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The relationship between age, blood serum cholesterol level and aortic calcification in the turtle
Comp. Biochem. Physiol., 1970, Vol. 35, pp. 491 to 493. Pergamon Press. Printed in Great Britain
T H E R E L A T I O N S H I P BETWEEN AGE, BLOOD SERUM CHOLESTEROL LEVEL AND AORTIC C A L C I F I C A T I O N IN THE T U R T L E C R A W F O R D G. J A C K S O N , JR., C H A R L I E M. H O L C O M B and M A R G U E R I T E M. J A C K S O N Department of Biological Sciences; Department of Physical Sciences; and Department of Biological Sciences, Mississippi State College for Women, Columbus, Mississippi 39701
(Received 14 January 1970) A b s t r a c t - - 1 . Determination by spectrophotometric methods of blood serum
cholesterol level and aortic calcium in an age series of the Suwarmee terrapin,
Pseudemys concinna suwanniensis, yielded total cholesterol values ranging from 210 to 300 mg/100 ml serum, and calcium values ranging from 27 to 62 mg/ 100 g dry wt. aorta. 2. There appears to be a definite increase in blood serum cholesterol level with increase in age (size), whereas there is no apparent correlation between the relative amount of aortic calcium and the size of the animal INTRODUCTION ADVANTAGEwas taken of an opportunity to determine the blood serum cholesterol level and the relative amount of aortic calcium in a size (age) series of thirteen individuals of the Suwanneeterrapin, Pseudemysconcinnasuwanniemis. Theanimals ranging in carapace length from 97 to 315 mm, represented very immature, juvenile-subadult and large mature individuals. All turtles were collected from the same river on 30 November 1969 and were utilized within the 11 days following; therefore, the data are assumed to reflect reasonably accurately the blood picture and aortic calcification condition existing in nature. None of the animals had been fed prior to bleeding. Although a rich literature exists with respect to the distribution of various sterols in the tissues of different animals (Cook, 1958), relatively little information is available regarding ectothermal vertebrates other than fishes. In the case of blood serum cholesterol levels in turtles, Jackson & LeGendre (1967) determined values for mature adults of a few species, all of which were freshly captured. Vastesaeger & Delcourt (1962) measured cholesterol level in three captive specimens. Stenroos & Bowman (1968) determined this parameter, along with several others, using small samples of four species of turtles which were obtained commercially and whose previous history (i.e. length of time in captivity, nutritional condition etc.) was presumably unknown. To the best of our knowledge, no previous investigations have dealt with either the association of aortic calcium to age (size) in the turtle or the relationship between blood serum cholesterol level and age in this animal. 491
492 CRAWFORD G. JACKSON, JR., CHARLIE M . HOLCOMB AND MARGUERITE ~'I. JACKSON
MATERIALS AND METHODS The animals were anesthetized by intraperitoneal injection of Nembutal (sodium pentobarbital) shortly before bleeding. Blood was obtained by cardiac puncture and needle aspiration. The sample consisted of eight males and five females. The smallest seven specimens were juveniles, six of which could only be sexed after dissection. Quantitative cholesterol determinations were made spectrophotometrically using a Beckman DB Spectrophotometer. The procedure followed that of Zlatkis et al. (1953) and consisted in the photometric measurement of the color developed in a diluted serum by a ferric chloride-sulfuric acid reagent. To determine aortic calcium, the proximal portion of the left aorta was removed, washed in distilled water, oven-dried to constant weight, then digested in a mixture of perchloric and nitric acids. Calcium concentration was then measured spectrophotometrically using the Beckman DB with an atomic absorption attachment. Due to the minute size of the aorta in the six smallest turtles, it was necessary to divide them into two size groups of three turtles each and pool the aortas. Specimen A of Table l consists of the pooled aortas of three turtles having carapace lengths of 97, 101 and 118 mm, while specimen B consists of the pooled aortas of turtles having carapace lengths of 140, 152 and 153 mm. T h e remaining seven individuals were large enough to be analyzed individually. RESULTS A s c a t t e r p l o t r e v e a l e d no a p p a r e n t c o r r e l a t i o n b e t w e e n size of t h e a n i m a l a n d r e l a t i v e a m o u n t of aortic c a l c i u m ; hence, t h e s e d a t a are s i m p l y l i s t e d in T a b l e 1. TABLE l--AoRTIC CALCIUM AS A FUNCTION OF TURTLE SIZE
Size class (ram)
Specimen
Carapace length (mm)
Calcium (mg]lO0 g dry wt. tissue)
100-179
A B C
X = 105 X = 148 165
62 40 27
180-249
D E F
200 210 225
28 60 37
250 and over
G H
257 280 315
28 61 49
I
A k h o u g h p e r h a p s f o r t u i t o u s c o n s i d e r i n g t h e s a m p l e size, it is of i n t e r e s t t h a t t h e e x t r e m e u p p e r a n d l o w e r v a l u e s w e r e a l m o s t i d e n t i c a l in t h e t h r e e size g r o u p s . W i t h r e s p e c t to b l o o d s e r u m c h o l e s t e r o l level, t h e s i t u a t i o n was q u i t e different, w i t h l a r g e r size b e i n g a s s o c i a t e d w i t h h i g h e r levels. A s i n d i c a t e d b y Fig. 1, t h e c o r r e l a t i o n is m o d e r a t e l y h i g h a n d c o n s i d e r e d ( F i s h e r & Yates, 1948) h i g h l y significant ( P = 0.001 w h e n d.f. = 9). T h e r e a s o n for this o n t o g e n e t i c c h a n g e in c h o l e s t e r o l p h y s i o l o g y is o b s c u r e . S i n c e C a r r (1952) has f o u n d this f o r m to b e essentially h e r b i v o r o u s , n o d i e t a r y
AGE, BLOOD SERUM CHOLESTEROLAND AORTICCALCIFICATIONIN TURTLE
493
shift in the life cycle is available as a possible explanation for the progressive rise in s e r u m cholesterol level. 3O0
J
28C
>-
0 0 ~ E
260
o
Y= 197 + 0"334 X ..~
r=+0-85
240
L)
/
220
200 50
I I00
r 150
I 200
Carapace length,
t 250
t 300
I 350
mm (X)
FIG. 1. Relationship between blood serum cholesterol level and size of the Suwannee terrapin (Pseudemys condnna suwanniensis). Regression line fitted by method of least squares. REFERENCES CARRA. F. (1962) Handbook of Turtles. Cornell University Press, Ithaca. Coo[( R. P. (1958) Cholesterol, Chemistry, Biochemistry and Pathology. Academic Press, New York. FISHER R. A. & YATES F. (1948) Statistical Tables for Biological and Medical Research, 3rd edn. Oliver & Boyd, Edinburgh. JACKSONC. G., JR. & LEG~'~DRS R. C. (1967) Blood serum cholesterol levels in turtles. Comp. Biochem. Physiol. 20, 311-312. STENROOS O. O. & BOWMAN W. M. (1968) Turtle b l o o d - - I . Concentrations of various constituents. Comp. Biochem. Physiol. 25, 219-222. VASTESAEGERM. M. & DELCOURT R. (1962) T h e natural history of atherosclerosis. Circulation 26, 841-855. ZLATKIS A., ZAK B. & BOYLE A. J. (1953) A new method for the direct determination of serum cholesterol.ft. Lab. clin. Med. 41,486-492.
Key Word Index--Calcium content in turtles' aorta; cholesterol in turtle serum; cholesterol level in turtles' blood; calcium in aortic tissue of turtle; ageing, effects on turtle serum cholesterol; ageing, effects on turtle aortic calcium; aortic calcium and blood cholesterol in reptiles.
T H E R E L A T I O N S H I P BETWEEN AGE, BLOOD SERUM CHOLESTEROL LEVEL AND AORTIC C A L C I F I C A T I O N IN THE T U R T L E C R A W F O R D G. J A C K S O N , JR., C H A R L I E M. H O L C O M B and M A R G U E R I T E M. J A C K S O N Department of Biological Sciences; Department of Physical Sciences; and Department of Biological Sciences, Mississippi State College for Women, Columbus, Mississippi 39701
(Received 14 January 1970) A b s t r a c t - - 1 . Determination by spectrophotometric methods of blood serum
cholesterol level and aortic calcium in an age series of the Suwarmee terrapin,
Pseudemys concinna suwanniensis, yielded total cholesterol values ranging from 210 to 300 mg/100 ml serum, and calcium values ranging from 27 to 62 mg/ 100 g dry wt. aorta. 2. There appears to be a definite increase in blood serum cholesterol level with increase in age (size), whereas there is no apparent correlation between the relative amount of aortic calcium and the size of the animal INTRODUCTION ADVANTAGEwas taken of an opportunity to determine the blood serum cholesterol level and the relative amount of aortic calcium in a size (age) series of thirteen individuals of the Suwanneeterrapin, Pseudemysconcinnasuwanniemis. Theanimals ranging in carapace length from 97 to 315 mm, represented very immature, juvenile-subadult and large mature individuals. All turtles were collected from the same river on 30 November 1969 and were utilized within the 11 days following; therefore, the data are assumed to reflect reasonably accurately the blood picture and aortic calcification condition existing in nature. None of the animals had been fed prior to bleeding. Although a rich literature exists with respect to the distribution of various sterols in the tissues of different animals (Cook, 1958), relatively little information is available regarding ectothermal vertebrates other than fishes. In the case of blood serum cholesterol levels in turtles, Jackson & LeGendre (1967) determined values for mature adults of a few species, all of which were freshly captured. Vastesaeger & Delcourt (1962) measured cholesterol level in three captive specimens. Stenroos & Bowman (1968) determined this parameter, along with several others, using small samples of four species of turtles which were obtained commercially and whose previous history (i.e. length of time in captivity, nutritional condition etc.) was presumably unknown. To the best of our knowledge, no previous investigations have dealt with either the association of aortic calcium to age (size) in the turtle or the relationship between blood serum cholesterol level and age in this animal. 491
492 CRAWFORD G. JACKSON, JR., CHARLIE M . HOLCOMB AND MARGUERITE ~'I. JACKSON
MATERIALS AND METHODS The animals were anesthetized by intraperitoneal injection of Nembutal (sodium pentobarbital) shortly before bleeding. Blood was obtained by cardiac puncture and needle aspiration. The sample consisted of eight males and five females. The smallest seven specimens were juveniles, six of which could only be sexed after dissection. Quantitative cholesterol determinations were made spectrophotometrically using a Beckman DB Spectrophotometer. The procedure followed that of Zlatkis et al. (1953) and consisted in the photometric measurement of the color developed in a diluted serum by a ferric chloride-sulfuric acid reagent. To determine aortic calcium, the proximal portion of the left aorta was removed, washed in distilled water, oven-dried to constant weight, then digested in a mixture of perchloric and nitric acids. Calcium concentration was then measured spectrophotometrically using the Beckman DB with an atomic absorption attachment. Due to the minute size of the aorta in the six smallest turtles, it was necessary to divide them into two size groups of three turtles each and pool the aortas. Specimen A of Table l consists of the pooled aortas of three turtles having carapace lengths of 97, 101 and 118 mm, while specimen B consists of the pooled aortas of turtles having carapace lengths of 140, 152 and 153 mm. T h e remaining seven individuals were large enough to be analyzed individually. RESULTS A s c a t t e r p l o t r e v e a l e d no a p p a r e n t c o r r e l a t i o n b e t w e e n size of t h e a n i m a l a n d r e l a t i v e a m o u n t of aortic c a l c i u m ; hence, t h e s e d a t a are s i m p l y l i s t e d in T a b l e 1. TABLE l--AoRTIC CALCIUM AS A FUNCTION OF TURTLE SIZE
Size class (ram)
Specimen
Carapace length (mm)
Calcium (mg]lO0 g dry wt. tissue)
100-179
A B C
X = 105 X = 148 165
62 40 27
180-249
D E F
200 210 225
28 60 37
250 and over
G H
257 280 315
28 61 49
I
A k h o u g h p e r h a p s f o r t u i t o u s c o n s i d e r i n g t h e s a m p l e size, it is of i n t e r e s t t h a t t h e e x t r e m e u p p e r a n d l o w e r v a l u e s w e r e a l m o s t i d e n t i c a l in t h e t h r e e size g r o u p s . W i t h r e s p e c t to b l o o d s e r u m c h o l e s t e r o l level, t h e s i t u a t i o n was q u i t e different, w i t h l a r g e r size b e i n g a s s o c i a t e d w i t h h i g h e r levels. A s i n d i c a t e d b y Fig. 1, t h e c o r r e l a t i o n is m o d e r a t e l y h i g h a n d c o n s i d e r e d ( F i s h e r & Yates, 1948) h i g h l y significant ( P = 0.001 w h e n d.f. = 9). T h e r e a s o n for this o n t o g e n e t i c c h a n g e in c h o l e s t e r o l p h y s i o l o g y is o b s c u r e . S i n c e C a r r (1952) has f o u n d this f o r m to b e essentially h e r b i v o r o u s , n o d i e t a r y
AGE, BLOOD SERUM CHOLESTEROLAND AORTICCALCIFICATIONIN TURTLE
493
shift in the life cycle is available as a possible explanation for the progressive rise in s e r u m cholesterol level. 3O0
J
28C
>-
0 0 ~ E
260
o
Y= 197 + 0"334 X ..~
r=+0-85
240
L)
/
220
200 50
I I00
r 150
I 200
Carapace length,
t 250
t 300
I 350
mm (X)
FIG. 1. Relationship between blood serum cholesterol level and size of the Suwannee terrapin (Pseudemys condnna suwanniensis). Regression line fitted by method of least squares. REFERENCES CARRA. F. (1962) Handbook of Turtles. Cornell University Press, Ithaca. Coo[( R. P. (1958) Cholesterol, Chemistry, Biochemistry and Pathology. Academic Press, New York. FISHER R. A. & YATES F. (1948) Statistical Tables for Biological and Medical Research, 3rd edn. Oliver & Boyd, Edinburgh. JACKSONC. G., JR. & LEG~'~DRS R. C. (1967) Blood serum cholesterol levels in turtles. Comp. Biochem. Physiol. 20, 311-312. STENROOS O. O. & BOWMAN W. M. (1968) Turtle b l o o d - - I . Concentrations of various constituents. Comp. Biochem. Physiol. 25, 219-222. VASTESAEGERM. M. & DELCOURT R. (1962) T h e natural history of atherosclerosis. Circulation 26, 841-855. ZLATKIS A., ZAK B. & BOYLE A. J. (1953) A new method for the direct determination of serum cholesterol.ft. Lab. clin. Med. 41,486-492.
Key Word Index--Calcium content in turtles' aorta; cholesterol in turtle serum; cholesterol level in turtles' blood; calcium in aortic tissue of turtle; ageing, effects on turtle serum cholesterol; ageing, effects on turtle aortic calcium; aortic calcium and blood cholesterol in reptiles.