Comp. Biochem. Physiol.,
1977, Vol. 57B,pp. 19 to 22. Pergamon
Press. Printed in Great Britain
CHOLESTERYL ESTERS OF PIGEON (COLUMBA LIVIA) AORTAS AS A FUNCTION OF AGE JEROME L. HOJNACKI, KATHLEEN D. CURWEN AND SAMUEL C. SMITH
Department of Animal Sciences and Department of Zoology, University of New Hampshire, Durham, NH 03824, U.S.A. (Received 3 September 1976) Abstract--1. The amounts of free and esterified cholesterol and the cholesteryl ester fatty acid composition of upper thoracic aortas and muscular foci at the celiac bifurcation in 1-day-old, 6-week-old and 6-month-old pigeons (Columba livia) were determined. 2. There were no significant age differences in the amounts of free and esterified cholesterol or in the cholesteryl ester fatty acid composition of upper thoracic aortas. 3. In celiac foci the amount of cholesteryl esters increased with age. Accumulation of saturated cholesteryl esters accounted for 70~o of the increase. 4. The pattern of cholesteryl ester accumulation in celiac foci of young pigeons resembles that seen in the aortas of other young animals including humans.
INTRODUCTION
MATERIALS A N D M E T H O D S
White Carneau pigeons have been widely used as an experimental animal system to study aortic lipid accumulation in atherosclerosis, and several extensive studies of lesion development between the ages of 1 day and 8 years have been reported (Santerre et al., 1972; Lauper et al., 1975). Paired muscular intimal thickenings occur at the celiac bifurcation of the aorta in embryos and squabs. By 4-6 months of age lipid becomes visible within cells in these muscular foci, and the free and esterified cholesterol content of the foci is much greater than that of the upper thoracic aorta (Santerre et al., 1972; Nicolosi et al., 1972). As lesion development progresses cholesteryl esters become a predominant lipid (Portman, 1970). The fatty acid composition of cholesteryl esters from aortas of 8-month, 1-year, and 5-8 year old White Carneau pigeons has been previously described (Young et al., 1964; Young & Middleton, 1966; St. Clair et al., 1968), and a recent report describes cholesteryl esters in White Carneau and Show Racer pigeons aged 6 months to 3 years (Subbiah et al., 1976). However, since the earliest visible lipid deposits appear in muscular foci in White Carneaux by 4-6 months of age (Santerre et al., 1972; Subbiah et al., 1976) it is important to examine aorta cholesteryl ester fatty acids of younger pigeons. Such data can define the developmental sequence of cholesteryl ester accumulation at the celiac bifurcation and perhaps indicate the origin of aorta cholesteryl esters (Smith & Slater, 1973). This communication describes the free and esterifled cholesterol content and the cholesteryl ester fatty acid composition of upper thoracic aortas and celiac foci from 1-day-old, 6-week-old and 6-month-old White Carneau pigeons. The developmental sequence of aorta cholesteryl ester accumulation is discussed in terms of processes which contribute to the accumulation of specific esters in pigeons, man and other species.
One-day-old (0-24hr posthatch), 6-week-old, and 6-month-old White Carneau pigeons were used in this study. Eggs obtained from our colonies were incubated to provide the source of 1-day-old birds. Six-week-old and 6-month-old pigeons were purchased from Palmetto Pigeon Plant, Sumter, SC. Birds from both sources were derived from the inbred line described by Clarkson et al. (1959). Birds were sacrificed by exsanguination, and samples of the upper thoracic aorta and celiac foci were excised and prepared as described previously (Nicolosi et al., 1972). Four pools of aorta tissue for each age-site combination were analyzed for lipid. Each pool contained pieces of aorta tissue from birds of both sexes to provide at least 9 mg wet weight. Following homogenization of aorta tissue in 1 ml of physiological saline (pH 7.0), aliquots were taken for DNA analysis (Prasad et al., 1972), and lipid was extracted by the method of Folch et al. (1957). The final lipid extract was dissolved in 10ml of methylene chloride:methanol (2:1 v/v). A 2 ml aliquot was separated into lipid classes by thin-layer chromatography (Hojnacki & Smith, 1974), and free and esterified cholesterol contents were determined by in situ fluorometry (Nicolosi et al., 1971). Cholesteryl esters in the remaining 8 ml of hpid extract were separated in a similar manner and eluted from the silica gel (Goldbrick & Hirsch, 1963). Methyl esters were prepared by direct micro-methanolysis using boron trichloride gas (Koes, 1971). Qualitative and quantitative analyses were performed according to established gas-liquid chromatographic procedures (McMullin et al., 1968; Koes, 1971). The column was calibrated with Applied Science (State College, Pennsylvania) fatty acid standard K-102. Standard fatty acid methyl esters showed a linear response over the range of sample sizes analyzed. Only 4 higher fatty acids (patmitate, C16; stearate, C18; oleate, Cts, t; and linoleate, C18,2) were quantitated since minor fatty acids eluted between laurate (C~ 2) and arachidonate (C2o~4) each constituted less than 1~ of the total fatty acids esterified to cholesterol. Arachidonate was occasionally detected in cholesteryl esters from large pools of aorta tissue. Since in these instances this acid also constituted less than 1~o of the total cholesteryl ester fatty acids, it was not included in tabulations of the data. 19
20
J . L . HOJNACKI, K. D. CURWEN AND S. C. SMITH
An analysis of variance was used to determine effects and interactions for age and aorta site. For each aorta site Duncan's multiple-range test was used to detect significantly different age means. For each age, differences between aorta site means were evaluated by t tests.
2.00
RESULTS
1.50
Figures 1 and 2 show the free and esterified cholesterol contents of celiac foci and upper thoracic aortas as a function of age. For each aorta site there are no significant age differences in the amount of free cholesterol (Fig. 1). Mean free cholesterol values for celiac foci at each age are, however, significantly different (P < 0.05) from corresponding values of upper thoracic aortas (2.34 #g/pg D N A vs 1.06, 1-day; 3:14 vs 1.24, 6-weeks; 2.58 vs 1.12, 6 months). While the amount of total cholesteryl ester in the upper thoracic aortas of 1-day-old, (0.36 #g/#g DNA), 6-week-old (0.28) and 6-month-old (0.36) pigeons remains essentially constant, there is an increase in total cholesteryl ester in celiac foci with age (Fig. 2). Mean values for cholesteryl ester in 1-day-old (0.43 #g/pg DNA), 6-week-old (1.03) and 6-month-old (1.88) celiac foci are significantly different (P
1977, Vol. 57B,pp. 19 to 22. Pergamon
Press. Printed in Great Britain
CHOLESTERYL ESTERS OF PIGEON (COLUMBA LIVIA) AORTAS AS A FUNCTION OF AGE JEROME L. HOJNACKI, KATHLEEN D. CURWEN AND SAMUEL C. SMITH
Department of Animal Sciences and Department of Zoology, University of New Hampshire, Durham, NH 03824, U.S.A. (Received 3 September 1976) Abstract--1. The amounts of free and esterified cholesterol and the cholesteryl ester fatty acid composition of upper thoracic aortas and muscular foci at the celiac bifurcation in 1-day-old, 6-week-old and 6-month-old pigeons (Columba livia) were determined. 2. There were no significant age differences in the amounts of free and esterified cholesterol or in the cholesteryl ester fatty acid composition of upper thoracic aortas. 3. In celiac foci the amount of cholesteryl esters increased with age. Accumulation of saturated cholesteryl esters accounted for 70~o of the increase. 4. The pattern of cholesteryl ester accumulation in celiac foci of young pigeons resembles that seen in the aortas of other young animals including humans.
INTRODUCTION
MATERIALS A N D M E T H O D S
White Carneau pigeons have been widely used as an experimental animal system to study aortic lipid accumulation in atherosclerosis, and several extensive studies of lesion development between the ages of 1 day and 8 years have been reported (Santerre et al., 1972; Lauper et al., 1975). Paired muscular intimal thickenings occur at the celiac bifurcation of the aorta in embryos and squabs. By 4-6 months of age lipid becomes visible within cells in these muscular foci, and the free and esterified cholesterol content of the foci is much greater than that of the upper thoracic aorta (Santerre et al., 1972; Nicolosi et al., 1972). As lesion development progresses cholesteryl esters become a predominant lipid (Portman, 1970). The fatty acid composition of cholesteryl esters from aortas of 8-month, 1-year, and 5-8 year old White Carneau pigeons has been previously described (Young et al., 1964; Young & Middleton, 1966; St. Clair et al., 1968), and a recent report describes cholesteryl esters in White Carneau and Show Racer pigeons aged 6 months to 3 years (Subbiah et al., 1976). However, since the earliest visible lipid deposits appear in muscular foci in White Carneaux by 4-6 months of age (Santerre et al., 1972; Subbiah et al., 1976) it is important to examine aorta cholesteryl ester fatty acids of younger pigeons. Such data can define the developmental sequence of cholesteryl ester accumulation at the celiac bifurcation and perhaps indicate the origin of aorta cholesteryl esters (Smith & Slater, 1973). This communication describes the free and esterifled cholesterol content and the cholesteryl ester fatty acid composition of upper thoracic aortas and celiac foci from 1-day-old, 6-week-old and 6-month-old White Carneau pigeons. The developmental sequence of aorta cholesteryl ester accumulation is discussed in terms of processes which contribute to the accumulation of specific esters in pigeons, man and other species.
One-day-old (0-24hr posthatch), 6-week-old, and 6-month-old White Carneau pigeons were used in this study. Eggs obtained from our colonies were incubated to provide the source of 1-day-old birds. Six-week-old and 6-month-old pigeons were purchased from Palmetto Pigeon Plant, Sumter, SC. Birds from both sources were derived from the inbred line described by Clarkson et al. (1959). Birds were sacrificed by exsanguination, and samples of the upper thoracic aorta and celiac foci were excised and prepared as described previously (Nicolosi et al., 1972). Four pools of aorta tissue for each age-site combination were analyzed for lipid. Each pool contained pieces of aorta tissue from birds of both sexes to provide at least 9 mg wet weight. Following homogenization of aorta tissue in 1 ml of physiological saline (pH 7.0), aliquots were taken for DNA analysis (Prasad et al., 1972), and lipid was extracted by the method of Folch et al. (1957). The final lipid extract was dissolved in 10ml of methylene chloride:methanol (2:1 v/v). A 2 ml aliquot was separated into lipid classes by thin-layer chromatography (Hojnacki & Smith, 1974), and free and esterified cholesterol contents were determined by in situ fluorometry (Nicolosi et al., 1971). Cholesteryl esters in the remaining 8 ml of hpid extract were separated in a similar manner and eluted from the silica gel (Goldbrick & Hirsch, 1963). Methyl esters were prepared by direct micro-methanolysis using boron trichloride gas (Koes, 1971). Qualitative and quantitative analyses were performed according to established gas-liquid chromatographic procedures (McMullin et al., 1968; Koes, 1971). The column was calibrated with Applied Science (State College, Pennsylvania) fatty acid standard K-102. Standard fatty acid methyl esters showed a linear response over the range of sample sizes analyzed. Only 4 higher fatty acids (patmitate, C16; stearate, C18; oleate, Cts, t; and linoleate, C18,2) were quantitated since minor fatty acids eluted between laurate (C~ 2) and arachidonate (C2o~4) each constituted less than 1~ of the total fatty acids esterified to cholesterol. Arachidonate was occasionally detected in cholesteryl esters from large pools of aorta tissue. Since in these instances this acid also constituted less than 1~o of the total cholesteryl ester fatty acids, it was not included in tabulations of the data. 19
20
J . L . HOJNACKI, K. D. CURWEN AND S. C. SMITH
An analysis of variance was used to determine effects and interactions for age and aorta site. For each aorta site Duncan's multiple-range test was used to detect significantly different age means. For each age, differences between aorta site means were evaluated by t tests.
2.00
RESULTS
1.50
Figures 1 and 2 show the free and esterified cholesterol contents of celiac foci and upper thoracic aortas as a function of age. For each aorta site there are no significant age differences in the amount of free cholesterol (Fig. 1). Mean free cholesterol values for celiac foci at each age are, however, significantly different (P < 0.05) from corresponding values of upper thoracic aortas (2.34 #g/pg D N A vs 1.06, 1-day; 3:14 vs 1.24, 6-weeks; 2.58 vs 1.12, 6 months). While the amount of total cholesteryl ester in the upper thoracic aortas of 1-day-old, (0.36 #g/#g DNA), 6-week-old (0.28) and 6-month-old (0.36) pigeons remains essentially constant, there is an increase in total cholesteryl ester in celiac foci with age (Fig. 2). Mean values for cholesteryl ester in 1-day-old (0.43 #g/pg DNA), 6-week-old (1.03) and 6-month-old (1.88) celiac foci are significantly different (P
