leukocytes. Finally, in experiments where distribution of label among the different classes of phospholipids was measured, lipids were spotted on Silica gel H plates which were developed with a solvent containing chloroform : methanol : acetone : acetic acid: water (30: 10 : 40 : 10: 5). In Table I it is evident that there is substantial incorporation of labeled oleate into erythrocyte total phospholipids and that, in control cells, the incorporation is elevated about 75% upon addition of calcium plus the bivalent cation ionophore, A23187. Incorporation into cells from Duchenne- and myotonic-dystrophic TABLE 1 INCORPORATIONOF [J4C]OLEATEINTO ERYTHROCYTEPHOSPHOLIPID Uptake (pmol/109 cells/min)
Normal (n = 8) Duchenne (n = 9) Myotonic (n = 4) a
1 mM CaCI2 + 2 #g A23187.
-Calcium
+Calcium + A23187a
A°70
1.6 _+ 0.1 1.4 _+ 0.1 1.3 _+ 0.2
2.8 +_ 0.2 1.7 _+ 0.1 1.7 _+ 0.2
+75 +21 +30
117
individuals is lower and, in particular, the enhancement due to internalized calcium is greatly diminished. In other experiments, incorporation of the fully-saturated fatty acid, palmitic acid (16:0), occurred at only about one-fifth of the rate with the unsaturated oleate (18:1) in either the presence or absence of calcium plus ionophore. Table I1 presents the distribution of oleate uptake among the several phospholipid classes. It is evident that substantial uptake occurs into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine + phosphatidylinositol and virtually none into sphingomyelin. Interestingly, substantial uptake occurs also into phosphatidate, a relatively minor membrane component (less than 2°7o of total phospholipids as compared to about 3007o for phosphatidylcholine - see ref. 5). In the absence of added calcium + ionophore, distribution of uptake into the several phospholipid fractions is similar in the Duchenne and control cases. However, internalized calcium induces a substantial increase in acyl labeling of phosphatidylcholine, phosphatidylethanolamine and phosphatidate in control cells; the increase is much diminished in cells from Duchenne-dystrophic subjects. This loss of calcium-induced stimulation is reflected in the more modest increase in uptake into total phospholipids of Duchenne cells indicated in Table I. A calcium-stimulated uptake of unsaturated fatty acids in normal (but not dystrophic) cells could account for the differential enrichment of palmitoleate (16: 1) noted earlier [4]. That this is indeed the case is suggested by recent unpublished experiments in which we failed to observe differences in palmitoleate content between control and Duchenne erythrocytes when blood was drawn into citrated, instead of heparin-coated, tubes. Thus, sequestering plasma calcium eliminated the difference, presumably by interfering with uptake of unsaturated fatty acids. Because palmitoleate is a relatively minor fatty acid in erythrocyte membranes, the presence (or absence) of calcium-induced uptake would be expected to be particularly apparent. T A B L E 1I INCORPORATION
OF [~4C]OLEATE INTO PHOSPHOLIPID
CLASSES
Uptake (pmol/109 cells/min)
P h o s p h o l i p i d class Normal
Duchenne dystrophy
- Calcium
+ Calcium a
- Calcium
+ Calcium
Sphingomyelin
0.04
0.04
0.05
0.05
Phosphatidylcholine
0.68 + 0.02
1.89 +_ 0 . 2
0.63 _+ 0 . 0 4
0.97 + 0.2
Phosphatidylserine (+ phosphatidylinositol) Phosphatidylethanolamine Phosphatidate
0.65 _+ 0 . 0 4 0 . 2 4 _+ 0 . 0 2 0 . 2 4 _+ 0.05
0.48 _+ 0 . 0 6 0 . 9 0 _+ 0.1 0.53 _+ 0 . 0 4
0.61 +_+_0 . 0 2 0 . 3 6 +_ 0 . 0 4 0.41 _+ 0.1
0 . 3 6 +_ 0.07 0 . 5 2 _+ 0 . 0 7 0 . 4 4 _+ 0 . 0 6
a 1.0 m M CaC12 + 2 ~zg A 2 3 1 8 7 .
118
It is likely that the uptake of acyl residues into erythrocytes occurs through acylation of lysophospholipids. Such a process has been extensively studied in the case of lysophosphatidylcholine, which is able to catalyze an acyl exchange in either direction [9]. Presumably a similar process can lead to incorporation into phosphatidate and there is evidence, at least in nucleated cells, that lysophosphatidate serves as an acceptor for unsaturated fatty acids in the subsequent synthesis of inositol-containing phospholipids [15]. Because lysophospholipids are known to be disruptive to membrane integrity, their production through the action of phospholipase A requires a capacity for reacylation as a repair function. If dystrophic cells are deficient in this function, then widespread membrane abnormalities should not be inexplicable. This investigation was Dystrophy Association of Maine Affiliate. We thank samples from patients with 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15
supported by research grants from The Muscular America, Inc. and The American Heart Association, Dr. Cairbre McCann for assistance in obtaining blood muscular dystrophies.
Barakat, H.A., Johnson, D.R. and Kerr, D.S., Changes in the phospholipid composition of microsomal membranes of dystrophic hamsters, Proc. Soc. exp. Biol. (N.Y.), 163 (1980) 167-170. Beyer, P., Bieth, R., Robert, J., Freysz, H. and Uhl, T., Phospholipides erythrocytaires darts la myopathie de Duchenne de Boulogne, Nouv. presse Med., 8 (1979) 4043-4044. Howland, J.L., Abnormal potassium conductance associated with genetic muscular dystrophy, Nature (Lond.), 251 (1974) 724-725. Howland, J.L. and lyer, S.L., Erythrocyte lipids in heterozygous carriers of Duchenne muscular dystrophy, Science, 198 (1977) 309 310. Kalofoutis, A., Jullien, G. and Spanos, V., Erythrocyte phospholipids in Duchenne muscular dystrophy, Clin. chim. Acta, 74 (1977) 85-87. Mawatari, S., Schonberg, M. and Olarte, M., AYPase and adeny[ cyclase in erythrocytes of patients with Duchenne muscular dystrophy, Arch. Neurol. (Chic.), 33 (1976) 489 493. McLaughlin, J. and Engel, W.K., Lipid composition of erythrocytes: findings in Duchenne's muscular dystrophy and myotonic atrophy, Arch. Neurol. (Chic.), 36 (1979) 351-354. Mollman, J., Cardenas, J. and Pleasure, D., Alteration in calcium transport by Duchenne erythrocytes, Neurology (Minneap.), 29 (1979) 557. Mulder, E. and van Deenen, L.L.M., Metabolism of red-celt lipids. Ill. Pathways for phospholipid renewal, Biochim. biophys. Acta (Amst.), 106 (1965) 348 356. Plishker, G.A. and Appel, S.H., Red blood cell alteration in muscular dystrophy: the role of lipids, Muscle and Nerve, 3 (1980) 70 81. Roses, A.D. and Appel, S.H., Erythrocyte spectrin peak II phosphorylation in Duchenne muscular dystrophy, J. neurol. Sci., 29 (1976) 185-193. Rowland, L.P., Pathogenesis of muscular dystrophies, Arch. Neurol. (Chic.), 33 (1976) 315-321. Ruitenbeek, W., The fatty acid composition of various lipid fraction isolated from erythrocytes and blood plasma of patients with Duchenne and congenital myotonic dystrophy, Clin. chim. Acta, 89 (1978) 99-110. Ruitenbeek, W., Membrane-bound enzymes of erythrocytes in human muscular dystrophy, J. neurol. Sci., 41 (1979) 71 80. Thompson, W., Cytidine diphosphate diglyceride of bovine brain and liver: isolation and characterization. In G. Porcellati, L. Amaducci and C. Galli (Eds.), Function and Metabolism of Phospholipids in the Central and Peripheral Nervous Systems, Plenum, New York, 1976, pp. 27-36.
115
INCORPORATION OF FATTY ACIDS INTO P H O S P H O L I P I D S E R Y T H R O C Y T E S FROM H U M A N S W I T H M U S C U L A R D Y S T R O P H Y
OF
A.P. SHERBLOM, D.J. McALLISTER, M.V. MACUL and J.L. HOWLAND Committee on Biochemistry, Bowdoin College, Brunswick, ME 04011 (U.S.A.) (Received May 14th, 1980; Accepted July 15th, 1980)
Erythrocytes from patients with either Duchenne or myotonic muscular dystrophy exhibit a diminished ability to incorporate exogenous 14C-labeled oleate into membrane phospholipids. In control cells, the incorporation is stimulation by calcium (plus the bivalent cation ionophore A23187); in dystrophic cells the stimulation is considerably lessened. A calcium-induced uptake of unsaturated fatty acids into normal erythrocytes, together with its diminution in dystrophic cells, may account for observations of reduced amounts of palmitoleate in erythrocytes from dystrophic individuals. Defective capacity for acylation of lysophospholipids could account for some of the membrane abnormalities reported to be associated with these diseases.
Muscular dystrophies are associated with membrane abnormalities affecting various tissues and it has been argued that a membrane defect underlies the pathogenesis of such diseases [12]. For example, erythrocytes from dystrophic individuals exhibit a wide variety of alterations, including abnormal activities of a number of membrane-bound enzymes [6, 11, 14] as well as abnormal ionic transport [3, 8]. The diversity of alterations in metabolically unrelated functions suggests a common underlying source in an abnormal hydrophobic (lipid) phase of the erythrocyte plasma membrane and alterations in the lipid composition of erythrocyte membranes have been reported [2, 4, 13]. For instance, we reported that cells from Duchenne-dystrophic and myotonic-dystrophic individuals contained less palmitoleic acid than did normal controls [4], and this observation was subsequently confirmed in other laboratories [2, 13]. Similarly, dystrophic hamsters exhibit significantly diminished palmitoleate in their intracellular membranes [1]. In contrast, Plishker and Appel [10] and McLaughlin and Engel [7] failed to confirm the finding of diminished palmitoleate and pointed out that our values for that fatty acid in normal cells were higher than many reported in the literature. These authors confirmed, moreover, the generally accepted observation [5] that phospholipid composition was identical to control values. We have therefore been concerned to seek both an explanation for the apparent discrepancy in palmitoleate content in control versus dystrophic erythrocyte membranes and a mechanism by which the hydrophobic region of the membrane might become altered (even in the face of
116 normal phospholipid composition) and so exert an abnormal influence of the several membrane-bound enzymes. We report here incorporation of unsaturated fatty acids into erythrocyte phospholipids, a process that is substantially diminished in cells from dystrophic individuals. This incorporation, together with its diminution in the dystrophic case, can, in principle, account for the observed differences in apparent palmitoleic acid content. Table I shows incorporation of 14C-labeled oleate (18:1) into total phospholipids from control and Duchenne-dystrophic cells. Blood was drawn into citratecontaining tubes, the buffy coat removed, and cells washed three times in isotonic saline solution containing 10 mM Tris. HC1, pH 7.4. Cells were incubated with shaking at 37°C for 1 h in a medium containing 8 mM glucose, 8 mM Tris. HC1, pH 7.4, 9 mM KC1, 130 mM NaC1, and 13 nmol of [~4C]oleate with approximately 105 cpm. The oleate was suspended in the medium prior to addition of cells using an ultrasonic probe for 30 sec. Labeled fatty acids were obtained from New England Nuclear Corp. The reaction volume was 0.25 ml. The reaction was terminated by rapid addition of 0.6 ml methanol plus 0.3 ml chloroform. After 30 min, 0.3 ml methanol plus 0.3 ml 2 M KC1 were added to each tube and the mixture was centrifuged at 1000 × g for 5 min. The chloroform layer was retained and evaporated to dryness under nitrogen. In experiments, such as in Table l, where incorporation into total phospholipids was measured, lipids were separated on a Silica gel H thin layer plate developed with hexane:diethyl ether: formic acid (70 : 30 : 2). This system separated a single phospholipid spot from various classes of neutral lipids. Virtually all label ran with the phospholipid spot; uptake into triglycerides, diglycerides, monoglycerides and cholesterol esters were entirely due to contaminating leukocytes. Finally, in experiments where distribution of label among the different classes of phospholipids was measured, lipids were spotted on Silica gel H plates which were developed with a solvent containing chloroform : methanol : acetone : acetic acid: water (30: 10 : 40 : 10: 5). In Table I it is evident that there is substantial incorporation of labeled oleate into erythrocyte total phospholipids and that, in control cells, the incorporation is elevated about 75% upon addition of calcium plus the bivalent cation ionophore, A23187. Incorporation into cells from Duchenne- and myotonic-dystrophic TABLE 1 INCORPORATIONOF [J4C]OLEATEINTO ERYTHROCYTEPHOSPHOLIPID Uptake (pmol/109 cells/min)
Normal (n = 8) Duchenne (n = 9) Myotonic (n = 4) a
1 mM CaCI2 + 2 #g A23187.
-Calcium
+Calcium + A23187a
A°70
1.6 _+ 0.1 1.4 _+ 0.1 1.3 _+ 0.2
2.8 +_ 0.2 1.7 _+ 0.1 1.7 _+ 0.2
+75 +21 +30
117
individuals is lower and, in particular, the enhancement due to internalized calcium is greatly diminished. In other experiments, incorporation of the fully-saturated fatty acid, palmitic acid (16:0), occurred at only about one-fifth of the rate with the unsaturated oleate (18:1) in either the presence or absence of calcium plus ionophore. Table I1 presents the distribution of oleate uptake among the several phospholipid classes. It is evident that substantial uptake occurs into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine + phosphatidylinositol and virtually none into sphingomyelin. Interestingly, substantial uptake occurs also into phosphatidate, a relatively minor membrane component (less than 2°7o of total phospholipids as compared to about 3007o for phosphatidylcholine - see ref. 5). In the absence of added calcium + ionophore, distribution of uptake into the several phospholipid fractions is similar in the Duchenne and control cases. However, internalized calcium induces a substantial increase in acyl labeling of phosphatidylcholine, phosphatidylethanolamine and phosphatidate in control cells; the increase is much diminished in cells from Duchenne-dystrophic subjects. This loss of calcium-induced stimulation is reflected in the more modest increase in uptake into total phospholipids of Duchenne cells indicated in Table I. A calcium-stimulated uptake of unsaturated fatty acids in normal (but not dystrophic) cells could account for the differential enrichment of palmitoleate (16: 1) noted earlier [4]. That this is indeed the case is suggested by recent unpublished experiments in which we failed to observe differences in palmitoleate content between control and Duchenne erythrocytes when blood was drawn into citrated, instead of heparin-coated, tubes. Thus, sequestering plasma calcium eliminated the difference, presumably by interfering with uptake of unsaturated fatty acids. Because palmitoleate is a relatively minor fatty acid in erythrocyte membranes, the presence (or absence) of calcium-induced uptake would be expected to be particularly apparent. T A B L E 1I INCORPORATION
OF [~4C]OLEATE INTO PHOSPHOLIPID
CLASSES
Uptake (pmol/109 cells/min)
P h o s p h o l i p i d class Normal
Duchenne dystrophy
- Calcium
+ Calcium a
- Calcium
+ Calcium
Sphingomyelin
0.04
0.04
0.05
0.05
Phosphatidylcholine
0.68 + 0.02
1.89 +_ 0 . 2
0.63 _+ 0 . 0 4
0.97 + 0.2
Phosphatidylserine (+ phosphatidylinositol) Phosphatidylethanolamine Phosphatidate
0.65 _+ 0 . 0 4 0 . 2 4 _+ 0 . 0 2 0 . 2 4 _+ 0.05
0.48 _+ 0 . 0 6 0 . 9 0 _+ 0.1 0.53 _+ 0 . 0 4
0.61 +_+_0 . 0 2 0 . 3 6 +_ 0 . 0 4 0.41 _+ 0.1
0 . 3 6 +_ 0.07 0 . 5 2 _+ 0 . 0 7 0 . 4 4 _+ 0 . 0 6
a 1.0 m M CaC12 + 2 ~zg A 2 3 1 8 7 .
118
It is likely that the uptake of acyl residues into erythrocytes occurs through acylation of lysophospholipids. Such a process has been extensively studied in the case of lysophosphatidylcholine, which is able to catalyze an acyl exchange in either direction [9]. Presumably a similar process can lead to incorporation into phosphatidate and there is evidence, at least in nucleated cells, that lysophosphatidate serves as an acceptor for unsaturated fatty acids in the subsequent synthesis of inositol-containing phospholipids [15]. Because lysophospholipids are known to be disruptive to membrane integrity, their production through the action of phospholipase A requires a capacity for reacylation as a repair function. If dystrophic cells are deficient in this function, then widespread membrane abnormalities should not be inexplicable. This investigation was Dystrophy Association of Maine Affiliate. We thank samples from patients with 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15
supported by research grants from The Muscular America, Inc. and The American Heart Association, Dr. Cairbre McCann for assistance in obtaining blood muscular dystrophies.
Barakat, H.A., Johnson, D.R. and Kerr, D.S., Changes in the phospholipid composition of microsomal membranes of dystrophic hamsters, Proc. Soc. exp. Biol. (N.Y.), 163 (1980) 167-170. Beyer, P., Bieth, R., Robert, J., Freysz, H. and Uhl, T., Phospholipides erythrocytaires darts la myopathie de Duchenne de Boulogne, Nouv. presse Med., 8 (1979) 4043-4044. Howland, J.L., Abnormal potassium conductance associated with genetic muscular dystrophy, Nature (Lond.), 251 (1974) 724-725. Howland, J.L. and lyer, S.L., Erythrocyte lipids in heterozygous carriers of Duchenne muscular dystrophy, Science, 198 (1977) 309 310. Kalofoutis, A., Jullien, G. and Spanos, V., Erythrocyte phospholipids in Duchenne muscular dystrophy, Clin. chim. Acta, 74 (1977) 85-87. Mawatari, S., Schonberg, M. and Olarte, M., AYPase and adeny[ cyclase in erythrocytes of patients with Duchenne muscular dystrophy, Arch. Neurol. (Chic.), 33 (1976) 489 493. McLaughlin, J. and Engel, W.K., Lipid composition of erythrocytes: findings in Duchenne's muscular dystrophy and myotonic atrophy, Arch. Neurol. (Chic.), 36 (1979) 351-354. Mollman, J., Cardenas, J. and Pleasure, D., Alteration in calcium transport by Duchenne erythrocytes, Neurology (Minneap.), 29 (1979) 557. Mulder, E. and van Deenen, L.L.M., Metabolism of red-celt lipids. Ill. Pathways for phospholipid renewal, Biochim. biophys. Acta (Amst.), 106 (1965) 348 356. Plishker, G.A. and Appel, S.H., Red blood cell alteration in muscular dystrophy: the role of lipids, Muscle and Nerve, 3 (1980) 70 81. Roses, A.D. and Appel, S.H., Erythrocyte spectrin peak II phosphorylation in Duchenne muscular dystrophy, J. neurol. Sci., 29 (1976) 185-193. Rowland, L.P., Pathogenesis of muscular dystrophies, Arch. Neurol. (Chic.), 33 (1976) 315-321. Ruitenbeek, W., The fatty acid composition of various lipid fraction isolated from erythrocytes and blood plasma of patients with Duchenne and congenital myotonic dystrophy, Clin. chim. Acta, 89 (1978) 99-110. Ruitenbeek, W., Membrane-bound enzymes of erythrocytes in human muscular dystrophy, J. neurol. Sci., 41 (1979) 71 80. Thompson, W., Cytidine diphosphate diglyceride of bovine brain and liver: isolation and characterization. In G. Porcellati, L. Amaducci and C. Galli (Eds.), Function and Metabolism of Phospholipids in the Central and Peripheral Nervous Systems, Plenum, New York, 1976, pp. 27-36.