- Email: [email protected]
In vivo estimation of fatty acid synthesis in the chicken (Gallus domesticus) utilizing 3H2O
Comp. Biochem. Physiol., 1976. Vol. 54B, pp. 403 to 407. Peryamon Press. Printed in Great Britain
IN VIVO ESTIMATION OF FATTY ACID SYNTHESIS IN THE CHICKEN (GALLUS DOMESTICUS) UTILIZING 3H20* LINDA BRADY, DALE R. ROMSOSt AND GILBERT A. LEVEILLE Department Food Science & Human Nutrition, Michigan State University, East Lansing, MI 48824, U.S.A. (Received 11 Auffust 1975)
Abstract--1. Tritiated water was found to rapidly equilibrate with the body water of chicks after injection. 2. The 3H also rapidly appeared in both liver and carcass fatty acids, with the greatest rates of incorporation occurring in the initial time periods of measurement. 3. Incorporation of 3H into fatty acids in the liver appeared to proceed primarily via de novo synthesis whereas incorporation of 3H into fatty acids in the carcass occurred primarily via chain elongation.
INTRODUCTION Most experimental evidence indicates that the liver is the primary site of fatty acid synthesis in the chicken. O ' H e a & Leveille (1968) found that isolated chick adipose tissue had a low capacity for fatty acid synthesis when [1-14C]acetate or [U-14C]glucose incorporation into fatty acids was measured. They also studied in vivo biosynthesis and transfer of fatty acids using the same tracers (O'Hea & Leveille, 1969) and estimated that 90-95~o of de novo fatty acid synthesis in the chick occurred.in the liver, with the adipose tissue appearing to function chiefly in lipid storage. Goodridge & Ball (1966) have done similar studies with pigeon liver and adipose tissue, both in vitro and in vivo. In vitro they found that synthesis of fatty acids in adipose tissue from labelled glucose or pyruvate was negligible. They also found low activities of the following lipogenic enzymes in adipose tissue: acetyl CoA carboxylase, citrate cleavage enzyme, malic enzyme and the hexose monophosphate dehydrogenases. In vivo work also showed that the liver was the major site of fatty acid synthesis in the pigeon (Goodridge & Ball, 1967). These studies (O'Hea & Leveille, 1968, 1969; Goodridge & Ball, 1966, 1967) examined the rates of fatty acid synthesis in liver and adipose tissue. However, Yeh & Leveille (1972) found significant incorporation of [1-14C]acetate into fatty acids in the carcasses of chicks (the carcass represented the hepatectomized animal). Because 15 min elapsed between tracer injection and removal of the liver, the possibility of transfer of newly synthesized labelled fatty acids from the liver to the carcass could not be ruled out. Another possibility explaining the high rate of [1-14C]acetate incorporation into fatty acids in chick
carcasses might have been chain elongation of fatty acids by extra-hepatic tissues. Our experiments were designed to estimate the rate of fatty acid synthesis, utilizing 3H20 , in the liver and carcass of chickens. Tritiated water was chosen as it estimates the rate of fatty acid synthesis from all carbon precursors rather than synthesis from a specific carbon precursor. The use of tritiated water also minimizes the problem of isotope dilution which exists with glucose and acetate tracers (Goodridge, 1968). A second purpose of our experiments was to obtain an estimate of de novo fatty acid synthesis vs chain elongation in several tissues of chickens. MATERIALS A N D M E T H O D S
Chicks and diets. Male broiler chicks (Fairview Farms,
Remington, Indiana, USA) were used for all experiments except experiment 3 where both males and females were used. Chicks were housed in electrically heated brooders with raised wire floors. They were fed a commercial highcarbohydrate diet (Master Mix Chick Starter and Grower, Central Soya, Fort Wayne, Indiana, USA). Water and food were available at all times. Experiment 1. The time for equilibration of 3H20 in the blood of young chicks was estimated. Seven chicks (250-400 g) were injected in a wing vein with 2 mCi of 3H20 in 2 ml of saline. After puncture of an opposite wing vein with a sterile microlance, 25 pl samples of blood were collected at 5, 10, 20, 30, 45 and 60 min after 3H20 injection. The samples were diluted and counted in a Packard Tri-Carb 3 channel scintillation counter. Counting efficiency was determined. In another group of chickens, blood samples were obtained at shorter time intervals after an injection of 250 #Ci 3H20 in 1 ml saline into a wing vein. Blood samples were drawn from 1000-1500g chickens at 1 min intervals between 1 and 10 min from either a catheter in the jugular vein or from a wing vein opposite the site of injection. The blood samples were centrifuged and 10 ~tl of plasma counted for radioactivity. * Supported in part by Public Health Service Grants Experiment 2. The rate of 3H incorporation from 3H20 HL 14677 and GMO 1818. D.R.R. is the recipient of Career into fatty acids in chick liver slices was determined. Two Development Award K04 AM 00112. Michigan Agricul- 500 g chicks were killed by cervical dislocation. The livers ture Experiment Station Journal Article No. 7341. were rapidly removed and chilled in saline. Fifteen slices f Address correspondence to: Dale R. Romsos, Food 006-200 rag) from the liver of each chick were prepared Science & Human Nutr. Michigan State University, East with a Stadie-Riggs hand microtome. Three slices from each chick were incubated for intervals of 30, 60, 90, 120 Lansing, MI 48824, U.S.A. 4O3
404
LINDA BRADY, DALE R ROMSOS AND GILBERT A. LEVEILLE
or 180 min in a Krebs-Ringer bicarbonate buffer containing per ml: 50pCi 3H20, 3 mg glucose and 0.1 unit porcine insulin (Eli Lilly, Indianapolis, IN). Incubation procedures and methods for counting radioactive fatty acids have been described (Leveille, 1966). Experiment 3. The in vivo rate of fatty acid synthesis was determined in 400-500 g chicks. Ad lib. fed birds were injected intravenously with 2 mCi 3H20 in 2 ml saline. After injection, birds were returned to their pens. Fresh food and water were available. Blood samples were taken and chicks were killed by cervical dislocation 5, 10 or 60 min after 3H20 injection. The livers were rapidly excised and frozen in dry ice-acetone. The remaining carcass was packed on ice and stored at -20°C until analyzed. The livers were homogenized with an equal weight of water and 0.5 ml of the slurry saponified and counted as above. The carcasses were weighed and dried at 100°C and ground twice. Two homogeneous samples of I g were extracted in 40 ml chloroform methanol (3:2) at 50°C for 30 rain, then filtered into tared aluminum dishes, dried, weighed and saponified as with the liver homogenates. Experiments 4 and 5. In experiment 4, 200-300 g chicks were injected intravenously with 1.5 mCi 3H20 in 2 ml saline and killed 1, 3, 5 or 10 min later. Experiment 5 was identical to experiment 4 except that times of sampling were expanded to 1, 3, 5, I0, 20 and 30 min after injection. Experiment 6. The in vivo rate of fatty acid synthesis was also determined in male rats for comparison with the chicks. Sprague-Dawley rats (Spartan Research Animals, Haslett, MI, USA) weighing 80-90 g were fed a high-carbohydrate semisynthetic diet (Romsos & Leveille, 1972). Tritiated water (1 mCi in 0.2 ml saline) was injected intraperitoneally and animals were killed 10, 20, or 30 min later. Epididymal fat pads, liver, and carcass were prepared as with the chicks. Experiment 7. Either 2.5 mCi 3H20 in 0.5 ml saline or 5/~Ci [1-14C]acetate in 0.5ml saline was injected into 300-400 g birds and after 5 rain they were killed to obtain estimates of the rates of fatty acid synthesis in various organs. Experiment 8. The amount of chain elongation occurring in various tissues was estimated by determining the dis/rain in total fatty acids: dis/rain in the carboxyl carbon after decarboxylation. Chickens were injected with 20/~Ci of [l-14C]acetate and killed 5 min later. RESULTS
The results of the study of equilibration of tritium in the blood of the chick after an injection of 3H20 are presented in Table 1. No significant differences were found among the values over time. The tritium specific activity in the blood had reached a plateau before the initial sample was taken. In the second part of experiment 1, therefore, blood samples were obtained from 1 to 10 min after injection of 3HzO. Sampling was from either the vena cava or a wing Table 1--Exp. 1. Blood specific activity in chicks following tritiated water iniection* Time after Pulse (min.)
5
, 1.58 L "Oft
I0 20 30 45 60
dpm3H x lO'/~ml plasma
1.53 1.56 1.57 1.59 "
L ~ + +
.11 .09 .10 .09
1.60 L . I 0
Table 2--Exp. 1. Plasma specific activity in chicks following tritiated water injection* Time after pulse (min.
Site of Sampling wing vein
vena cava
I
1.46 L -15f
2
1.36 + .15
1.31 + .Ig
3
1.34 L .06
1.34 + .16
4
1.34 L .11
1.31 + .16
5
1.32 L .10
1.21 + .15
6
1.32 + .13
I.ii
7
1.22 + .18
1.30 L .16
8
1.23 ~ .OS
1.19 ~ .12
9
1.29 ? .06 1.34 ~ .06
1.22 ~ .09 1.18 ~ .10
10
1.29 L .29
L .08
*250 ~Ci 3H20 injected into 1000 g chicks, +Mean~++SEM f~r 3-4 chicks. Results expressed as dpm ~Hx 10- /ml plasma.
vein opposite the site of injection (Table 2). No significant differences were found between sites at each time tested, nor over time for each site. Thus, the blood plasma 3H20 had equilibrated by 1 min in these chicks and rapid homogeneous mixing was obtained. An in vitro experiment was conducted, utilizing 3 H 2 0 , to determine the rate of fatty acid synthesis in chick liver (Table 3). The in vitro incorporation of 3H from 3H20 into fatty acids of chick liver slices appeared to be linear from 30 to 120 min, but leveled off between 120 and 180 min of incubation. Several experiments were conducted to determine the in vivo rate of fatty acid synthesis in the chick. The results of experiment 3 are presented in Table 4. The rate of 3H incorporation from 3H20 into fatty acids in chick liver was most rapid initially. Between 5 and 10 min and between 10 and 60 min, the rates of incorporation slowed and, at 60 min incorporation was not as great as would be predicted if synthesis were linear with time. This is contrary to results obtained with rats (Sullivan et al., 1974; Lowenstein, 1971). Fatty acid synthesis measured by 3H incorporation was linear for 60-90 min in the rat. The concentration of labelled fatty acids in chick liver was 23-91 times greater than observed in the carcass, but the carcass represented a greater mass. Consequently, the incorporation of 3H into fatty acids in the carcass was significant at each time. Tritium incorporation into fatty acids did not increase linearly with time in the carcass either. Because the in vivo rate of fatty acid synthesis in the chick did not appear linear with time after a 3 H 2 0 injection, another experiment was conducted. Table 3--Exp. 2. In vitro incorporation of 3H into fatty acids by chick liver slices* Incubation time (min.)
Fatty acid synthesis
30 60 90
5,501 + 684 ~ 13,134 T 548 23,536 V 2704
120 180
4"
36,784 + 2592 33,978 ~ 3102
*Two mCi 3H20 injected into 300-400 g chicks.
*Dpm tritium from 3HpO incorporated into fatty acids per gram Iive~.
fMean ~ SEM for 6 chicks.
÷Mean + SEM for 15 liver slices,
Tritiated water and fatty acid synthesis Table 4---Exp. 3. Tritium incorporation into chick liver and carcass fatty acids*t Minutes after injection
5 I0 60
SO00
Fatty acid synthesis per gram Liver
Carcass
1159 + 153t 1899 + 561 6247 ¥ 794
51 + I0 41 ¥ 5 69 T 12
Fatty acid synthesis per total Liver 5 I0 60
405
Carcass
18,503 + 2101 26,791+ 5305 28,207 ¥ 8604 20,824¥ 2585 96,488 ~ 13,325 35,068~ 6378
~ 200( E
t~ "o
~:~ L~ IOOC
*Two mCi 3H20 injected into 400-600 g chicks. fResults expressed as dpm 3H incorporated into fatty acids.
Table 5--Exp. 4. Total 3H incorporation into fatty acids in liver and carcass of chieks*t
iO
I
I
8
I0
rain
Fig. 1. Tritium incorporation into fatty acids/g liver and carcass of the chick, - - carcass, ---O-- liver. Each point represents the mean of eight 200-300 g chicks. cass 3H incorporation into fatty acids was lower than either liver or adipose tissue, and as with the chick did not increase significantly over time. The rates of incorporation of 3H from 3H20 and of [lr14C]acetate into various tissues of the chick were determined in experiment 7. With 3H, the liver, as expected, exhibited from 5 to 14 times more incorporation than other tissues (Table 6). In the same size birds, the relative incorporation of [1-14C]acetate was different than for 3H from 3H20. Liver 14C incorporation into fatty acids was from 11 to 48 times the kidney and muscle respectively. The kidney, intestine, and adipose tissue all exhibited approximately the same amount of incorporation/g. The differences in 3H and 1'~C incorporation might have been due to the changing specific activity of acetate during the experiment. Because the apparent incorporation of 3H from 3H20 into carcass fatty acids was significant in all experiments, an attempt was made to differentiate
7000
~ 5000
E
~
/
t
T
3000 +/,~
7,894 L 11,954 L 17,208 + 37,499 +
Carcass 10791: 36,333L 2860 82,672~ 2874 143,280~ 4279 116,584~
2768 5989 15,191 12,191
"1.5 mCi 3H20 injected into 200-300 g chicks. +Results expressed as total dpm 3H incorporated into fatty acids. ~MeanL SEMfor 8 chicks.
I
6
4
Fatty Acid Synthesis Liver
I 3 5
I
2
Time,
In this experiment (experiment 4) chicks were killed 1, 3, 5 or 10 min after injection of 3H20. In the liver the most rapid 3H incorporation into fatty acids occurred initially, although 3H incorporation into fatty acids increased for the 10 min of the experiment (Table 5 and Fig. 1). Carcass 3H incorporation from 3H20 to fatty acids rose initially, then remained constant between 5 and 10 min, much as in experiment 3 where carcass values did not increase significantly from 5 to 60 min. Again the carcass appeared to contribute a sizeable proportion of labelled fatty acids. Experiment 5 was identical to experiment 4, except that times of 20 and 30 min were added. The liver incorporation of 3H into fatty acids again showed the greatest rate initially (Fig. 2). Although 3H incorporation increased in the liver throughout the experiment, the rates were not linear. The carcass 3H incorporation into fatty acids (not shown) remained relatively constant. Studies of 3H incorporation into fatty acids were also done with rats to compare the two species and to determine if rates were linear in rat liver, carcass and adipose according to our methods. As mentioned, previous reports have found hepatic fatty acid synthesis in the rat to be linear for up to 90 min. Our results (Fig. 3) showed essentially the same results over 30 min in adipose tissue of the rat. A tendency for slower rates of fatty acid synthesis after 10 min was observed in rat liver; however, the effect was not as marked as in the chick. Per g, liver and adipose 3H incorporation into fatty acids were relatively close, with liver values slightly higher at each time. The car-
Minutes after injection
i
0
~'Mean + SEMfor eight chicks.
I000 l '~'
t
I
6
I
12 Time,
I
18
I
24
I
:50
rain
Fig. 2. Experiment 5. Tritium incorporation into fatty acids in chick liver. Each point represents the mean + S.E.M. for eight 200-300 g chicks.
406
LINDA BRADY, DALE R. ROMSOS AND GILBERT A. LEVEILLE
I0000
ation of 14C into fatty acids in the muscle remained constant. Jungas (1968) has shown that de novo fatty acid synthesis as measured by 3H incorporation from 3H20 in rat adipose tissue dropped in the fasted state. Adipose tissue is a major site of fatty acid synthesis in the rat.
j/
8000
// // / / / / /"I1/" j
6000
n 4000
_
DISCUSSION
ii/I/
200(~
I 6
12 Time,
I
I
I
18 min
24
30
Fig. 3. Experiment 6. Tritium incorporation into rat liver, adipose, and carcass of the rat. -. . . . . liver, - - adipose, . . . . . . . . carcass. between de novo fatty acid synthesis and chain elongation in carcass tissues in vivo. Theoretically, in vitro, the ratio of total fatty acid dis per min:carboxyl carbon dis/min should be 8 or 9. A ratio of 1 or 2 would indicate chain elongation in vitro. Thus, de novo synthesis would result in a higher ratio than chain elongation by a factor of 4-8. Tables 7 and 8 present the results obtained in vivo. Ratios of total fatty acid dis/min:dis/min in COOH in the liver of fed chicks was slightly higher than typical results obtained in vitro (Goodridge, 1970; Joshi & Sidbury, 1975), a fact which may be related to declining specific activity of acetate with time after injection in vivo. Based on the ratios obtained, chain elongation primarily occurs in kidney, skin, intestine, muscle, heart and adipose tissue. Microsomal or mitochondrial chain elongation would incorporate tritium atoms also via NADH and NADPH. Table 8 shows the ratios of total dis/min in fatty acids:dis/min in COOH in liver and muscle of both fed and fasted birds. The mean ratio in the muscle did not change with state of nutrition, but in liver the ratio decreased by almost 75~o, which indicates a drop in de novo synthesis with fasting. Incorporation of 14C was much greater for liver than muscle in both the fed and the fasted states, but I4C incorporation into fatty acids in livers from fasted chicks dropped to 16~o of the fed state while incorporTable 6--Exp. 7. 3H and 14C incorporation into fatty acids in various chick organs* T-issue
3H-dpm/gwet weight
Liver Skin Adipose Intestine Heart Muscle Kidney
4713 ~ 1108@ 352 + 97 510 ~ 30 951 ~ 68 534 + 84 710 L 201 921 ~ 182
An assumption of in vivo tracer experiments is that the tracer that is injected rapidly mixes with the plasma pool, and subsequently with other body pools. Theoretically, tracer specific activity should be constant in the body pools during the time its incorporation into other compounds is being measured. In practice, the time of plasma equilibration is often not as rapid as theory demands. Katz et al. (1974) found plasma 3HzO equilibration in the rabbit to occur from 25 to 30 min after injection. In the chick, however, the assumption of a rapid mixing of 3H20 with the body water pool appears to be met. In our earliest reported sampling after injection (1 min) it was clear that plasma specific activity had equilibrated. This appears reasonable in view of the fact that the chick's frequency of heartbeat is 350~50/min and cardiac output 218 ml/min per kg (Akester, 1971). The chick thus appears to meet one of the requirements for in vivo tracer experiments. However, we made one further assumption: that the aH20 in the plasma rapidly equilibrated with the immediate precursors of the fatty acids. This assumption was not tested in our experiments. The rapidity of plasma 3H20 equilibration was also reflected in the appearance of 3H in the fatty acids. Based on earlier studies with rats (Sullivan et al., 1974; Lowenstein, 1971), it was expected that a linear rate of 3H incorporation into fatty acids would exist for up to an hour. However, in all the studies reported here, the initial rate of 3H incorporation into fatty acids in both liver and carcass of the birds was the greatest rate observed. This fact has significance because if rates of synthesis are to be compared among treatments, they should be compared during linear rates of synthesis. In this case, waiting beyond 5 min does not appear advisable in the young chick as 3H incorporation is not always linear beyond this point. Almost all experimental evidence has indicated that the liver is the main site of fatty acid synthesis in the chick and our experiments appear to support the fact that most of the de novo fatty acid synthesis Table 7--Exp. 8. Ratio of total fatty acid DPM/carboxyl carbon DPM in fed chicks*
14C-dpm/gwet weight Tissue 81,105 L 3,320 L 6,621 ~ 5,142 ~ 3,152 L 1,688 ~ 7,060 L
37,684 1,636 3,700 2,232 1,238 471 2,916
*5 minutes after injection of 2.5 mCi 3H20or 5 uCi 14C acetate into 300-400 g chicks. fMean ~ SEMfor 5 chicks.
Liver Kidney Skin Intestine Muscle Heart Adipose
Total dpm/COOHdpm 15 2 1 1 i 1 I
*20 uCi acetate-1-14C in 2 m] saline injected into 300-400 g chicks.
Tritiated water and fatty acid synthesis Table 8--Exp. 8. Comparison of liver and muscle chain elongation in fed and fasted chicks* Fed
Fasted
dpm 14C in FA FA dpm/COOHdpm ~ q tissue g tlssue Liver Muscle
FA dpm/COOHdpm
96,281
15
15,178
4
1,924
1
1,811
I
*Eight 300-400 ~4fed chicks and 8 chicks fasted 24 hr. with 20 uCi I- C-acetate and 2 ml saline.
407
liver and carcass. The rate of incorporation was always greater initially than at later time intervals. The liver appeared to synthesize fatty acids de novo while the carcass appeared to elongate fatty acids, both processes incorporating tritium into the fatty acid molecule. The content of labelled fatty acids in the carcass did not increase with time, suggesting that fatty acid oxidation was occurring simultaneously in some carcass tissues.
Chicks injected
occurs in chick liver. However, tritium from 3H20 may also be incorporated into fatty acids undergoing chain elongation in the carcass. We consistently found the presence of tritium in carcass fatty acids at very short times after injection. This does not rule out transfer of fatty acids from the liver to the carcass, but the rapidity and magnitude of tritium appearance in the carcass fatty acids suggests significant de novo synthesis or chain elongation in some carcass tissues. Two other studies (Yeh & Leveille, 1972, 1973) have also found significant apparent carcass synthesis of fatty acids in vivo, although glucose and acetate were used as tracers. Another observation of the tritium incorporation into carcass tissues was the lack of accumulation of labelled fatty acids in the carcass with time. This suggests rapid oxidation in some carcass tissues simultaneously with de novo synthesis or chain elongation. Muscle represents a great mass of the body and has been shown to utilize fatty acids as an energy source (Owen & Reichard, 1971; Newsholme & Start, 1973). The incorporation of tritium into fatty acids in rat liver, adipose and carcass differed from that observed in the chick. The rat adipose tissue showed linear rates of synthesis from 10 to 30 min. The liver had a tendency for most rapid incorporation to occur initially, but the rate differences with time were not as marked as in the chick. Some of the tritium of the carcass and adipose tissue fatty acids could have come from the liver and might explain a tendency for rates of tritium incorporation into fatty acids in the liver to slow slightly. However, carcass values did not increase with time, again suggesting rapid utilization of fatty acids by the carcass. With tritiated water, chain elongation cannot be differentiated from de novo fatty acid synthesis, and the tritium incorporation into carcass fatty acids could be substantially through chain elongation. Using [l-14C]acetate we found that chick muscle, intestine, adipose, heart, and skin appeared to synthesize fatty acids via chain elongation. Joshi & Sidbury (1975) found that in vitro, the ability of the heart mitochondrial chain elongation system increased with age in chicks. Their results indicated that the major type of label incorporation in in vitro embryo and adult heart was by mitochondrial chain elongation, while that of the liver was by de novo synthesis. Donaldson & Mueller (1971) found some microsomal and mitochondrial chain elongation capability in vitro in liver, but found primarily cytosolic synthesis in carbohydrate-fed chicks. In conclusion, our results indicate that 3 H 2 0 rapidly equilibrated with the body water and was rapidly incorporated into fatty acids in both chick
REFERENCES
AKES~R A. R. (1971) The Heart. In Physiology and Biochemistry of the Domestic Fowl (Edited by BELL D. J. & FREEMANB. M.), p. 746, 777. DONALDSON W. E. & MUELLER N. S. (1971) Synthesis of unsaturated fatty acids in chick embryo liver. Can. J. Biochem. 49, 563-567. GOODRIDOE A. G. (1968) Conversion of fU-~4C]glucose into CO2, glycogen, cholesterol and fatty acids in liver slices from embryonic and growing chicks. Biochem. J. 108, 655-661. GOODRIDGEA. G. (1970) Regulation of lipogenesis: stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick. Biochem. J. 118, 259-263. GOODRIDGEA. G. & BALL E. G. (1966) Lipogenesis in the pigeon: in vitro studies. Am. J. Physiol. 211, 803-808. GOODRIDGEA. G. & BALL E. G. (1967) Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. 213, 245-249. JOSHI V. C. & SIDBURY J. B. (1975) Fatty acid synthesis in chick embryonic heart and liver during development. Devl Biol. 42, 282-291. JUNGASR. L. (1968) Fatty acid synthesis in adipose tissue incubated in tritiated water. Biochemistry 7, 3708-3717. KATZ J., DUNN A. & CHENOWETHM. (1974) Determination of synthesis, recycling, and body mass of glucose in rats and rabbits with 3H and 14C-labelled glucose. Biochem. J. 142, 171-183. LEVEILLE G. A. (1966) Glycogen metabolism in meal fed rats and chicks and the time sequence of lipogenic and enzymatic adaptive changes. J. Nutr. 90, 449-460. LOWENSTEINJ. M. (1971) Effect of hydroxycitrate on fatty acid synthesis by rat liver in vivo. J. biol. Chem. 246, 629-632. NEWSHOLME E. A. & START C. (1973) In Regulation in Metabolism, p. 213 Wiley, New York. O'HEA E. K. & LEVEILLEG. A. (1968) Lipogenesis in isolated adipose tissue of the domestic chick (Gallus Domesticus). Comp. Biochem. Physiol. 26, 111-120. O'HEA E. K. & LEVEILLEG. A. (1969) Lipid biosynthesis and transport in the domestic chick. Comp. Biochem. Physiol. 30, 149-159. OWEN O. E. & RE1CHARO G. A. (1971) Fuels consumed by man: the interplay between carbohydrate and fatty acids. In Biochemistry and Pharmacology of Free Fatty Acids (Edited by HOLMES W. L., BORTZ W. M. & KARGER S.), pp. 199-200. Basel, Switzerland. ROMSOS D. R. & LEVEmLE G. A. (1972) Cellularity and in vitro fatty acid biosynthesis in adipose tissue of meal fed and nibbling rats. Proc. Soc. exp. Biol. Med. 139, 868-871. SULLIVANA. C., TRISCARI J., HAMILTON J. G., MILLER O. N. ~,~ WHEATLEY V. R.
(1974) Effect
of
hydroxycitrate
upon the accumulation of lipid in the rat--I. Lipogenesis Lipids 9, 121-128. YEH S. C. & LEVEILLEG. A. (1972) Cholesterol and fatty acid synthesis in chicks fed different levels of protein. J. Nutr. 102, 349-358. YEn S. C. & LEVE1LLEG. A. (1973) Significance of skin as a site of fatty acid and cholesterol synthesis in the chick. Proc. Soc. exp. Biol. Med. 142, 115-119.
IN VIVO ESTIMATION OF FATTY ACID SYNTHESIS IN THE CHICKEN (GALLUS DOMESTICUS) UTILIZING 3H20* LINDA BRADY, DALE R. ROMSOSt AND GILBERT A. LEVEILLE Department Food Science & Human Nutrition, Michigan State University, East Lansing, MI 48824, U.S.A. (Received 11 Auffust 1975)
Abstract--1. Tritiated water was found to rapidly equilibrate with the body water of chicks after injection. 2. The 3H also rapidly appeared in both liver and carcass fatty acids, with the greatest rates of incorporation occurring in the initial time periods of measurement. 3. Incorporation of 3H into fatty acids in the liver appeared to proceed primarily via de novo synthesis whereas incorporation of 3H into fatty acids in the carcass occurred primarily via chain elongation.
INTRODUCTION Most experimental evidence indicates that the liver is the primary site of fatty acid synthesis in the chicken. O ' H e a & Leveille (1968) found that isolated chick adipose tissue had a low capacity for fatty acid synthesis when [1-14C]acetate or [U-14C]glucose incorporation into fatty acids was measured. They also studied in vivo biosynthesis and transfer of fatty acids using the same tracers (O'Hea & Leveille, 1969) and estimated that 90-95~o of de novo fatty acid synthesis in the chick occurred.in the liver, with the adipose tissue appearing to function chiefly in lipid storage. Goodridge & Ball (1966) have done similar studies with pigeon liver and adipose tissue, both in vitro and in vivo. In vitro they found that synthesis of fatty acids in adipose tissue from labelled glucose or pyruvate was negligible. They also found low activities of the following lipogenic enzymes in adipose tissue: acetyl CoA carboxylase, citrate cleavage enzyme, malic enzyme and the hexose monophosphate dehydrogenases. In vivo work also showed that the liver was the major site of fatty acid synthesis in the pigeon (Goodridge & Ball, 1967). These studies (O'Hea & Leveille, 1968, 1969; Goodridge & Ball, 1966, 1967) examined the rates of fatty acid synthesis in liver and adipose tissue. However, Yeh & Leveille (1972) found significant incorporation of [1-14C]acetate into fatty acids in the carcasses of chicks (the carcass represented the hepatectomized animal). Because 15 min elapsed between tracer injection and removal of the liver, the possibility of transfer of newly synthesized labelled fatty acids from the liver to the carcass could not be ruled out. Another possibility explaining the high rate of [1-14C]acetate incorporation into fatty acids in chick
carcasses might have been chain elongation of fatty acids by extra-hepatic tissues. Our experiments were designed to estimate the rate of fatty acid synthesis, utilizing 3H20 , in the liver and carcass of chickens. Tritiated water was chosen as it estimates the rate of fatty acid synthesis from all carbon precursors rather than synthesis from a specific carbon precursor. The use of tritiated water also minimizes the problem of isotope dilution which exists with glucose and acetate tracers (Goodridge, 1968). A second purpose of our experiments was to obtain an estimate of de novo fatty acid synthesis vs chain elongation in several tissues of chickens. MATERIALS A N D M E T H O D S
Chicks and diets. Male broiler chicks (Fairview Farms,
Remington, Indiana, USA) were used for all experiments except experiment 3 where both males and females were used. Chicks were housed in electrically heated brooders with raised wire floors. They were fed a commercial highcarbohydrate diet (Master Mix Chick Starter and Grower, Central Soya, Fort Wayne, Indiana, USA). Water and food were available at all times. Experiment 1. The time for equilibration of 3H20 in the blood of young chicks was estimated. Seven chicks (250-400 g) were injected in a wing vein with 2 mCi of 3H20 in 2 ml of saline. After puncture of an opposite wing vein with a sterile microlance, 25 pl samples of blood were collected at 5, 10, 20, 30, 45 and 60 min after 3H20 injection. The samples were diluted and counted in a Packard Tri-Carb 3 channel scintillation counter. Counting efficiency was determined. In another group of chickens, blood samples were obtained at shorter time intervals after an injection of 250 #Ci 3H20 in 1 ml saline into a wing vein. Blood samples were drawn from 1000-1500g chickens at 1 min intervals between 1 and 10 min from either a catheter in the jugular vein or from a wing vein opposite the site of injection. The blood samples were centrifuged and 10 ~tl of plasma counted for radioactivity. * Supported in part by Public Health Service Grants Experiment 2. The rate of 3H incorporation from 3H20 HL 14677 and GMO 1818. D.R.R. is the recipient of Career into fatty acids in chick liver slices was determined. Two Development Award K04 AM 00112. Michigan Agricul- 500 g chicks were killed by cervical dislocation. The livers ture Experiment Station Journal Article No. 7341. were rapidly removed and chilled in saline. Fifteen slices f Address correspondence to: Dale R. Romsos, Food 006-200 rag) from the liver of each chick were prepared Science & Human Nutr. Michigan State University, East with a Stadie-Riggs hand microtome. Three slices from each chick were incubated for intervals of 30, 60, 90, 120 Lansing, MI 48824, U.S.A. 4O3
404
LINDA BRADY, DALE R ROMSOS AND GILBERT A. LEVEILLE
or 180 min in a Krebs-Ringer bicarbonate buffer containing per ml: 50pCi 3H20, 3 mg glucose and 0.1 unit porcine insulin (Eli Lilly, Indianapolis, IN). Incubation procedures and methods for counting radioactive fatty acids have been described (Leveille, 1966). Experiment 3. The in vivo rate of fatty acid synthesis was determined in 400-500 g chicks. Ad lib. fed birds were injected intravenously with 2 mCi 3H20 in 2 ml saline. After injection, birds were returned to their pens. Fresh food and water were available. Blood samples were taken and chicks were killed by cervical dislocation 5, 10 or 60 min after 3H20 injection. The livers were rapidly excised and frozen in dry ice-acetone. The remaining carcass was packed on ice and stored at -20°C until analyzed. The livers were homogenized with an equal weight of water and 0.5 ml of the slurry saponified and counted as above. The carcasses were weighed and dried at 100°C and ground twice. Two homogeneous samples of I g were extracted in 40 ml chloroform methanol (3:2) at 50°C for 30 rain, then filtered into tared aluminum dishes, dried, weighed and saponified as with the liver homogenates. Experiments 4 and 5. In experiment 4, 200-300 g chicks were injected intravenously with 1.5 mCi 3H20 in 2 ml saline and killed 1, 3, 5 or 10 min later. Experiment 5 was identical to experiment 4 except that times of sampling were expanded to 1, 3, 5, I0, 20 and 30 min after injection. Experiment 6. The in vivo rate of fatty acid synthesis was also determined in male rats for comparison with the chicks. Sprague-Dawley rats (Spartan Research Animals, Haslett, MI, USA) weighing 80-90 g were fed a high-carbohydrate semisynthetic diet (Romsos & Leveille, 1972). Tritiated water (1 mCi in 0.2 ml saline) was injected intraperitoneally and animals were killed 10, 20, or 30 min later. Epididymal fat pads, liver, and carcass were prepared as with the chicks. Experiment 7. Either 2.5 mCi 3H20 in 0.5 ml saline or 5/~Ci [1-14C]acetate in 0.5ml saline was injected into 300-400 g birds and after 5 rain they were killed to obtain estimates of the rates of fatty acid synthesis in various organs. Experiment 8. The amount of chain elongation occurring in various tissues was estimated by determining the dis/rain in total fatty acids: dis/rain in the carboxyl carbon after decarboxylation. Chickens were injected with 20/~Ci of [l-14C]acetate and killed 5 min later. RESULTS
The results of the study of equilibration of tritium in the blood of the chick after an injection of 3H20 are presented in Table 1. No significant differences were found among the values over time. The tritium specific activity in the blood had reached a plateau before the initial sample was taken. In the second part of experiment 1, therefore, blood samples were obtained from 1 to 10 min after injection of 3HzO. Sampling was from either the vena cava or a wing Table 1--Exp. 1. Blood specific activity in chicks following tritiated water iniection* Time after Pulse (min.)
5
, 1.58 L "Oft
I0 20 30 45 60
dpm3H x lO'/~ml plasma
1.53 1.56 1.57 1.59 "
L ~ + +
.11 .09 .10 .09
1.60 L . I 0
Table 2--Exp. 1. Plasma specific activity in chicks following tritiated water injection* Time after pulse (min.
Site of Sampling wing vein
vena cava
I
1.46 L -15f
2
1.36 + .15
1.31 + .Ig
3
1.34 L .06
1.34 + .16
4
1.34 L .11
1.31 + .16
5
1.32 L .10
1.21 + .15
6
1.32 + .13
I.ii
7
1.22 + .18
1.30 L .16
8
1.23 ~ .OS
1.19 ~ .12
9
1.29 ? .06 1.34 ~ .06
1.22 ~ .09 1.18 ~ .10
10
1.29 L .29
L .08
*250 ~Ci 3H20 injected into 1000 g chicks, +Mean~++SEM f~r 3-4 chicks. Results expressed as dpm ~Hx 10- /ml plasma.
vein opposite the site of injection (Table 2). No significant differences were found between sites at each time tested, nor over time for each site. Thus, the blood plasma 3H20 had equilibrated by 1 min in these chicks and rapid homogeneous mixing was obtained. An in vitro experiment was conducted, utilizing 3 H 2 0 , to determine the rate of fatty acid synthesis in chick liver (Table 3). The in vitro incorporation of 3H from 3H20 into fatty acids of chick liver slices appeared to be linear from 30 to 120 min, but leveled off between 120 and 180 min of incubation. Several experiments were conducted to determine the in vivo rate of fatty acid synthesis in the chick. The results of experiment 3 are presented in Table 4. The rate of 3H incorporation from 3H20 into fatty acids in chick liver was most rapid initially. Between 5 and 10 min and between 10 and 60 min, the rates of incorporation slowed and, at 60 min incorporation was not as great as would be predicted if synthesis were linear with time. This is contrary to results obtained with rats (Sullivan et al., 1974; Lowenstein, 1971). Fatty acid synthesis measured by 3H incorporation was linear for 60-90 min in the rat. The concentration of labelled fatty acids in chick liver was 23-91 times greater than observed in the carcass, but the carcass represented a greater mass. Consequently, the incorporation of 3H into fatty acids in the carcass was significant at each time. Tritium incorporation into fatty acids did not increase linearly with time in the carcass either. Because the in vivo rate of fatty acid synthesis in the chick did not appear linear with time after a 3 H 2 0 injection, another experiment was conducted. Table 3--Exp. 2. In vitro incorporation of 3H into fatty acids by chick liver slices* Incubation time (min.)
Fatty acid synthesis
30 60 90
5,501 + 684 ~ 13,134 T 548 23,536 V 2704
120 180
4"
36,784 + 2592 33,978 ~ 3102
*Two mCi 3H20 injected into 300-400 g chicks.
*Dpm tritium from 3HpO incorporated into fatty acids per gram Iive~.
fMean ~ SEM for 6 chicks.
÷Mean + SEM for 15 liver slices,
Tritiated water and fatty acid synthesis Table 4---Exp. 3. Tritium incorporation into chick liver and carcass fatty acids*t Minutes after injection
5 I0 60
SO00
Fatty acid synthesis per gram Liver
Carcass
1159 + 153t 1899 + 561 6247 ¥ 794
51 + I0 41 ¥ 5 69 T 12
Fatty acid synthesis per total Liver 5 I0 60
405
Carcass
18,503 + 2101 26,791+ 5305 28,207 ¥ 8604 20,824¥ 2585 96,488 ~ 13,325 35,068~ 6378
~ 200( E
t~ "o
~:~ L~ IOOC
*Two mCi 3H20 injected into 400-600 g chicks. fResults expressed as dpm 3H incorporated into fatty acids.
Table 5--Exp. 4. Total 3H incorporation into fatty acids in liver and carcass of chieks*t
iO
I
I
8
I0
rain
Fig. 1. Tritium incorporation into fatty acids/g liver and carcass of the chick, - - carcass, ---O-- liver. Each point represents the mean of eight 200-300 g chicks. cass 3H incorporation into fatty acids was lower than either liver or adipose tissue, and as with the chick did not increase significantly over time. The rates of incorporation of 3H from 3H20 and of [lr14C]acetate into various tissues of the chick were determined in experiment 7. With 3H, the liver, as expected, exhibited from 5 to 14 times more incorporation than other tissues (Table 6). In the same size birds, the relative incorporation of [1-14C]acetate was different than for 3H from 3H20. Liver 14C incorporation into fatty acids was from 11 to 48 times the kidney and muscle respectively. The kidney, intestine, and adipose tissue all exhibited approximately the same amount of incorporation/g. The differences in 3H and 1'~C incorporation might have been due to the changing specific activity of acetate during the experiment. Because the apparent incorporation of 3H from 3H20 into carcass fatty acids was significant in all experiments, an attempt was made to differentiate
7000
~ 5000
E
~
/
t
T
3000 +/,~
7,894 L 11,954 L 17,208 + 37,499 +
Carcass 10791: 36,333L 2860 82,672~ 2874 143,280~ 4279 116,584~
2768 5989 15,191 12,191
"1.5 mCi 3H20 injected into 200-300 g chicks. +Results expressed as total dpm 3H incorporated into fatty acids. ~MeanL SEMfor 8 chicks.
I
6
4
Fatty Acid Synthesis Liver
I 3 5
I
2
Time,
In this experiment (experiment 4) chicks were killed 1, 3, 5 or 10 min after injection of 3H20. In the liver the most rapid 3H incorporation into fatty acids occurred initially, although 3H incorporation into fatty acids increased for the 10 min of the experiment (Table 5 and Fig. 1). Carcass 3H incorporation from 3H20 to fatty acids rose initially, then remained constant between 5 and 10 min, much as in experiment 3 where carcass values did not increase significantly from 5 to 60 min. Again the carcass appeared to contribute a sizeable proportion of labelled fatty acids. Experiment 5 was identical to experiment 4, except that times of 20 and 30 min were added. The liver incorporation of 3H into fatty acids again showed the greatest rate initially (Fig. 2). Although 3H incorporation increased in the liver throughout the experiment, the rates were not linear. The carcass 3H incorporation into fatty acids (not shown) remained relatively constant. Studies of 3H incorporation into fatty acids were also done with rats to compare the two species and to determine if rates were linear in rat liver, carcass and adipose according to our methods. As mentioned, previous reports have found hepatic fatty acid synthesis in the rat to be linear for up to 90 min. Our results (Fig. 3) showed essentially the same results over 30 min in adipose tissue of the rat. A tendency for slower rates of fatty acid synthesis after 10 min was observed in rat liver; however, the effect was not as marked as in the chick. Per g, liver and adipose 3H incorporation into fatty acids were relatively close, with liver values slightly higher at each time. The car-
Minutes after injection
i
0
~'Mean + SEMfor eight chicks.
I000 l '~'
t
I
6
I
12 Time,
I
18
I
24
I
:50
rain
Fig. 2. Experiment 5. Tritium incorporation into fatty acids in chick liver. Each point represents the mean + S.E.M. for eight 200-300 g chicks.
406
LINDA BRADY, DALE R. ROMSOS AND GILBERT A. LEVEILLE
I0000
ation of 14C into fatty acids in the muscle remained constant. Jungas (1968) has shown that de novo fatty acid synthesis as measured by 3H incorporation from 3H20 in rat adipose tissue dropped in the fasted state. Adipose tissue is a major site of fatty acid synthesis in the rat.
j/
8000
// // / / / / /"I1/" j
6000
n 4000
_
DISCUSSION
ii/I/
200(~
I 6
12 Time,
I
I
I
18 min
24
30
Fig. 3. Experiment 6. Tritium incorporation into rat liver, adipose, and carcass of the rat. -. . . . . liver, - - adipose, . . . . . . . . carcass. between de novo fatty acid synthesis and chain elongation in carcass tissues in vivo. Theoretically, in vitro, the ratio of total fatty acid dis per min:carboxyl carbon dis/min should be 8 or 9. A ratio of 1 or 2 would indicate chain elongation in vitro. Thus, de novo synthesis would result in a higher ratio than chain elongation by a factor of 4-8. Tables 7 and 8 present the results obtained in vivo. Ratios of total fatty acid dis/min:dis/min in COOH in the liver of fed chicks was slightly higher than typical results obtained in vitro (Goodridge, 1970; Joshi & Sidbury, 1975), a fact which may be related to declining specific activity of acetate with time after injection in vivo. Based on the ratios obtained, chain elongation primarily occurs in kidney, skin, intestine, muscle, heart and adipose tissue. Microsomal or mitochondrial chain elongation would incorporate tritium atoms also via NADH and NADPH. Table 8 shows the ratios of total dis/min in fatty acids:dis/min in COOH in liver and muscle of both fed and fasted birds. The mean ratio in the muscle did not change with state of nutrition, but in liver the ratio decreased by almost 75~o, which indicates a drop in de novo synthesis with fasting. Incorporation of 14C was much greater for liver than muscle in both the fed and the fasted states, but I4C incorporation into fatty acids in livers from fasted chicks dropped to 16~o of the fed state while incorporTable 6--Exp. 7. 3H and 14C incorporation into fatty acids in various chick organs* T-issue
3H-dpm/gwet weight
Liver Skin Adipose Intestine Heart Muscle Kidney
4713 ~ 1108@ 352 + 97 510 ~ 30 951 ~ 68 534 + 84 710 L 201 921 ~ 182
An assumption of in vivo tracer experiments is that the tracer that is injected rapidly mixes with the plasma pool, and subsequently with other body pools. Theoretically, tracer specific activity should be constant in the body pools during the time its incorporation into other compounds is being measured. In practice, the time of plasma equilibration is often not as rapid as theory demands. Katz et al. (1974) found plasma 3HzO equilibration in the rabbit to occur from 25 to 30 min after injection. In the chick, however, the assumption of a rapid mixing of 3H20 with the body water pool appears to be met. In our earliest reported sampling after injection (1 min) it was clear that plasma specific activity had equilibrated. This appears reasonable in view of the fact that the chick's frequency of heartbeat is 350~50/min and cardiac output 218 ml/min per kg (Akester, 1971). The chick thus appears to meet one of the requirements for in vivo tracer experiments. However, we made one further assumption: that the aH20 in the plasma rapidly equilibrated with the immediate precursors of the fatty acids. This assumption was not tested in our experiments. The rapidity of plasma 3H20 equilibration was also reflected in the appearance of 3H in the fatty acids. Based on earlier studies with rats (Sullivan et al., 1974; Lowenstein, 1971), it was expected that a linear rate of 3H incorporation into fatty acids would exist for up to an hour. However, in all the studies reported here, the initial rate of 3H incorporation into fatty acids in both liver and carcass of the birds was the greatest rate observed. This fact has significance because if rates of synthesis are to be compared among treatments, they should be compared during linear rates of synthesis. In this case, waiting beyond 5 min does not appear advisable in the young chick as 3H incorporation is not always linear beyond this point. Almost all experimental evidence has indicated that the liver is the main site of fatty acid synthesis in the chick and our experiments appear to support the fact that most of the de novo fatty acid synthesis Table 7--Exp. 8. Ratio of total fatty acid DPM/carboxyl carbon DPM in fed chicks*
14C-dpm/gwet weight Tissue 81,105 L 3,320 L 6,621 ~ 5,142 ~ 3,152 L 1,688 ~ 7,060 L
37,684 1,636 3,700 2,232 1,238 471 2,916
*5 minutes after injection of 2.5 mCi 3H20or 5 uCi 14C acetate into 300-400 g chicks. fMean ~ SEMfor 5 chicks.
Liver Kidney Skin Intestine Muscle Heart Adipose
Total dpm/COOHdpm 15 2 1 1 i 1 I
*20 uCi acetate-1-14C in 2 m] saline injected into 300-400 g chicks.
Tritiated water and fatty acid synthesis Table 8--Exp. 8. Comparison of liver and muscle chain elongation in fed and fasted chicks* Fed
Fasted
dpm 14C in FA FA dpm/COOHdpm ~ q tissue g tlssue Liver Muscle
FA dpm/COOHdpm
96,281
15
15,178
4
1,924
1
1,811
I
*Eight 300-400 ~4fed chicks and 8 chicks fasted 24 hr. with 20 uCi I- C-acetate and 2 ml saline.
407
liver and carcass. The rate of incorporation was always greater initially than at later time intervals. The liver appeared to synthesize fatty acids de novo while the carcass appeared to elongate fatty acids, both processes incorporating tritium into the fatty acid molecule. The content of labelled fatty acids in the carcass did not increase with time, suggesting that fatty acid oxidation was occurring simultaneously in some carcass tissues.
Chicks injected
occurs in chick liver. However, tritium from 3H20 may also be incorporated into fatty acids undergoing chain elongation in the carcass. We consistently found the presence of tritium in carcass fatty acids at very short times after injection. This does not rule out transfer of fatty acids from the liver to the carcass, but the rapidity and magnitude of tritium appearance in the carcass fatty acids suggests significant de novo synthesis or chain elongation in some carcass tissues. Two other studies (Yeh & Leveille, 1972, 1973) have also found significant apparent carcass synthesis of fatty acids in vivo, although glucose and acetate were used as tracers. Another observation of the tritium incorporation into carcass tissues was the lack of accumulation of labelled fatty acids in the carcass with time. This suggests rapid oxidation in some carcass tissues simultaneously with de novo synthesis or chain elongation. Muscle represents a great mass of the body and has been shown to utilize fatty acids as an energy source (Owen & Reichard, 1971; Newsholme & Start, 1973). The incorporation of tritium into fatty acids in rat liver, adipose and carcass differed from that observed in the chick. The rat adipose tissue showed linear rates of synthesis from 10 to 30 min. The liver had a tendency for most rapid incorporation to occur initially, but the rate differences with time were not as marked as in the chick. Some of the tritium of the carcass and adipose tissue fatty acids could have come from the liver and might explain a tendency for rates of tritium incorporation into fatty acids in the liver to slow slightly. However, carcass values did not increase with time, again suggesting rapid utilization of fatty acids by the carcass. With tritiated water, chain elongation cannot be differentiated from de novo fatty acid synthesis, and the tritium incorporation into carcass fatty acids could be substantially through chain elongation. Using [l-14C]acetate we found that chick muscle, intestine, adipose, heart, and skin appeared to synthesize fatty acids via chain elongation. Joshi & Sidbury (1975) found that in vitro, the ability of the heart mitochondrial chain elongation system increased with age in chicks. Their results indicated that the major type of label incorporation in in vitro embryo and adult heart was by mitochondrial chain elongation, while that of the liver was by de novo synthesis. Donaldson & Mueller (1971) found some microsomal and mitochondrial chain elongation capability in vitro in liver, but found primarily cytosolic synthesis in carbohydrate-fed chicks. In conclusion, our results indicate that 3 H 2 0 rapidly equilibrated with the body water and was rapidly incorporated into fatty acids in both chick
REFERENCES
AKES~R A. R. (1971) The Heart. In Physiology and Biochemistry of the Domestic Fowl (Edited by BELL D. J. & FREEMANB. M.), p. 746, 777. DONALDSON W. E. & MUELLER N. S. (1971) Synthesis of unsaturated fatty acids in chick embryo liver. Can. J. Biochem. 49, 563-567. GOODRIDOE A. G. (1968) Conversion of fU-~4C]glucose into CO2, glycogen, cholesterol and fatty acids in liver slices from embryonic and growing chicks. Biochem. J. 108, 655-661. GOODRIDGEA. G. (1970) Regulation of lipogenesis: stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick. Biochem. J. 118, 259-263. GOODRIDGEA. G. & BALL E. G. (1966) Lipogenesis in the pigeon: in vitro studies. Am. J. Physiol. 211, 803-808. GOODRIDGEA. G. & BALL E. G. (1967) Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. 213, 245-249. JOSHI V. C. & SIDBURY J. B. (1975) Fatty acid synthesis in chick embryonic heart and liver during development. Devl Biol. 42, 282-291. JUNGASR. L. (1968) Fatty acid synthesis in adipose tissue incubated in tritiated water. Biochemistry 7, 3708-3717. KATZ J., DUNN A. & CHENOWETHM. (1974) Determination of synthesis, recycling, and body mass of glucose in rats and rabbits with 3H and 14C-labelled glucose. Biochem. J. 142, 171-183. LEVEILLE G. A. (1966) Glycogen metabolism in meal fed rats and chicks and the time sequence of lipogenic and enzymatic adaptive changes. J. Nutr. 90, 449-460. LOWENSTEINJ. M. (1971) Effect of hydroxycitrate on fatty acid synthesis by rat liver in vivo. J. biol. Chem. 246, 629-632. NEWSHOLME E. A. & START C. (1973) In Regulation in Metabolism, p. 213 Wiley, New York. O'HEA E. K. & LEVEILLEG. A. (1968) Lipogenesis in isolated adipose tissue of the domestic chick (Gallus Domesticus). Comp. Biochem. Physiol. 26, 111-120. O'HEA E. K. & LEVEILLEG. A. (1969) Lipid biosynthesis and transport in the domestic chick. Comp. Biochem. Physiol. 30, 149-159. OWEN O. E. & RE1CHARO G. A. (1971) Fuels consumed by man: the interplay between carbohydrate and fatty acids. In Biochemistry and Pharmacology of Free Fatty Acids (Edited by HOLMES W. L., BORTZ W. M. & KARGER S.), pp. 199-200. Basel, Switzerland. ROMSOS D. R. & LEVEmLE G. A. (1972) Cellularity and in vitro fatty acid biosynthesis in adipose tissue of meal fed and nibbling rats. Proc. Soc. exp. Biol. Med. 139, 868-871. SULLIVANA. C., TRISCARI J., HAMILTON J. G., MILLER O. N. ~,~ WHEATLEY V. R.
(1974) Effect
of
hydroxycitrate
upon the accumulation of lipid in the rat--I. Lipogenesis Lipids 9, 121-128. YEH S. C. & LEVEILLEG. A. (1972) Cholesterol and fatty acid synthesis in chicks fed different levels of protein. J. Nutr. 102, 349-358. YEn S. C. & LEVE1LLEG. A. (1973) Significance of skin as a site of fatty acid and cholesterol synthesis in the chick. Proc. Soc. exp. Biol. Med. 142, 115-119.