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The lipid composition and triglyceride structure of eggs and fat bodies of the lizard Sceloporus jarrovi
Comp. Biochem. Physiol., 1974, Vol. 48B, pp. 275 to 284. PergamonPress. Printed in Great Britain
THE L I P I D C O M P O S I T I O N AND T R I G L Y C E R I D E STRUCTURE OF EGGS AND FAT BODIES OF THE LIZARD SCELOPORUS JARROVI N E I L F. H A D L E Y x and W I L L I A M W. C H R I S T I E 2 1Department of Zoology, Arizona State University, Tempe, Arizona 85281 ; and ZHannah Research Institute, Ayr, Scotland KA6 5HL, U.K. (Received 13 ffuly 1973)
Abstract--1. The lipid composition and triglyceride structure of eggs and fat bodies of the viviparous montane lizard Sceloporus jarrovi were determined by thin-layer and gas-chromatographic procedures. The structures of the triglycerides were obtained by a stereospecific analysis procedure. 2. Lipids comprised approximately 36 per cent of the dry weight of the eggs and 91 per cent of that of the fat bodies. 3. Triglyceride was the major lipid class in the eggs (86.9-90.2 per cent) and in the fat bodies (92"6-94"9 per cent); other lipid classes included phospholipids, cholesteryl esters, free fatty acids, diglycerides and free cholesterol. Alkyldiglycerides were also present in fat bodies from older lizards. Phosphatidyl choline and phosphatidyl ethanolamine were always the major egg phospholipids comprising 76"5-83.1 per cent and 7"7-13"8 per cent, respectively, of this lipid class. 4. Oleic acid (18:1) was the major component of egg and fat body triglycerides followed by palmitic (16:0) and linoleic acid (18:2). 5. Structures of the triglycerides from fat bodies were similar to those in adipose tissue of higher vertebrates; however, egg triglycerides differed considerably from those of avian eggs.
INTRODUCTION VERY little is known of the lipid composition of lizard eggs and adipose tissue, and it is not yet possible to make valid comparisons with similar data for other vertebrate species. Lizard fat bodies, possibly because of their role in the reproductive cycle and as an energy store, have received the most attention (Hahn & Tinkle, 1965). The percentage weight of component fatty acids of depot fat of the lizard Varanus salvator was reported in two separate studies (Klenk et al., 1935 ; Hilditch & Paul, 1937). Zain & Zain-UI-Abedin (1967) chemically analyzed the abdominal fat pads of the lizard Uromastix and determined the proportionate amounts of esterified fatty acids, cholesterol, phospholipids, glycogen and carotenoids. Information on this species was expanded to include the seasonal changes in individual lipid classes in adipose tissue over a period of 11 months as well as the fatty acid composition during hibernation, arousal and activity (Afroz et aL, 1971). 275
276
NEIL F. HADLEY AND WILLIAM W . CHRISTIE
T h e lipid content of lizard eggs apparently has only been investigated m o r p h o logically (Grodzinski, 1949), and the triglyceride structures do not appear to have b e e n d e t e r m i n e d for any reptilian tissue. I n the present study, the a m o u n t s and fatty acid composition of the m a i n lipid classes f r o m the eggs and fat bodies (male and female) of the viviparous m o n t a n e lizard Sceloporus jarrovi were d e t e r m i n e d b y c o m b i n e d m e t h o d s of thin-layer and gas-liquid c h r o m a t o g r a p h y . T h e structures of the triglycerides f r o m these tissues were also d e t e r m i n e d b y a stereospecific analysis procedure. MATERIALS AND METHODS
Samples Egg-bearing female S. jarrovi were collected from Mt. Graham, Safford, Arizona, on 16 December 1972 and 28 January 1973. A small group of adult males was also taken on 28 January. Lizards were weighed and measured (snout-vent) before being sacrificed. Eggs and fat bodies were removed, weighed and then freeze-dried for 72 hr. Samples were maintained deep-frozen under a nitrogen atmosphere in sealed glass vials prior to analysis. Dry weights were recorded for all samples immediately before lipid extraction. Eggs and fat bodies from lizards collected on 16 December were pooled into corresponding groups; samples from 28 January animals were processed and maintained separately.
Lipid extraction and isolation of classes Lipids were extracted with chloroform-methanol (2 : 1, v/v; Ways & Hanahan, 1964). Neutral lipid classes were separated by thin-layer chromatography (TLC) on 10 x 20 cm glass plates coated with layers 0"5 m m thick of Silica Gel G (E. Merck, A.G., Darmstadt); the solvent system was hexane-diethyl ether-formic acid (80 : 20 : 2 by vol.). Phospholipids were separated on Silica Gel H (Camag, Muttenz) coated plates; a developing solvent system consisting of chloroform-methanol-acetic acid-water (25 : 15 : 4 • 2 by vol.) was used. Lipid bands were detected under u.v. light after spraying the plates with 2',7'dichlorofluorescein (0"1%, wt/vol.) and identified against known standards. Bands were either scraped into small chromatographic columns and eluted with chloroform (neutral lipids) or chloroform-methanol-water (5 : 5 : 1 by vol.) (complex lipids), or were scraped directly into 15-ml test-tubes and methylated according to the procedures described by Christie (1972). Methyl pentadecanoate standard solution was added to each band prior to methylation, and the amount of each class was determined by relating the combined areas of the peaks for the components in the gas-liquid chromatography (GLC) trace to those of the internal standard (Christie et al., 1970).
Gas-liquid chromatography Fatty acid analyses were performed on columns (7 ft x 0-25 in.) of 15~o E G S S - X or EGSS-Y on Gas-chrom P (100-120 mesh; Applied Sciences Labs., Inc., State College, Pa.) isothermally at 180°C (EGSS-X) or 194°C (EGSS-Y) in a Pye 104 chromatograph (PyeUnicam Ltd., Cambridge, U.K.). Components were identified by their retention times relative to authentic standards and by their chromatographic behaviour on thin layers of silica gel-G impregnated with 10% (w/w) silver nitrate. The amount of each ester present (wt %) was calculated from the product of the peak height and retention time. Results for phospholipid classes were converted to mole % by multiplying these values by appropriate factors (Christie et al., 1970).
Stereospecific analysis of triglycerides Triglycerides were isolated by preparative T L C as described above. A procedure based on that devised by Broekerhoff (1965) was used for the stereospecifie analysis of triglycerides
LIPIDS AND TRIGLYCERIDES IN LIZARD EGGS
277
(Christie & Moore, 1969). ct,fl-Diglycerideswere prepared by the action of ethyl magnesium bromide on triglycerides and converted synthetically to phospholipids which were hydrolyzed by the stereospecific phospholipase A of snake venom. Results for position 1 were obtained by analysis of the lysophosphatide produced in the final stage of the procedure, those for position 2 were obtained by pancreatic lipase hydrolysis and those for position 3 were calculated by difference from the known triglyceride composition. Checks were made on the results for positions 2 and 3; analyses were accepted only when they conformed to the accuracy standards described by Christie & Moore (1969). RESULTS Data on lizard size and weights of eggs, fat bodies and their corresponding lipids for animals processed and analyzed individually are given in Table 1. The mean water content of fat bodies, based on dry weight-wet weight ratios, was approximately 10 per cent higher than the mean egg water content, although variation was greater in the fat bodies. Lipid content was significantly higher for fat TABLE 1--WEIGHTS (g) AND WEIGHT-RATIOS FOR EGGS, FAT BODIES AND THE CORRESPONDING LIPIDS FROM SIX FEMALE LIZARDS* COLLECTED ON 28 JANUARY 1973 (MEANS AND STANDARD ERRORS)
Wtwt. Dry wt. Dry wt/wet wt. Lipid wt/dry wt.
Eggs
Fat bodies
2.39+0.14 1.43_+0.08 0.60 + 0-01 0"36 -+0.02
0.15_+0-03 0.11 _+0.02 0"70 4-0.03 0'91 + 0.12
* Fresh wt. = 16"13 +0"85 g; snout-vent length = 80-3 + 1.91 mm. bodies (91.1 vs 35.6%, based on dry weights) and less variable in comparison to eggs. Generally, the percentage of egg lipid increased as the egg dry weight increased; however, a similar correlation was not apparent between lipid percentage and dry weight of fat bodies. Lipids were extracted from lizard eggs and fat bodies, and the proportions of the main lipid classes determined by thin-layer and gas-chromatographic techniques. The results are listed in Table 2. In addition to triglycerides and phospholipids, all eggs contained free fatty acids, diglycerides and cholesterol esters. The latter three classes when combined did not exceed 6 per cent of the total lipid content. Free cholesterol was also detected in the egg samples (Maclntyre & Ralston 1954), but was quantified only for the January females which contained 0.8 + 0.1 per cent. Triglyceride was again the major lipid class in lizard fat bodies, comprising an even greater proportion than found for eggs (92.6-94.9 per cent vs 86"9-90.2 per cent). Phospholipid levels were correspondingly less in fat bodies, which also included comparable amounts of diglycerides, free fatty acids and cholesterol esters. Alkyl diglycerides were also present (approx. 2 per cent) in male and female fat bodies from lizards collected in January, but were absent in December females.
Not determined Not determined
1"8
10"1±1'9
1-0 + 0"2
81"1±1"8
10"8
Not determined
5"0±1"0
6"7±0"8
88-2 ± 1-0
76"5
Phosphatidyl ethanolamine
1-3
6"2
Phosphatidyl choline
Principal phospholipids (mole %)
Sphingomyelin
9"0
Phospholipids
86"9
Triglycerides
Fat bodies: December collection (5 females ; 94"0 samples pooled) January collection (3 females ; 94"0 ± 0"7 and S.E.) January collection (6 males ; 94'9 samples pooled)
Eggs: December collection (5 lizards; samples pooled) January collection (3 lizards ; and S.E.)
Sample
Principal lipid classes (wt %)
TABLE 2--LIPID COMPOSITIONOF LIZARDEGGS AND FAT BODIES
2-1±0"8
4"0
Cardiolipin
m
P
>
m >
Z m t~
to Oo
L I P I D S AND TRIGLYCERIDES I N LIZARD EGGS
279
Neutral plasmalogens were not detected. Trace amounts of cholesterol were present in all fat body samples but the amounts did not exceed 0.6 per cent per animal in individuals collected in January. Egg phospholipids were also separated into individual classes by T L C and the results are listed in Table 2. Values for eggs from lizards collected in December and January were quite similar. Phosphatidyl choline was by far the most abundant class comprising 76.5-83.1 per cent of the total phospholipids. Phosphatidyl ethanolamine varied from 7.7 to 13.8 per cent, while sphingomyelin and cardiolipins were present in amounts less than 10 per cent of the total. Trace amounts ( < 2.0 per cent) of lysophosphatidyl choline were also present in all samples. The fatty acid compositions of the triglycerides in eggs and fat bodies (male and female) and phosphatidyl choline in eggs were also determined by gas chromatography. Results are listed in Tables 3 and 4 respectively. Oleic acid (18:1) was the major component of the triglycerides, ranging from 44.8 to 54.6 per cent in egg triglycerides and from 57.3 to 73-5 per cent in fat body triglycerides. Following oleic acid in abundance were palmitic acid (16:0) and the essential fatty acid, linoleic acid (18:2). Linoleic acid amounts varied considerably, ranging from 5.8 to 23.7 per cent in egg triglycerides to between 5.9 and 13.0 per cent in fat body triglycerides. Amounts of 16:0 were more constant with values of 16.9-18.6 per cent in egg triglycerides and 12-3-17-6 per cent in fat body triglycerides. In addition to the remaining fatty acid components listed in Table 3, small amounts of the following were invariably present: myristic acid (14:0), eicosadienoic acid (20:2) and eicosatrienoic acid (20:3). Two observations perhaps warrant mention at this point. First, an unusually high percentage (16.9 per cent) of palmitoleic acid (16:1) was found in the egg triglycerides of one of the lizards from the January collection. This acid, the presence of which was confirmed by silver nitrate chromatography, also comprised an unusually high percentage of the fatty acids in phosphatidyl choline in eggs from the same female (see Table 4), but not in the fat bodies. Secondly, there was a definite increase in oleic acid (18:1) and a concomitant decrease in palmitic acid (16:0) when fat body triglycerides in January-collected lizards were compared with fat body triglycerides in December animals. The fatty acids in phosphatidyl choline isolated from the eggs were very similar to those found in triglycerides, except that in phosphatidyl choline there were greater numbers and/or larger amounts of C20 and C~2 polyunsaturated fatty acids (Table 4). Trace amounts of 20:1, 20:2, 20:3, 20:5 and 22:4 were also present. There were some marked differences in the percentage composition of the major fatty acids in phosphatidyl choline when compared to that of the triglycerides in the eggs. Palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2) were approximately equal in overall abundance and together comprised between 76.0-83.3 per cent of the total fatty acids. Small but relatively consistent amounts of stearic acid (18:0), linolenic acid (18:3) and arachidonic acid (20:4) were always present. Palmitoleic acid (16:1) comprised only a small percentage of the total except for the individual described above.
3--FATTY
28"7 24"9 + 0'4
D e c e m b e r collection* J a n u a r y collection ( m e a n s a n d s t a n d a r d errors)
* Eggs f r o m 5 females pooled.
16:0
Sample
2"4 + 0'3 3"4
12"5 + 0"2 12"5
-3"1 + 1 '9
16:1 3"6 4-6 _+0"4
18:0
ACID COMPOSITION
4-7
2'8 8'5+_4"1
16:1
73-5
5"9
9'6 _+ 1 "8
10"7
15-3 13'8+_5.2
18:2
22'2 23"8 + 3'1
32"4 28-9 + 3-9
18:2
2"5 1 "4 + 0'8
18:3
F a t t y acid c o m p o s i t i o n (mole ~o) 18:1
1 "3
0'7 _+0'4
2"7
1"2 2'9+2.0
18:3
4-7 3"1 + 0"3
20:4
C H O L I N E O F L I Z A R D EGGS
67'9 + 2-7
57' 3
54"6 47'5+_1'6
18:1
OF THE PHOSPHATIDYL
3"5
4"6 + 0 ' 4
6"0
2"8 2-8+0"4
18:0
F a t t y acid c o m p o s i t i o n (wt % )
O F T H E T R I G L Y C E R I D E S O F L I Z A R D EGGS A N D F A T B O D I E S
17"6
18"6 17"6+0'5
16:0
ACID COMPOSITION
TABLE 4--FATTY
* Eggs from 5 females pooled.
F a t bodies : D e c e m b e r collection (female; n = 5) J a n u a r y collection (female) (means a n d s t a n d a r d errors) J a n u a r y collection (male ; n = 6)
Eggs : D e c e m b e r collection (n = 5)* J a n u a r y collection ( m e a n s a n d s t a n d a r d errors)
Sample
TABLE
2"0 1 "5 + 0"2
22:5/6
0" 1
1-1 + 0'3
0'6
2"6 3'8+_0"2
20:1
3-9 8"6 + 0"6
Remainder
--
--
--
1"6 1"1+_0-6
20:4
(3
t~
00
Fat bodies (male) : • January collection
Fat bodies (female) : December collection
Eggs: December collection
Fat bodies (male) : January collection
Fat bodies (female) : December collection
Eggs: December collection
Sample
TABLE
1 2 3
1 2 3
1 2 3
TG 1 2 3
TG 1 2 3
TG 1 2 3
70 7 23
72 11 17
66 6 28
13.3 28-1 2-6 9.2
18.0 38"8 5-8 9.4
19.4 38.3 3-5 16.3
LIZARD
EGGS AND FAT BODIES
4.2 10"0 1.6 1.0
5"9 11.0 2.8 3"9
3.6 8-4 1.4 0"9
18:0
68-9 49.3 82.2 75"2
55.8 33.6 68-2 65"6
50-7 35.8 61-8 54"8
18:1
7"3 4.5 8-6 8"8
10.6 5.7 13.1 13"0
17.0 7.7 27-2 16"0
18:2
Fatty acid composition (mole %)
FROM
1.1 0.8 1.2 1.3
2.8 1-8 3.7 2-9
2"2 1.2 2"4 3.1
18:3
44 24 32
46 27 27
32 15 53
79 13 8
62 16 22
78 13 9
24 40 36
20 41 39
24 41 35
21 39 40
18 41 41
15 53 32
24 36 40
21 44 35
18 36 46
Proportion (%) of the available fatty acids in each position
4.4 5.8 3-2 4.2
5.1 7-0 4.2 4"1
4.3 4-1 1.9 7"0
16:1
OF TRIGLYCERIDES
16:0
ANALYSIS
Position
5--STEREOSPECIFIC
--
m
m
_
0-9 1"5 0"6 0.6
20:4
t~ oo
~
~rj
282
NEIL F. HADLEY AND WILLIAM W. CHRISTIE
Preparative TLC was used to isolate triglycerides from the lipids of the eggs and fat bodies of females (December collection) and fat bodies of the males (January collection). The distribution of fatty acids in positions 1, 2 and 3 of the L-glycerol moiety was then determined by a stereospecific analysis procedure. The results are listed in Table 5. Saturated fatty acids were concentrated in position 1, although there were also appreciable amounts of 16:0 in position 3, while the C18 unsaturated fatty acids were in greatest concentration in positions 2 and 3. DISCUSSION The lipid composition of the fat bodies of S.jarrovi was similar to that reported for Varanus salvator depot fat (Hilditch & Williams, 1964) and the adipose tissue of Uromastix hardwickii (Zain & Zain-U1-Abedin, 1967; Afroz et al., 1971); however, the different analytical procedures and methods of data presentation made accurate comparisons difficult. The study by Afroz et al., (1971) provided the best opportunity for meaningful comparison. They found that triglycerides comprised approximately 90 per cent of the total lipid content of the adipose tissues of U. hardwickii, with the remainder consisting of diglycerides, monoglycerides, free fatty acids and cholesterol. The fatty acid composition of the triglycerides was also similar to results obtained on S. jarrovi fat bodies, although the proportionate amounts of the major constituents varied slightly. In U. hardwickii adipose tissue, the major fatty acids were oleic and palmitic, comprising 50-53 per cent and 20-24 per cent respectively in comparison to 57-74 per cent oleic acid and 12-18 per cent palmitic acid in S. jarrovi fat bodies. In both species the proportion of unsaturated fatty acids (65 per cent in U. hardwickii, 76-84 per cent in S. jarrovi) was greater than saturated ones. The fact that alkyl diglycerides were found in small amounts in the fat bodies of S. jarrovi in the January collection but not in those of the December collection may possibly be associated with the difference in maturity of the animals. Using snout-vent length as an age indicator, December lizards (snout-vent -- 51-61 ram) were approximately 1 year old, while lizards collected in January (snout-vent = 76-85 ram) were approximately 2 years or older (Tinkle & Hadley, 1973). Ether bonds are known to be formed and metabolized much more slowly than ester bonds and compounds containing these can accumulate with age (Snyder, 1970). The extremely high concentration of 16:1 at the expense of 18:2 in the eggs of the individual from the January collection may reflect variation in the diet of this animal or it may be that it has a highly active fatty acid desaturation system. Similarly, we can only speculate as to the cause of the apparent increased unsaturation of fat bodies in January-collected lizards (83 per cent) vs December-collected animals (76 per cent). Controlled experiments over a several-month period would be necessary to determine if there is a selective utilization of saturated over unsaturated fatty acids or if there is continuous desaturation of fatty acids already present with no fat deposition. In U. hardwickii there was no change in the total unsaturation of adipose tissue during hibernation, arousal and activity, in spite of alteration in the fatty acid pattern of triglycerides (Afroz et al., 1971).
L I P I D S AND TRIGLYCERIDES I N LIZARD EGGS
283
The influence of environmental temperature on the lipid composition of eggs and fat bodies of S. jarrovi warrants further study. Ovulation takes place in late autumn and eggs are carried over the winter (Tinkle & Hadley, 1973). Many females and males are active above ground during this time even though subfreezing temperatures are common during the night. Lizards seek shelter in shallow rock crevices and between boulders; however, this exposed winter microhabitat affords a maximum thermal buffering of only 5-6°C (Lowe et al., 1971). To meet these thermal demands, S. jarrovi are capable of internal supercooling to - 5.5°C (Lowe et al., 1971). It is likely that the relatively high degree of unsaturation of the total lipids in eggs and fat bodies reflected these low temperatures, but confirmation awaits comparative studies on lizards with different modes of reproduction and temperature r6gimes. Additional experimentation is also necessary to establish if the unsaturation is a result of increased deposition of dietary polyunsaturated fatty acids or if an increased de novo synthesis of unsaturated fatty acids takes place in response to lower temperatures. In fish (Malins & Wekell, 1970) and birds (Fisher et al., 1962) there was an increase in the concentration of unsaturated fatty acids in the depot fat with decrease in temperature, but this was the result of increased deposition of dietary unsaturated fatty acids rather than increased desaturation of endogenous saturated fatty acids. Although the triglycerides from fat bodies from males and females and from eggs of S. jarrovi differed considerably in fatty acid composition, the available fatty acids were distributed among the three positions of the glycerol moiety in a similar manner. This can best be seen if the proportions rather than the absolute amounts of each fatty acid in each position are calculated (Table 5). As an example, although the absolute amount of 18:1 in the triglycerides varied between 51 and 69 per cent, the proportion of this in position 1 only varied between 20 and 24 per cent, in position 2 between 40 and 41 per cent and in position 3 between 35 and 39 per cent. Of the major components, only 18:2 differed in the way it was distributed in the triglycerides of the eggs and fat bodies in that in the former there was somewhat more in position 2 and correspondingly less in position 3 than in the latter. In addition, the structures of the triglycerides from the fat bodies were similar to those in adipose tissue from most mammalian species (Brockerhoff, 1966) and also to that of adipose tissue in the domestic chicken (Christie & Moore, 1972a). The structures of the egg triglycerides differed considerably from those of avian eggs (Christie & Moore, 1972b), which contain the most asymmetric triglycerides analyzed to date, but no other analyses of reptilian eggs are available for comparison. Acknowledgement--The authors acknowledge the skilled technical assistance of Miss M. Hunter and Mrs. P. Paterson. REFERENCES
AFROZH., ISHAQM. & ALI S. S. (1971) Seasonal changes in the lipids of adipose tissue in a hibernating lizard (Uroraastix hardwickii). Proc. Soc. exp. Biol. Med. 136, 894-898. BROCKERHOFFH. (1965) A stereospecific analysis of triglycerides. 07. Lipid Res. 6, 10-15. BROCKEm-IOFFH. (1966) Fatty acid distribution patterns of animal depot fats. Comp. Biochem. Physiol. 19, 1-12.
284
NEIL F. HADLEY AND WILLIAM W. CHRISTIE
CHRISTIE W. W. (1972) Quantitative esterification of lipids on thin-layer adsorbents. Analyst 97, 221-223. CHRISTIE W. W. & MOORE J. H. (1969) A semimicro method for the stereospecific analysis of triglycerides. Biochim. biophys. Acta 210, 46-56. CHRISTIE W. W. & MOORE J. H. (1972a) T h e lipid components of the plasma, liver and ovarian follicles in the domestic chicken (Gallus gallus). Comp. Biochem. Physiol. 41B, 287-295. CHRISTIE W. W. & MOORE J. H. (1972b) The lipid composition and triglyceride structure of eggs from several avian species. Comp. Biochem. Physiol. 41B, 297-306. CHRISTIE W. W., NOBLE R. C. & MOORE J. H. (1970) Determination of lipid classes by a gas chromatographic procedure. Analyst 95, 940-944. FISHER H., HOLLANDSK. G. & WEISS H. S. (1962) Environmental temperature and composition of body fat. Proc. Soc. exp. Biol. Med. 110, 832-833. GRODZINSKI Z. (1949) T h e fat in the yolk of the sand-lizard Lacerta agilis L. Bull. Acad. pol. sci. Ser. B, II, 368-381. HAHN W. E. & TINKLE D. W. (1965) Fat body cycling and experimental evidence for its adaptive significance to ovarian follicle development in the lizard Uta stansburiana. J. exp. Zool. 158, 79-86. HILDITCH T. P. & PAVL H. (1937) The depot fat of Varanus salvator (Laur.). Biochem. J. 31, 227-228. HILDITCH T. P. & WILLIAMS P. N. (1964) The Chemical Constitution of Natural Fats, 4th Edn. J. Wiley & Sons, New York. KLENK E., DITT F. & DIEBOLD W. (1935) Uber das Depotfett der Wirbeltiere. HoppeSeyler's Z. physiol. Chem. 232, 54-63. LowE C. H., LARDNER P. J. & HALPERN E. A. (1971) Supercooling in reptiles and other vertebrates. Comp. B iochem. Physiol., 39A, 125-135. MACINTYRE I. & RALSTON M. (1954) Direct determination of serum cholesterol. Biochem.ff., 56, xiii. MALINS D. C. & WEKELL J. C. (1970) The lipid biochemistry of marine organisms. In Progress in the Chemistry of Fats and Other Lipids (Edited by HOLMAN R. T.), Vol. 10, pp. 337-363. Pergamon Press, Oxford. SNYDER F. (1970) T h e biochemistry of lipids containing ether bonds. In Progress in the Chemistry of Fats and Other Lipids (Edited by HOLMAN R. T.), Vol. 10, pp. 287-336. Pergamon Press, Oxford. TINKLE D. W. & HADLEY N. F. (1973) Reproductive effort and winter activity in the viviparous montane lizard Sceloporus jarrovi. Copeia (1973), 272-277. WAYS P. & HANAHAN D. J. (1964) Characterization and quantification of red cell lipids in normal man. ft. Lipid Res. 5, 318-328. ZAIN B. K. & ZAIN-uL-ABEDIN M. (1967) Characterization of the abdominal fat pads of a lizard. Comp. Bioehem. Physiol. 23, 173-177.
Key Word Index--Eggs ; fat bodies; adipose tissue ; lizard ; reptile ; Sceloporus jarrovi; lipid composition; triglyceride structure; triglycerides; stereospecific analysis from lizard eggs; egg lipids; fat body lipids.
THE L I P I D C O M P O S I T I O N AND T R I G L Y C E R I D E STRUCTURE OF EGGS AND FAT BODIES OF THE LIZARD SCELOPORUS JARROVI N E I L F. H A D L E Y x and W I L L I A M W. C H R I S T I E 2 1Department of Zoology, Arizona State University, Tempe, Arizona 85281 ; and ZHannah Research Institute, Ayr, Scotland KA6 5HL, U.K. (Received 13 ffuly 1973)
Abstract--1. The lipid composition and triglyceride structure of eggs and fat bodies of the viviparous montane lizard Sceloporus jarrovi were determined by thin-layer and gas-chromatographic procedures. The structures of the triglycerides were obtained by a stereospecific analysis procedure. 2. Lipids comprised approximately 36 per cent of the dry weight of the eggs and 91 per cent of that of the fat bodies. 3. Triglyceride was the major lipid class in the eggs (86.9-90.2 per cent) and in the fat bodies (92"6-94"9 per cent); other lipid classes included phospholipids, cholesteryl esters, free fatty acids, diglycerides and free cholesterol. Alkyldiglycerides were also present in fat bodies from older lizards. Phosphatidyl choline and phosphatidyl ethanolamine were always the major egg phospholipids comprising 76"5-83.1 per cent and 7"7-13"8 per cent, respectively, of this lipid class. 4. Oleic acid (18:1) was the major component of egg and fat body triglycerides followed by palmitic (16:0) and linoleic acid (18:2). 5. Structures of the triglycerides from fat bodies were similar to those in adipose tissue of higher vertebrates; however, egg triglycerides differed considerably from those of avian eggs.
INTRODUCTION VERY little is known of the lipid composition of lizard eggs and adipose tissue, and it is not yet possible to make valid comparisons with similar data for other vertebrate species. Lizard fat bodies, possibly because of their role in the reproductive cycle and as an energy store, have received the most attention (Hahn & Tinkle, 1965). The percentage weight of component fatty acids of depot fat of the lizard Varanus salvator was reported in two separate studies (Klenk et al., 1935 ; Hilditch & Paul, 1937). Zain & Zain-UI-Abedin (1967) chemically analyzed the abdominal fat pads of the lizard Uromastix and determined the proportionate amounts of esterified fatty acids, cholesterol, phospholipids, glycogen and carotenoids. Information on this species was expanded to include the seasonal changes in individual lipid classes in adipose tissue over a period of 11 months as well as the fatty acid composition during hibernation, arousal and activity (Afroz et aL, 1971). 275
276
NEIL F. HADLEY AND WILLIAM W . CHRISTIE
T h e lipid content of lizard eggs apparently has only been investigated m o r p h o logically (Grodzinski, 1949), and the triglyceride structures do not appear to have b e e n d e t e r m i n e d for any reptilian tissue. I n the present study, the a m o u n t s and fatty acid composition of the m a i n lipid classes f r o m the eggs and fat bodies (male and female) of the viviparous m o n t a n e lizard Sceloporus jarrovi were d e t e r m i n e d b y c o m b i n e d m e t h o d s of thin-layer and gas-liquid c h r o m a t o g r a p h y . T h e structures of the triglycerides f r o m these tissues were also d e t e r m i n e d b y a stereospecific analysis procedure. MATERIALS AND METHODS
Samples Egg-bearing female S. jarrovi were collected from Mt. Graham, Safford, Arizona, on 16 December 1972 and 28 January 1973. A small group of adult males was also taken on 28 January. Lizards were weighed and measured (snout-vent) before being sacrificed. Eggs and fat bodies were removed, weighed and then freeze-dried for 72 hr. Samples were maintained deep-frozen under a nitrogen atmosphere in sealed glass vials prior to analysis. Dry weights were recorded for all samples immediately before lipid extraction. Eggs and fat bodies from lizards collected on 16 December were pooled into corresponding groups; samples from 28 January animals were processed and maintained separately.
Lipid extraction and isolation of classes Lipids were extracted with chloroform-methanol (2 : 1, v/v; Ways & Hanahan, 1964). Neutral lipid classes were separated by thin-layer chromatography (TLC) on 10 x 20 cm glass plates coated with layers 0"5 m m thick of Silica Gel G (E. Merck, A.G., Darmstadt); the solvent system was hexane-diethyl ether-formic acid (80 : 20 : 2 by vol.). Phospholipids were separated on Silica Gel H (Camag, Muttenz) coated plates; a developing solvent system consisting of chloroform-methanol-acetic acid-water (25 : 15 : 4 • 2 by vol.) was used. Lipid bands were detected under u.v. light after spraying the plates with 2',7'dichlorofluorescein (0"1%, wt/vol.) and identified against known standards. Bands were either scraped into small chromatographic columns and eluted with chloroform (neutral lipids) or chloroform-methanol-water (5 : 5 : 1 by vol.) (complex lipids), or were scraped directly into 15-ml test-tubes and methylated according to the procedures described by Christie (1972). Methyl pentadecanoate standard solution was added to each band prior to methylation, and the amount of each class was determined by relating the combined areas of the peaks for the components in the gas-liquid chromatography (GLC) trace to those of the internal standard (Christie et al., 1970).
Gas-liquid chromatography Fatty acid analyses were performed on columns (7 ft x 0-25 in.) of 15~o E G S S - X or EGSS-Y on Gas-chrom P (100-120 mesh; Applied Sciences Labs., Inc., State College, Pa.) isothermally at 180°C (EGSS-X) or 194°C (EGSS-Y) in a Pye 104 chromatograph (PyeUnicam Ltd., Cambridge, U.K.). Components were identified by their retention times relative to authentic standards and by their chromatographic behaviour on thin layers of silica gel-G impregnated with 10% (w/w) silver nitrate. The amount of each ester present (wt %) was calculated from the product of the peak height and retention time. Results for phospholipid classes were converted to mole % by multiplying these values by appropriate factors (Christie et al., 1970).
Stereospecific analysis of triglycerides Triglycerides were isolated by preparative T L C as described above. A procedure based on that devised by Broekerhoff (1965) was used for the stereospecifie analysis of triglycerides
LIPIDS AND TRIGLYCERIDES IN LIZARD EGGS
277
(Christie & Moore, 1969). ct,fl-Diglycerideswere prepared by the action of ethyl magnesium bromide on triglycerides and converted synthetically to phospholipids which were hydrolyzed by the stereospecific phospholipase A of snake venom. Results for position 1 were obtained by analysis of the lysophosphatide produced in the final stage of the procedure, those for position 2 were obtained by pancreatic lipase hydrolysis and those for position 3 were calculated by difference from the known triglyceride composition. Checks were made on the results for positions 2 and 3; analyses were accepted only when they conformed to the accuracy standards described by Christie & Moore (1969). RESULTS Data on lizard size and weights of eggs, fat bodies and their corresponding lipids for animals processed and analyzed individually are given in Table 1. The mean water content of fat bodies, based on dry weight-wet weight ratios, was approximately 10 per cent higher than the mean egg water content, although variation was greater in the fat bodies. Lipid content was significantly higher for fat TABLE 1--WEIGHTS (g) AND WEIGHT-RATIOS FOR EGGS, FAT BODIES AND THE CORRESPONDING LIPIDS FROM SIX FEMALE LIZARDS* COLLECTED ON 28 JANUARY 1973 (MEANS AND STANDARD ERRORS)
Wtwt. Dry wt. Dry wt/wet wt. Lipid wt/dry wt.
Eggs
Fat bodies
2.39+0.14 1.43_+0.08 0.60 + 0-01 0"36 -+0.02
0.15_+0-03 0.11 _+0.02 0"70 4-0.03 0'91 + 0.12
* Fresh wt. = 16"13 +0"85 g; snout-vent length = 80-3 + 1.91 mm. bodies (91.1 vs 35.6%, based on dry weights) and less variable in comparison to eggs. Generally, the percentage of egg lipid increased as the egg dry weight increased; however, a similar correlation was not apparent between lipid percentage and dry weight of fat bodies. Lipids were extracted from lizard eggs and fat bodies, and the proportions of the main lipid classes determined by thin-layer and gas-chromatographic techniques. The results are listed in Table 2. In addition to triglycerides and phospholipids, all eggs contained free fatty acids, diglycerides and cholesterol esters. The latter three classes when combined did not exceed 6 per cent of the total lipid content. Free cholesterol was also detected in the egg samples (Maclntyre & Ralston 1954), but was quantified only for the January females which contained 0.8 + 0.1 per cent. Triglyceride was again the major lipid class in lizard fat bodies, comprising an even greater proportion than found for eggs (92.6-94.9 per cent vs 86"9-90.2 per cent). Phospholipid levels were correspondingly less in fat bodies, which also included comparable amounts of diglycerides, free fatty acids and cholesterol esters. Alkyl diglycerides were also present (approx. 2 per cent) in male and female fat bodies from lizards collected in January, but were absent in December females.
Not determined Not determined
1"8
10"1±1'9
1-0 + 0"2
81"1±1"8
10"8
Not determined
5"0±1"0
6"7±0"8
88-2 ± 1-0
76"5
Phosphatidyl ethanolamine
1-3
6"2
Phosphatidyl choline
Principal phospholipids (mole %)
Sphingomyelin
9"0
Phospholipids
86"9
Triglycerides
Fat bodies: December collection (5 females ; 94"0 samples pooled) January collection (3 females ; 94"0 ± 0"7 and S.E.) January collection (6 males ; 94'9 samples pooled)
Eggs: December collection (5 lizards; samples pooled) January collection (3 lizards ; and S.E.)
Sample
Principal lipid classes (wt %)
TABLE 2--LIPID COMPOSITIONOF LIZARDEGGS AND FAT BODIES
2-1±0"8
4"0
Cardiolipin
m
P
>
m >
Z m t~
to Oo
L I P I D S AND TRIGLYCERIDES I N LIZARD EGGS
279
Neutral plasmalogens were not detected. Trace amounts of cholesterol were present in all fat body samples but the amounts did not exceed 0.6 per cent per animal in individuals collected in January. Egg phospholipids were also separated into individual classes by T L C and the results are listed in Table 2. Values for eggs from lizards collected in December and January were quite similar. Phosphatidyl choline was by far the most abundant class comprising 76.5-83.1 per cent of the total phospholipids. Phosphatidyl ethanolamine varied from 7.7 to 13.8 per cent, while sphingomyelin and cardiolipins were present in amounts less than 10 per cent of the total. Trace amounts ( < 2.0 per cent) of lysophosphatidyl choline were also present in all samples. The fatty acid compositions of the triglycerides in eggs and fat bodies (male and female) and phosphatidyl choline in eggs were also determined by gas chromatography. Results are listed in Tables 3 and 4 respectively. Oleic acid (18:1) was the major component of the triglycerides, ranging from 44.8 to 54.6 per cent in egg triglycerides and from 57.3 to 73-5 per cent in fat body triglycerides. Following oleic acid in abundance were palmitic acid (16:0) and the essential fatty acid, linoleic acid (18:2). Linoleic acid amounts varied considerably, ranging from 5.8 to 23.7 per cent in egg triglycerides to between 5.9 and 13.0 per cent in fat body triglycerides. Amounts of 16:0 were more constant with values of 16.9-18.6 per cent in egg triglycerides and 12-3-17-6 per cent in fat body triglycerides. In addition to the remaining fatty acid components listed in Table 3, small amounts of the following were invariably present: myristic acid (14:0), eicosadienoic acid (20:2) and eicosatrienoic acid (20:3). Two observations perhaps warrant mention at this point. First, an unusually high percentage (16.9 per cent) of palmitoleic acid (16:1) was found in the egg triglycerides of one of the lizards from the January collection. This acid, the presence of which was confirmed by silver nitrate chromatography, also comprised an unusually high percentage of the fatty acids in phosphatidyl choline in eggs from the same female (see Table 4), but not in the fat bodies. Secondly, there was a definite increase in oleic acid (18:1) and a concomitant decrease in palmitic acid (16:0) when fat body triglycerides in January-collected lizards were compared with fat body triglycerides in December animals. The fatty acids in phosphatidyl choline isolated from the eggs were very similar to those found in triglycerides, except that in phosphatidyl choline there were greater numbers and/or larger amounts of C20 and C~2 polyunsaturated fatty acids (Table 4). Trace amounts of 20:1, 20:2, 20:3, 20:5 and 22:4 were also present. There were some marked differences in the percentage composition of the major fatty acids in phosphatidyl choline when compared to that of the triglycerides in the eggs. Palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2) were approximately equal in overall abundance and together comprised between 76.0-83.3 per cent of the total fatty acids. Small but relatively consistent amounts of stearic acid (18:0), linolenic acid (18:3) and arachidonic acid (20:4) were always present. Palmitoleic acid (16:1) comprised only a small percentage of the total except for the individual described above.
3--FATTY
28"7 24"9 + 0'4
D e c e m b e r collection* J a n u a r y collection ( m e a n s a n d s t a n d a r d errors)
* Eggs f r o m 5 females pooled.
16:0
Sample
2"4 + 0'3 3"4
12"5 + 0"2 12"5
-3"1 + 1 '9
16:1 3"6 4-6 _+0"4
18:0
ACID COMPOSITION
4-7
2'8 8'5+_4"1
16:1
73-5
5"9
9'6 _+ 1 "8
10"7
15-3 13'8+_5.2
18:2
22'2 23"8 + 3'1
32"4 28-9 + 3-9
18:2
2"5 1 "4 + 0'8
18:3
F a t t y acid c o m p o s i t i o n (mole ~o) 18:1
1 "3
0'7 _+0'4
2"7
1"2 2'9+2.0
18:3
4-7 3"1 + 0"3
20:4
C H O L I N E O F L I Z A R D EGGS
67'9 + 2-7
57' 3
54"6 47'5+_1'6
18:1
OF THE PHOSPHATIDYL
3"5
4"6 + 0 ' 4
6"0
2"8 2-8+0"4
18:0
F a t t y acid c o m p o s i t i o n (wt % )
O F T H E T R I G L Y C E R I D E S O F L I Z A R D EGGS A N D F A T B O D I E S
17"6
18"6 17"6+0'5
16:0
ACID COMPOSITION
TABLE 4--FATTY
* Eggs from 5 females pooled.
F a t bodies : D e c e m b e r collection (female; n = 5) J a n u a r y collection (female) (means a n d s t a n d a r d errors) J a n u a r y collection (male ; n = 6)
Eggs : D e c e m b e r collection (n = 5)* J a n u a r y collection ( m e a n s a n d s t a n d a r d errors)
Sample
TABLE
2"0 1 "5 + 0"2
22:5/6
0" 1
1-1 + 0'3
0'6
2"6 3'8+_0"2
20:1
3-9 8"6 + 0"6
Remainder
--
--
--
1"6 1"1+_0-6
20:4
(3
t~
00
Fat bodies (male) : • January collection
Fat bodies (female) : December collection
Eggs: December collection
Fat bodies (male) : January collection
Fat bodies (female) : December collection
Eggs: December collection
Sample
TABLE
1 2 3
1 2 3
1 2 3
TG 1 2 3
TG 1 2 3
TG 1 2 3
70 7 23
72 11 17
66 6 28
13.3 28-1 2-6 9.2
18.0 38"8 5-8 9.4
19.4 38.3 3-5 16.3
LIZARD
EGGS AND FAT BODIES
4.2 10"0 1.6 1.0
5"9 11.0 2.8 3"9
3.6 8-4 1.4 0"9
18:0
68-9 49.3 82.2 75"2
55.8 33.6 68-2 65"6
50-7 35.8 61-8 54"8
18:1
7"3 4.5 8-6 8"8
10.6 5.7 13.1 13"0
17.0 7.7 27-2 16"0
18:2
Fatty acid composition (mole %)
FROM
1.1 0.8 1.2 1.3
2.8 1-8 3.7 2-9
2"2 1.2 2"4 3.1
18:3
44 24 32
46 27 27
32 15 53
79 13 8
62 16 22
78 13 9
24 40 36
20 41 39
24 41 35
21 39 40
18 41 41
15 53 32
24 36 40
21 44 35
18 36 46
Proportion (%) of the available fatty acids in each position
4.4 5.8 3-2 4.2
5.1 7-0 4.2 4"1
4.3 4-1 1.9 7"0
16:1
OF TRIGLYCERIDES
16:0
ANALYSIS
Position
5--STEREOSPECIFIC
--
m
m
_
0-9 1"5 0"6 0.6
20:4
t~ oo
~
~rj
282
NEIL F. HADLEY AND WILLIAM W. CHRISTIE
Preparative TLC was used to isolate triglycerides from the lipids of the eggs and fat bodies of females (December collection) and fat bodies of the males (January collection). The distribution of fatty acids in positions 1, 2 and 3 of the L-glycerol moiety was then determined by a stereospecific analysis procedure. The results are listed in Table 5. Saturated fatty acids were concentrated in position 1, although there were also appreciable amounts of 16:0 in position 3, while the C18 unsaturated fatty acids were in greatest concentration in positions 2 and 3. DISCUSSION The lipid composition of the fat bodies of S.jarrovi was similar to that reported for Varanus salvator depot fat (Hilditch & Williams, 1964) and the adipose tissue of Uromastix hardwickii (Zain & Zain-U1-Abedin, 1967; Afroz et al., 1971); however, the different analytical procedures and methods of data presentation made accurate comparisons difficult. The study by Afroz et al., (1971) provided the best opportunity for meaningful comparison. They found that triglycerides comprised approximately 90 per cent of the total lipid content of the adipose tissues of U. hardwickii, with the remainder consisting of diglycerides, monoglycerides, free fatty acids and cholesterol. The fatty acid composition of the triglycerides was also similar to results obtained on S. jarrovi fat bodies, although the proportionate amounts of the major constituents varied slightly. In U. hardwickii adipose tissue, the major fatty acids were oleic and palmitic, comprising 50-53 per cent and 20-24 per cent respectively in comparison to 57-74 per cent oleic acid and 12-18 per cent palmitic acid in S. jarrovi fat bodies. In both species the proportion of unsaturated fatty acids (65 per cent in U. hardwickii, 76-84 per cent in S. jarrovi) was greater than saturated ones. The fact that alkyl diglycerides were found in small amounts in the fat bodies of S. jarrovi in the January collection but not in those of the December collection may possibly be associated with the difference in maturity of the animals. Using snout-vent length as an age indicator, December lizards (snout-vent -- 51-61 ram) were approximately 1 year old, while lizards collected in January (snout-vent = 76-85 ram) were approximately 2 years or older (Tinkle & Hadley, 1973). Ether bonds are known to be formed and metabolized much more slowly than ester bonds and compounds containing these can accumulate with age (Snyder, 1970). The extremely high concentration of 16:1 at the expense of 18:2 in the eggs of the individual from the January collection may reflect variation in the diet of this animal or it may be that it has a highly active fatty acid desaturation system. Similarly, we can only speculate as to the cause of the apparent increased unsaturation of fat bodies in January-collected lizards (83 per cent) vs December-collected animals (76 per cent). Controlled experiments over a several-month period would be necessary to determine if there is a selective utilization of saturated over unsaturated fatty acids or if there is continuous desaturation of fatty acids already present with no fat deposition. In U. hardwickii there was no change in the total unsaturation of adipose tissue during hibernation, arousal and activity, in spite of alteration in the fatty acid pattern of triglycerides (Afroz et al., 1971).
L I P I D S AND TRIGLYCERIDES I N LIZARD EGGS
283
The influence of environmental temperature on the lipid composition of eggs and fat bodies of S. jarrovi warrants further study. Ovulation takes place in late autumn and eggs are carried over the winter (Tinkle & Hadley, 1973). Many females and males are active above ground during this time even though subfreezing temperatures are common during the night. Lizards seek shelter in shallow rock crevices and between boulders; however, this exposed winter microhabitat affords a maximum thermal buffering of only 5-6°C (Lowe et al., 1971). To meet these thermal demands, S. jarrovi are capable of internal supercooling to - 5.5°C (Lowe et al., 1971). It is likely that the relatively high degree of unsaturation of the total lipids in eggs and fat bodies reflected these low temperatures, but confirmation awaits comparative studies on lizards with different modes of reproduction and temperature r6gimes. Additional experimentation is also necessary to establish if the unsaturation is a result of increased deposition of dietary polyunsaturated fatty acids or if an increased de novo synthesis of unsaturated fatty acids takes place in response to lower temperatures. In fish (Malins & Wekell, 1970) and birds (Fisher et al., 1962) there was an increase in the concentration of unsaturated fatty acids in the depot fat with decrease in temperature, but this was the result of increased deposition of dietary unsaturated fatty acids rather than increased desaturation of endogenous saturated fatty acids. Although the triglycerides from fat bodies from males and females and from eggs of S. jarrovi differed considerably in fatty acid composition, the available fatty acids were distributed among the three positions of the glycerol moiety in a similar manner. This can best be seen if the proportions rather than the absolute amounts of each fatty acid in each position are calculated (Table 5). As an example, although the absolute amount of 18:1 in the triglycerides varied between 51 and 69 per cent, the proportion of this in position 1 only varied between 20 and 24 per cent, in position 2 between 40 and 41 per cent and in position 3 between 35 and 39 per cent. Of the major components, only 18:2 differed in the way it was distributed in the triglycerides of the eggs and fat bodies in that in the former there was somewhat more in position 2 and correspondingly less in position 3 than in the latter. In addition, the structures of the triglycerides from the fat bodies were similar to those in adipose tissue from most mammalian species (Brockerhoff, 1966) and also to that of adipose tissue in the domestic chicken (Christie & Moore, 1972a). The structures of the egg triglycerides differed considerably from those of avian eggs (Christie & Moore, 1972b), which contain the most asymmetric triglycerides analyzed to date, but no other analyses of reptilian eggs are available for comparison. Acknowledgement--The authors acknowledge the skilled technical assistance of Miss M. Hunter and Mrs. P. Paterson. REFERENCES
AFROZH., ISHAQM. & ALI S. S. (1971) Seasonal changes in the lipids of adipose tissue in a hibernating lizard (Uroraastix hardwickii). Proc. Soc. exp. Biol. Med. 136, 894-898. BROCKERHOFFH. (1965) A stereospecific analysis of triglycerides. 07. Lipid Res. 6, 10-15. BROCKEm-IOFFH. (1966) Fatty acid distribution patterns of animal depot fats. Comp. Biochem. Physiol. 19, 1-12.
284
NEIL F. HADLEY AND WILLIAM W. CHRISTIE
CHRISTIE W. W. (1972) Quantitative esterification of lipids on thin-layer adsorbents. Analyst 97, 221-223. CHRISTIE W. W. & MOORE J. H. (1969) A semimicro method for the stereospecific analysis of triglycerides. Biochim. biophys. Acta 210, 46-56. CHRISTIE W. W. & MOORE J. H. (1972a) T h e lipid components of the plasma, liver and ovarian follicles in the domestic chicken (Gallus gallus). Comp. Biochem. Physiol. 41B, 287-295. CHRISTIE W. W. & MOORE J. H. (1972b) The lipid composition and triglyceride structure of eggs from several avian species. Comp. Biochem. Physiol. 41B, 297-306. CHRISTIE W. W., NOBLE R. C. & MOORE J. H. (1970) Determination of lipid classes by a gas chromatographic procedure. Analyst 95, 940-944. FISHER H., HOLLANDSK. G. & WEISS H. S. (1962) Environmental temperature and composition of body fat. Proc. Soc. exp. Biol. Med. 110, 832-833. GRODZINSKI Z. (1949) T h e fat in the yolk of the sand-lizard Lacerta agilis L. Bull. Acad. pol. sci. Ser. B, II, 368-381. HAHN W. E. & TINKLE D. W. (1965) Fat body cycling and experimental evidence for its adaptive significance to ovarian follicle development in the lizard Uta stansburiana. J. exp. Zool. 158, 79-86. HILDITCH T. P. & PAVL H. (1937) The depot fat of Varanus salvator (Laur.). Biochem. J. 31, 227-228. HILDITCH T. P. & WILLIAMS P. N. (1964) The Chemical Constitution of Natural Fats, 4th Edn. J. Wiley & Sons, New York. KLENK E., DITT F. & DIEBOLD W. (1935) Uber das Depotfett der Wirbeltiere. HoppeSeyler's Z. physiol. Chem. 232, 54-63. LowE C. H., LARDNER P. J. & HALPERN E. A. (1971) Supercooling in reptiles and other vertebrates. Comp. B iochem. Physiol., 39A, 125-135. MACINTYRE I. & RALSTON M. (1954) Direct determination of serum cholesterol. Biochem.ff., 56, xiii. MALINS D. C. & WEKELL J. C. (1970) The lipid biochemistry of marine organisms. In Progress in the Chemistry of Fats and Other Lipids (Edited by HOLMAN R. T.), Vol. 10, pp. 337-363. Pergamon Press, Oxford. SNYDER F. (1970) T h e biochemistry of lipids containing ether bonds. In Progress in the Chemistry of Fats and Other Lipids (Edited by HOLMAN R. T.), Vol. 10, pp. 287-336. Pergamon Press, Oxford. TINKLE D. W. & HADLEY N. F. (1973) Reproductive effort and winter activity in the viviparous montane lizard Sceloporus jarrovi. Copeia (1973), 272-277. WAYS P. & HANAHAN D. J. (1964) Characterization and quantification of red cell lipids in normal man. ft. Lipid Res. 5, 318-328. ZAIN B. K. & ZAIN-uL-ABEDIN M. (1967) Characterization of the abdominal fat pads of a lizard. Comp. Bioehem. Physiol. 23, 173-177.
Key Word Index--Eggs ; fat bodies; adipose tissue ; lizard ; reptile ; Sceloporus jarrovi; lipid composition; triglyceride structure; triglycerides; stereospecific analysis from lizard eggs; egg lipids; fat body lipids.