Clin. Biochem. 12 (3) 98-99 (1979)
A Study of Erythrocyte Fatty Acids, Adenosinetriphosphatase and Acetylcholinesterase in Cystic Fibrosis K.M. KUTTY, A.K. GUHA, R.K. CHANDRA and D. VAZE Departments of Biochemistry and Pediatrics, The Dr. Charles A. Janeway Child Health Centre, St. John's, Newfoundland (Accepted December 18, 1978)
CLBIA, 12 (3) 98-99 (1979)
Clin. Biochem. Kutty, K.M., Guha, A.K., Chandra, R.K., and Vaze, D.
Departments of Biochemistry and Pediatrics, The Dr. Charles A. Janeway Child Health Centre, St. John's Newfoundland.
m e m b r a n e s t r u c t u r e and f u n c t i o n , it is also a n imp o r t a n t p r e c u r s o r of p r o s t a g l a n d i n s '3). T h i s s t u d y was u n d e r t a k e n in order to f i n d out w h e t h e r f a t t y acids and m e m b r a n e b o u n d enzymes like A T P a s e a n d acetylcholinesterase are d i f f e r e n t in the e r y t h r o c y t e s in C F p a t i e n t s a n d controls.
METHODS
A STUDY OF ERYTHROCYTE F A T T Y ACIDS, ADENOS1NETR1PHOSPHATASE AND ACETYLC H O L I N E S T E R A S E IN CYSTIC FIBROSIS 1. The proportions of both saturated and polyunsaturated fatty acids were measured in the erythrocytes in Cystic Fibrosis (CF) patients along with two membrane bound enzymes ATPase and acetylcholinesterase. Linoleic acid was found to be significantly decreased and arachidonic acid increased in CF patients. The proportion of saturated fatty acids were not significantly different from the controls. Only adenosinetriphosphatase activity was found to be reduced and not acetylcholinesterase in - F patients.
CYSTIC FIBROSIS ( C F ) IS A DISEASE t h a t p r i m a r i l y affects exocrine g l a n d u l a r secretions. One of the m a i n s y m p t o m s associated with such an a b n o r m a l secretion is p a n c r e a t i c i n s u f f i c i e n c y . As a result, severe m a l a b s o r p t i o n problems arise, p a r t i c u l a r l y of lipids. P o l y u n s a t u r a t e d f a t t y acids ( P U F A ) are not synthesized in the h u m a n body and hence had to be o b t a i n e d t h r o u g h d i e t a r y sources. Since C F is associated with f a t m a l a b s o r p t i o n , one would expect decreased levels of p o l y u n s a t u r a t e d f a t t y acids ( P U F A ) in the blood a n d tissues of C F p a t i e n t s . P U F A t h r o u g h its association w i t h phospholipids play a m a j o r role in the s t r u c t u r e a n d f u n c t i o n of plasma membranes('.zL Since some of the enzymes like A T P a s e a n d acetylchoIinesterase are embedded in the lipoprotein m a t r i x of the m e m b r a n e , a l t e r a t i o n in the m e m b r a n e s t r u c t u r e due to the lack of P U F A m i g h t also a f f e c t the a c t i v i t y of the above enzymes in CF. I n a d d i t i o n to the role of P U F A in
For the analysis of fatty acids in the erythrocytes of CF patients and controls, blood was collected in "Vacutainer" containing EDTA. The diagnosis of CF was made on the basis of sweat chlorides and well established clinical findings. The red cells were isolated by washing in 0.85% saline three times. The packed red cells were then extracted for lipids with chloroform-methanol(2:l) and fatty acid methyl esters were prepared using boron trifluoride in methanol as described before (4) Gas liquid chromatography of the fatty acid methyl esters was performed on a Pye Unicam Gas Chromatograph using Flame Ionization Detector. The fatty acid peaks were identified using known standards and the areas under the peaks were calculated as percentage proportions from the total peak areas of all the fatty acids in the chromatogram. ATPase in the erythrocytes was measured according to the method of Dunham et al (5) and acetylcholinesterase by Ache - Tel method ~).
d* 40'
20"
400
18" 350
16"
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14' • CONTROL 0 CF " - ~ ME AN 2SEM 20"
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10' ~ ~
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INOLEIC ARAC HIDONIC ATP~SE AC I E) AC ID ACTIVITY
Correspondence: Dr. K.M. Kutty, Director of Biochemistry, The Dr. Charles A. Janeway Child Health Centre, St. John's, Newfoundland. Canada A1A 1R8
~oO
ACE TYLCHOLINESTERASE ACT IVI TY
Fig. 1 - - Cystic Fibrosis patients and controls: Column 1 Percentage proportion of Linoleic and Arachidonic acid: Column 2 ATPase activity (Nanomoles of inorganic phosphate liberated~rain~rag protein) Column 3 Acetylcholinesterase activity (Nanomoles of Thiocholine liberated/ min/mg protein).
ADENOSINETRIPHOSPHATASE AND ACETYLCHOLINESTERASE IN CYSTIC FIBROSIS RESULTS The saturated f a t t y acids like myristate (C,,:0), palmitate (C,6:0) and stearate (C,8:0) in the erythrocytes revealed no differences between CF patients and controls. These results are therefore not included here. The mean of linoleic acid in 16 CF patients was 9.3% and 15 controls 11.9%. Statistical analysis indicated a significant decrease (P < 0.001) in the percentage proportion of linoleic acid in CF patients. On the other hand, arachidonic acid in CF patients showed a mean value of 15.9% and in controls 12.6%. The increase of arachidonic acid in CF patients is also highly significant (P < 0.005). Erythrocyte activity for ATPase in 15 CF patients was 5.6 ~moles and 21 controls 8.5 ~amoles. Statistical analysis (P < 0.05) indicates a significant decrease of ATPase in CF patients. AchE activity showed wide individual variation and the difference between CF patients and controls is not statistically significant. Although the increase in arachidonic acid and decrease in linoleic acid and ATPase is statistically significant, these differences do not exist in all the patients. Therefore the results are presented in Fig. 1 showing individual values. DISCUSSION Decreased levels of linoleic acid in the erythrocytes of CF patients is consistent with pancreatic insufficiency and fat malabsorption. The deficiency of polyunsaturated f a t t y acid in CF patients' erythrocytes has been demonstrated earlier ~7~. However, a significant increase in arachidonic acid has not been clearly shown before in CF patients. The exact mechanism for the increased ratio of arachidonic acid in relation to other f a t t y acids in CF is not yet clear. It could be either due to increased conversion of linoleic acid into arachidonic acid or a decreased conversion of arachidonic acid into prostaglandins. Arachidonic acid is an important precursor for prostaglandin synthesis TM. The various clinical manifestations in CF like increased mucous secretion
99
from exocrine glands and increased sweat chlorides may be due to a defect in the membrane structure or decreased prostaglandin synthesis. The decrease in ATPase in the erythrocytes of CF is an interesting finding because AChE which is another membrane bound enzyme is not any different in CF compared to the controls. Decreased levels of sodium potassium dependent. ATPase have been reported before in CF patients '8~. It may be also interesting to study metabolism of prostaglandins in CF with various degrees of clinical severity to understand whether these compounds have any relationship to the pathophysiology of the disease. ACKNOWLEDGEMENTS
This work was supported by Canadian Cystic Fibrosis Foundation. We thank Mrs. B. English for the secretarial assistance. REFERENCES
1. Lehinger A.L., (1975). The Molecular Basis of Cell Structure and Function. Biochemistry 2nd Ed. Worth Publishers Inc., New York, N.Y. pp. 669-670. 2. Rothfield, Lawrence I., (1971) Structure and Function o/ Biological Membranes. Academic Press, New York: London pp. 196-198. 3. Lee, James B. (1973) Perspectives on the Prostaglan. dins, Medcan Medical Update Series, Medcan Press, New York. pp. 8-17. 4. Bonelli, Ernest J. (1968). Lipid Analysis by Gas Chromatograph. Varian Aerography, Walnut Creek, California. 5. Dunham, E.T. and Glynn, L.M. (1961) Adenosine Triphosphatase Activity and other active movement of Alkali Metal Ions, J. Physiol. 156, pp. 274-293. 6. AchE - ChE tel Kit Pfizer Diagnostics. 7. McEvoy, F.A., Aug. 2 1975 (letter) Essential Fatty Acids rind Cystic Fibrosis, 2, pp. 236. Lancet. 8. Balfe, J.W., Cole, C., Welt, L.G., (1968) Red-Cell Transport Defect in Patients with Cystic Fibrosis and Defect in Patients with Cystic Fibrosis and in their Parents Science 162, pp. 689-690.
A Study of Erythrocyte Fatty Acids, Adenosinetriphosphatase and Acetylcholinesterase in Cystic Fibrosis K.M. KUTTY, A.K. GUHA, R.K. CHANDRA and D. VAZE Departments of Biochemistry and Pediatrics, The Dr. Charles A. Janeway Child Health Centre, St. John's, Newfoundland (Accepted December 18, 1978)
CLBIA, 12 (3) 98-99 (1979)
Clin. Biochem. Kutty, K.M., Guha, A.K., Chandra, R.K., and Vaze, D.
Departments of Biochemistry and Pediatrics, The Dr. Charles A. Janeway Child Health Centre, St. John's Newfoundland.
m e m b r a n e s t r u c t u r e and f u n c t i o n , it is also a n imp o r t a n t p r e c u r s o r of p r o s t a g l a n d i n s '3). T h i s s t u d y was u n d e r t a k e n in order to f i n d out w h e t h e r f a t t y acids and m e m b r a n e b o u n d enzymes like A T P a s e a n d acetylcholinesterase are d i f f e r e n t in the e r y t h r o c y t e s in C F p a t i e n t s a n d controls.
METHODS
A STUDY OF ERYTHROCYTE F A T T Y ACIDS, ADENOS1NETR1PHOSPHATASE AND ACETYLC H O L I N E S T E R A S E IN CYSTIC FIBROSIS 1. The proportions of both saturated and polyunsaturated fatty acids were measured in the erythrocytes in Cystic Fibrosis (CF) patients along with two membrane bound enzymes ATPase and acetylcholinesterase. Linoleic acid was found to be significantly decreased and arachidonic acid increased in CF patients. The proportion of saturated fatty acids were not significantly different from the controls. Only adenosinetriphosphatase activity was found to be reduced and not acetylcholinesterase in - F patients.
CYSTIC FIBROSIS ( C F ) IS A DISEASE t h a t p r i m a r i l y affects exocrine g l a n d u l a r secretions. One of the m a i n s y m p t o m s associated with such an a b n o r m a l secretion is p a n c r e a t i c i n s u f f i c i e n c y . As a result, severe m a l a b s o r p t i o n problems arise, p a r t i c u l a r l y of lipids. P o l y u n s a t u r a t e d f a t t y acids ( P U F A ) are not synthesized in the h u m a n body and hence had to be o b t a i n e d t h r o u g h d i e t a r y sources. Since C F is associated with f a t m a l a b s o r p t i o n , one would expect decreased levels of p o l y u n s a t u r a t e d f a t t y acids ( P U F A ) in the blood a n d tissues of C F p a t i e n t s . P U F A t h r o u g h its association w i t h phospholipids play a m a j o r role in the s t r u c t u r e a n d f u n c t i o n of plasma membranes('.zL Since some of the enzymes like A T P a s e a n d acetylchoIinesterase are embedded in the lipoprotein m a t r i x of the m e m b r a n e , a l t e r a t i o n in the m e m b r a n e s t r u c t u r e due to the lack of P U F A m i g h t also a f f e c t the a c t i v i t y of the above enzymes in CF. I n a d d i t i o n to the role of P U F A in
For the analysis of fatty acids in the erythrocytes of CF patients and controls, blood was collected in "Vacutainer" containing EDTA. The diagnosis of CF was made on the basis of sweat chlorides and well established clinical findings. The red cells were isolated by washing in 0.85% saline three times. The packed red cells were then extracted for lipids with chloroform-methanol(2:l) and fatty acid methyl esters were prepared using boron trifluoride in methanol as described before (4) Gas liquid chromatography of the fatty acid methyl esters was performed on a Pye Unicam Gas Chromatograph using Flame Ionization Detector. The fatty acid peaks were identified using known standards and the areas under the peaks were calculated as percentage proportions from the total peak areas of all the fatty acids in the chromatogram. ATPase in the erythrocytes was measured according to the method of Dunham et al (5) and acetylcholinesterase by Ache - Tel method ~).
d* 40'
20"
400
18" 350
16"
o
30
3OO
14' • CONTROL 0 CF " - ~ ME AN 2SEM 20"
o
12.
250
10' ~ ~
200
o
co
o o
50
10'
~ o Qo • o
loo
,4.
o
2.
~o
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so
INOLEIC ARAC HIDONIC ATP~SE AC I E) AC ID ACTIVITY
Correspondence: Dr. K.M. Kutty, Director of Biochemistry, The Dr. Charles A. Janeway Child Health Centre, St. John's, Newfoundland. Canada A1A 1R8
~oO
ACE TYLCHOLINESTERASE ACT IVI TY
Fig. 1 - - Cystic Fibrosis patients and controls: Column 1 Percentage proportion of Linoleic and Arachidonic acid: Column 2 ATPase activity (Nanomoles of inorganic phosphate liberated~rain~rag protein) Column 3 Acetylcholinesterase activity (Nanomoles of Thiocholine liberated/ min/mg protein).
ADENOSINETRIPHOSPHATASE AND ACETYLCHOLINESTERASE IN CYSTIC FIBROSIS RESULTS The saturated f a t t y acids like myristate (C,,:0), palmitate (C,6:0) and stearate (C,8:0) in the erythrocytes revealed no differences between CF patients and controls. These results are therefore not included here. The mean of linoleic acid in 16 CF patients was 9.3% and 15 controls 11.9%. Statistical analysis indicated a significant decrease (P < 0.001) in the percentage proportion of linoleic acid in CF patients. On the other hand, arachidonic acid in CF patients showed a mean value of 15.9% and in controls 12.6%. The increase of arachidonic acid in CF patients is also highly significant (P < 0.005). Erythrocyte activity for ATPase in 15 CF patients was 5.6 ~moles and 21 controls 8.5 ~amoles. Statistical analysis (P < 0.05) indicates a significant decrease of ATPase in CF patients. AchE activity showed wide individual variation and the difference between CF patients and controls is not statistically significant. Although the increase in arachidonic acid and decrease in linoleic acid and ATPase is statistically significant, these differences do not exist in all the patients. Therefore the results are presented in Fig. 1 showing individual values. DISCUSSION Decreased levels of linoleic acid in the erythrocytes of CF patients is consistent with pancreatic insufficiency and fat malabsorption. The deficiency of polyunsaturated f a t t y acid in CF patients' erythrocytes has been demonstrated earlier ~7~. However, a significant increase in arachidonic acid has not been clearly shown before in CF patients. The exact mechanism for the increased ratio of arachidonic acid in relation to other f a t t y acids in CF is not yet clear. It could be either due to increased conversion of linoleic acid into arachidonic acid or a decreased conversion of arachidonic acid into prostaglandins. Arachidonic acid is an important precursor for prostaglandin synthesis TM. The various clinical manifestations in CF like increased mucous secretion
99
from exocrine glands and increased sweat chlorides may be due to a defect in the membrane structure or decreased prostaglandin synthesis. The decrease in ATPase in the erythrocytes of CF is an interesting finding because AChE which is another membrane bound enzyme is not any different in CF compared to the controls. Decreased levels of sodium potassium dependent. ATPase have been reported before in CF patients '8~. It may be also interesting to study metabolism of prostaglandins in CF with various degrees of clinical severity to understand whether these compounds have any relationship to the pathophysiology of the disease. ACKNOWLEDGEMENTS
This work was supported by Canadian Cystic Fibrosis Foundation. We thank Mrs. B. English for the secretarial assistance. REFERENCES
1. Lehinger A.L., (1975). The Molecular Basis of Cell Structure and Function. Biochemistry 2nd Ed. Worth Publishers Inc., New York, N.Y. pp. 669-670. 2. Rothfield, Lawrence I., (1971) Structure and Function o/ Biological Membranes. Academic Press, New York: London pp. 196-198. 3. Lee, James B. (1973) Perspectives on the Prostaglan. dins, Medcan Medical Update Series, Medcan Press, New York. pp. 8-17. 4. Bonelli, Ernest J. (1968). Lipid Analysis by Gas Chromatograph. Varian Aerography, Walnut Creek, California. 5. Dunham, E.T. and Glynn, L.M. (1961) Adenosine Triphosphatase Activity and other active movement of Alkali Metal Ions, J. Physiol. 156, pp. 274-293. 6. AchE - ChE tel Kit Pfizer Diagnostics. 7. McEvoy, F.A., Aug. 2 1975 (letter) Essential Fatty Acids rind Cystic Fibrosis, 2, pp. 236. Lancet. 8. Balfe, J.W., Cole, C., Welt, L.G., (1968) Red-Cell Transport Defect in Patients with Cystic Fibrosis and Defect in Patients with Cystic Fibrosis and in their Parents Science 162, pp. 689-690.
