Fat pad triacylglycerol fatty acid loss and oxidation as indices of total body triacylglycerol fatty acid mobilization and oxidation in starving mice

We tested our hypothesis that, kinetically, triacylglycerol fatty acids in heterogeneously labeled adipocytes behave similarly to the whole fat pad triacylglycerol fatty acid during starvation in mice. Adipose triacylglycerol fatty acids were labeled with [1-14C]palmitate (complexed to albumin) by injection of a small bolus (2-5 microliter) into either epididymal or inguinal fat pads. Both 14C-labeled triacylglycerol fatty acid spec. act. and breath 14CO2 spec. act. were monitored 30 min after tracer injection and after 24-72 h starvation. Adipose triacylglycerol fatty acid spec. act. remained approximately constant during fasting, i.e., tracer and mass disappeared at similar rates. Negligible translocation of labeled triacylglycerol fatty acid from the injection site to other parts of the same fat pad or to distant fat pads occurred. Triacylglycerol fatty acid was mobilized more slowly from epididymal than from inguinal fat pads in two of three studies. Triacylglycerol fatty acid disappearance (loss) from inguinal fat pads was more replicable than from epididymal fat pads and more closely reflected the fall in whole body total lipid during starvation. The estimated percent of breath CO2-carbon derived from adipose triacylglycerol fatty acid increased from an average of approx. 32% in the postabsorptive state to about 77% after 48 h starvation. The data help to validate the direct tracer injection technique as a means of studying adipose triacylglycerol fatty acid turnover and oxidation. This approach should be particularly useful for studying the fate of adipose triacylglycerol fatty acid when it is mobilized. e.g., during states of inanition and starvation and in response to hormones and cancer-induced cachexia.     

Use of the doubly labeled water method for measurement of energy expenditure, total body water, water intake, and metabolizable energy intake in humans and small animals

The basis of the doubly labeled water method is measurement of the differential rates of disappearance of two isotopes of water (H2 18O and either 2H2O or 3H2O, administered at the start of the study) from body water. Published studies indicate that, in its current forms, this technique can be used to provide accurate and reasonably precise information on carbon dioxide production, total body water, and water intake in free-living humans and many small animals. Total energy expenditure can be calculated from carbon dioxide production with little loss of precision. Metabolizable energy intake can also be predicted, as the sum of total energy expenditure plus an estimate for the change in body energy stores during the measurement, but this prediction is unlikely to be accurate and precise unless the subject is in approximate energy balance.     

Gas chromatography-mass spectrometry assay of the (18)o enrichment of water as trimethyl phosphate

We have developed an assay for determining the 18O enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the 2H enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our 18O method and the 2H method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water (2H(2)O + H(2)18O), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the 18O enrichment of body water can be monitored down to 0.025%.     

Influence of diet on the modeling of adipose tissue triglycerides during growth

We have studied the accretion of lipids in growing mice. We measured the rates of synthesis and degradation of triglycerides in epididymal fat pads of mice maintained for 44 days on a low-fat, high-carbohydrate diet (I) or a high-fat, low-carbohydrate diet (II). 2H2O was added to the drinking water for 14 days. Rates of incorporation/washout of 2H to/from C1 of triglyceride-glycerol showed that triglyceride synthesis was greater than triglyceride degradation (net triglyceride balance was approximately 2.5 times greater in II than in I). The data also show that the contribution of de novo lipogenesis to triglyceride-bound palmitate was approximately 3 times greater in I than in II. This was consistent with a greater relative intake of carbohydrate in I vs. II. The rates of incorporation and washout of newly synthesized (2H-labeled) palmitate into and from triglycerides were also measured. Those data suggested a remodeling of triglyceride-bound fatty acids. On measuring the profile of triglyceride-bound fatty acids, we observed a decrease in the relative abundance of triglyceride-bound palmitate and stearate and an increase in triglyceride-bound oleate and linoleate. This was observed in I and II. In summary, diet substantially affects the deposition and modeling of triglycerides in adipose tissue during growth. 2H2O can be used to examine the mechanisms responsible for the accumulation of triglycerides, e.g., factors that affect 1) triglyceride synthesis and degradation and 2) the source of fatty acids that are used in esterification.     

A comparison of extrahepatic lipogenesis from a small glucose meal in obob and gold thioglucose obese mice fed low- or high-fat diets with or without the addition of Delta22-5beta-taurocholenic acid

Extrahepatic fatty acid synthesis from a 250 mg meal of [U-(14)C]-glucose was measured in epidymal fat pads and the remaining carcass of hyperglycemic obese (obob), gold thioglucose obese, and nonobese controls under conditions of maximum and minimum lipogenesis. Also assessed was the effect of Delta(22)-5beta-taurocholenic acid, previously shown to inhibit hepatic fatty acid synthesis. Both types of obese and nonobese mice were fed for 6 weeks glucose-based diets containing either 1% corn oil or 40% lard with or without the addition of 0.05% taurocholenic acid. In mice fed 1% corn oil, incorporation of labeled glucose into carcass fatty acids was 25% greater in nonobese than obese mice of either type of obesity. On this diet incorporation of labeled glucose into epididymal fatty acids was reduced 83% in hyperglycemic obese mice compared with nonobese littermates. The corresponding reduction in lipogenesis in gold thioglucose obese mice was only 23% compared with nonobese controls. Feeding 40% lard reduced incorporation of labeled glucose into epididymal and carcass fatty acid 67 to 95% compared with mice fed 1% corn oil in both types of obese and nonobese mice whether or not taurocholenic acid had been fed. Both types of obesity or feeding 40% lard reduced lipogenesis in fat pads to a greater extent than glucose uptake by the pads with the reductions additive. Feeding taurocholenic acid reduced pad weight 30% across strain and obesity status, increased uptake of labeled glucose into epididymal fat pads and increased the percentage of the labeled glucose in the pad recovered as fatty acid in both types of obese and nonobese mice when the diet was 1% corn oil. Similarities and differences between the two obesity models are discussed.     

Compensation for partial lipectomy in mice with genetic alterations of leptin and its receptor subtypes

One hypothesis for the regulation of total body fat suggests that leptin is a lipostatic feedback signal that acts at brain sites involved in regulation of energy balance. The importance of leptin in recovery from partial surgical lipectomy was tested by performing bilateral epididymal lipectomy or sham surgery on wild-type and leptin-deficient ob/ob mice. Eight weeks later, nonexcised pads of lipectomized mice were increased but total carcass fat was lower than in sham-operated ob/ob mice. In experiment 2, ob/ob mice, wild-type mice, and two db/db mutants, C57BL/6J db(Lepr)/db(Lepr) (BL/6J) mice possessing short-form and circulating leptin receptors and C57BL/6J db(3J)/db(3J) (BL/3J) mice expressing only circulating receptors, were lipectomized or sham operated. Sixteen weeks later, body mass and carcass lipid were not different between sham and lipectomized ob/ob mice, wild-type mice, or BL/6J db/db mice, whereas there was incomplete (decreased carcass fat) but suggestive recovery (increased retroperitoneal fat mass and cell number) in lipectomized BL/3J db/db mice. These data indicate that leptin is not required for the regulation of total body fat.     

Doubly labeled water measurement of human energy expenditure during strenuous exercise

The energy expenditures (EE) of 23 adult male Marines were measured during a strenuous 11-day cold-weather field exercise at 2,200- to 2,550-m elevation by both doubly labeled water (2H2 18O, DLW) and intake balance methods. The DLW EE calculations were corrected for changes in baseline isotopic abundances in a control group that did not receive 2H2 18O. Intake balance EE was estimated from the change in body energy stores and food intake. Body energy-store changes were calculated from anthropometric [-1,574 +/- 144 (SE) kcal/day] and isotope dilution (-1,872 +/- 293 kcal/day) measurements made before and after the field exercise. The subjects kept daily logbook records of ration consumption (3,132 +/- 165 kcal/day). Mean DLW EE (4,919 +/- 190 kcal/day) did not differ significantly from intake balance EE estimated from food intake and either anthropometric (4,705 +/- 181 kcal/day) or isotope dilution (5,004 +/- 240 kcal/day) estimates of the change in body energy stores. The DLW method can be used with at least the same degree of confidence as the intake balance method to measure the EE of active free-living humans.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X. Effect of glucose transport stimuli on the efflux of isotopically labelled calcium and 3-O-methylglucose from soleus muscles and epididymal fat pads of the rat

(1) The relationship between Ca2+ and sugar transport has been studied by comparing the washout of 45Ca and 3-O-[14C]methylglucose from preloaded isolated rat soleus muscles and whole epididymal fat pads. (2) In soleus muscle, nine different agents with well established stimulating effects on glucose transport were all found to produce a marked increase in 3-O-[14C]methylglucose washout, which in each instance was preceded by or coincided with a rise in the washout of 45Ca. (3) Trypsin, 2,4-dinitrophenol, p-chloromercuriphenylsulfonic acid, H2O2 and hyperosmolarity all produced dose-dependent stimulation of the washout of 45Ca and 3-O-[3H]methylglucose. Regression analysis showed a highly significant correlation between the increases in the two parameters (P < 0.001). (4) Depolarization and Na+ influx induced by veratrine were found to be associated with a marked rise in 45Ca release followed by stimulation of 3-O-[14C]methylglucose washout. (5) In epididymal fat pads, six different agents known to stimulate glucose transport were found to produce a highly significant (P < 0.001) increase in the washout of 45Ca and 3-O-[14C]methylglucose. (6) It is concluded that in the major targets for insulin action, activation of the glucose transport system can be elicited by a rise in cytoplasmic Ca2+ concentration brought about by mobilization of Ca2+ from endogenous cellular pools.     

Measurement of energy expenditure in humans by doubly labeled water method

The utility of the doubly labeled water method for the determination of energy expenditure and water output was investigated in humans. Approximately 10 g of 18O and 0.5 g of 2H as water was orally administered to four healthy adults. Total body water was determined from the isotope dilution, and the ensuing 18O and 2H disappearance rates from body water were determined for 13 days by mass spectrometric isotope ratio analysis of the urinary water. During this period, subjects were maintained on a measured diet to determine energy and water intake. The energy expenditure from the doubly labeled water method differed from dietary intake plus change in body composition by an average of 2%, with a coefficient of variation of 6%. The water outputs determined by the two methods differed by 1%, with a coefficient of variation of 7%. The doubly labeled water method is noninvasive, and the subjects could maintain their daily activities without restriction.     

The CB1 Receptor Antagonist SR141716A Reverses Adult Male Mice Overweight and Metabolic Alterations Induced by Early Stress

Perinatal stress may cause metabolic and hormonal disruptions during adulthood. The aim of this study was to evaluate the effects of early postnatal nociceptive stimulation (NS) on body weight and other metabolic parameters during adulthood and to determine whether CB₁ endocannabinoid receptors (CB₁Rs) may be involved in these effects. Male mice were subjected to NS during lactation with a daily subcutaneous injection of saline solution. Subsequently, both control and NS‐mice were treated from day 40 to 130, with an oral dose (1 µg/g body weight) of SR141716A, a specific CB₁R antagonist/inverse agonist. Mice body weight and food intake was periodically evaluated. Adult animals were then killed to evaluate epididymal fat pads and metabolic parameters. NS did not influence food intake in adult animals, but caused significant increases in body weight, epididymal fat pads, and circulating levels of leptin, corticosterone, and triglycerides (TGs). Chronic treatment with SR141716A normalized these parameters, with the exception of corticosterone levels. This treatment also reduced plasma levels of glucose, insulin, and total cholesterol in both adult control and NS‐mice. In addition, fatty acid (FA) amide hydrolase (FAAH) activity (the enzyme able to hydrolyze endocannabinoids) from liver and epididymal fat of adult NS‐mice was decreased by 40–50% in comparison to activities found in same tissues of control mice. Results suggest that overactive liver and epididymal fat CB₁R due to early NS may be involved in late metabolic alterations, which are sensitive to chronic treatment with SR141716A.     

Sympathetic denervation does not prevent a reduction in fat pad size of rats or mice treated with peripherally administered leptin

Leptin increases sympathetic nervous system (SNS) activity in brown adipose tissue and renal nerves. Experiments described here tested whether SNS innervation is required for peripheral, physiological concentrations of leptin to reduce body fat. In experiment 1, one epididymal (EPI) fat pad was sympathectomized by local injection of 6-hydroxydopamine (6OHDA) in C57BL/6 mice that were then infused for 13 days with PBS or 10 microg leptin/day from an intraperitoneal miniosmotic pump. Surprisingly, EPI denervation increased total body fat of PBS-infused mice but leptin decreased the size of both injected and noninjected EPI pads in 6OHDA mice. Experiment 2 was identical except for the use of male Sprague-Dawley rats that were infused with 50 microg leptin/day. Leptin had little effect on EPI weight or norepinephrine (NE) content, but denervation of one EPI pad decreased the effect of leptin on intact EPI, inguinal and retroperitoneal (RP) fat and increased the size of the mesenteric fat pad. Experiment 3 included groups in which either one EPI or one RP pad was denervated. RP denervation reduced RP NE content but did not prevent a leptin-induced reduction in fat pad mass. Therefore, the SNS is not required for low doses of leptin to reduce body fat. EPI denervation significantly increased adipocyte number in contralateral EPI and RP fat pads and this was prevented by leptin. These changes in intact pads of rats with one denervated fat pad imply communication between fat depots and suggest that both leptin and the SNS regulate the size of individual depots.     

Exogenous hypercortisolism and epididymal fat cell count in young rats.

The influence of two different grades of exogenous hypercortisolism on body weight, epididymal fat pad weight and the total number of fat cells in epididymal fat pads was investigated in young, growing rats. Cortisol, 4-5 mg/kg/day orally from the 7th to the 9th week, reduced body weight gain, whereas epididymal fat pad weight and fat cell content did not differ from those of the control rats. Cortisone acetate, 2.5 mg per 100 g, given subcutaneously for 2 weeks to rats 4-11 week of age caused in the young rat (4-6 weeks) a partial inhibition of the normal increase in body weight, whereas in the young-adult rat (6 weeks and older) an actual decrease of body weight was seen. At both dose levels and - with respect to the higher dose level- in all age groups studied, the weight and fat cell content of the epididymal fat pad were not changed by the cortisone (cortisol) treatment.     

白藜芦醇对成年期追赶生长大鼠体成分的影响及机制

目的:观察白藜芦醇对成年期追赶生长大鼠体成分的影响及可能机制。方法:8周龄雄性 SD 大鼠分为6组(共2个时间点),即4周时间点3组:正常饮食4周 (NC4)组、热卡限制4周(R4)组,热卡限制同时白藜芦醇治疗(R4E)组;12周时间点3组:正常饮食12周(NC12)组,追赶生长(CUG)组,追赶生长白藜芦醇治疗(CUGE)组。每组含6只大鼠,白藜芦醇用生理盐水配制成一定浓度悬浊液,按100 mg/(kg·d)剂量予实验动物灌胃治疗。实验第4周、12周检测体重、躯干和全身的肌肉及脂肪含量、躯干与全身脂肪比例,实验第12周检测骨骼肌与附睾脂肪组织SIRT1的表达,附睾脂肪组织PPARγ的表达。结果:与NC12组相比,CUG组躯干及全身的脂肪含量、躯干与全身脂肪比例、附睾脂肪组织PPARγ的表达均明显升高(P＜0.05),肌肉含量、骨骼肌与附睾脂肪组织SIRT1的表达显著降低(P＜0.05或P＜0.01);与CUG组相比,经白藜芦醇干预后的CUGE组全身的脂肪含量、躯干与全身脂肪比例、附睾脂肪组织PPARγ的表达均明显降低(P＜0.05),肌肉含量、骨骼肌与附睾脂肪组织SIRT1的表达较CUG组显著提高(P＜0.05)。结论:白藜芦醇降低成年期追赶生长大鼠体脂含量,增加肌肉含量,改善腹部脂肪堆积,其机制可能与增加骨骼肌及内脏脂肪组织SIRT1表达,抑制内脏脂肪PPARγ的表达有关。     

The expression, activity and localisation of the secretory pathway Ca2+ -ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

Field use of D2 18O to measure energy expenditure of soldiers at different energy intakes

To test the application of doubly labeled water under adverse field conditions, energy expenditures of 16 special operations soldiers were measured during a 28-day field training exercise. Subjects were matched by fat-free mass and divided equally between an ad libitum ready-to-eat meal diet and a 2,000 kcal/day lightweight ration. Subjects recorded intakes daily, and body composition was measured before and after the exercise. At the beginning of the study, subjects moved to a new northerly location and, therefore, a new water supply. To compensate for this, a group of soldiers who did not receive heavy water was followed to measure isotopic base-line changes. Energy expenditure by doubly labeled water was in agreement with intake/balance (3,400 +/- 260 vs. 3,230 +/- 520 kcal/day). The overall coefficient of variation of energy expenditure by doubly labeled water was half that of intake/balance (7.6 vs. 16.1%). The coefficient of variation of repeat measures with doubly labeled water was 7.3%. Energy expenditure of the ready-to-eat meal group, 3,540 +/- 180 kcal/day, was not significantly different from the lightweight ration group, 3,330 +/- 301 kcal/day. Doubly labeled water was valid under field conditions.     

Studies on lipogenesis in vivo. Lipogenesis during extended periods of re-feeding after starvation

1. Lipogenesis was studied in mice re-fed for up to 21 days after starvation. At appropriate times [U-(14)]glucose was given by stomach tube and incorporation of (14)C into various lipid fractions measured. 2. In mice starved for 48hr. and then re-fed for 4 days with a diet containing 1% of corn oil, incorporation of (14)C from [U-(14)C]glucose into liver fatty acids and cholesterol was respectively threefold and eightfold higher than in controls fed ad libitum. The percentages by weight of fatty acids and cholesterol in the liver also increased and reached peaks after 7 days. Both the radioactivity and weights of the fractions returned to control values after 10-14 days' re-feeding. These changes could be diminished by re-feeding the mice with a diet containing 20% of corn oil. Incorporation of (14)C from [U-(14)C]glucose into extrahepatic fatty acids (excluding those of the epididymal fat pads) was not elevated during re-feeding with a diet containing either 1% or 20% of corn oil. However, incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads was increased in mice re-fed with either diet, as compared with non-starved controls. 3. Lipogenesis was also studied in mice alternately fed and starved. Mice given a diet containing 1% of corn oil for 6hr./day for 4 weeks lost weight initially and never attained the weight or carcass fat content of controls fed ad libitum. Incorporation of (14)C from dietary [U-(14)C]-glucose into the fatty acids of the epididymal fat pads was elevated threefold in the mice allowed limited access to food, although the incorporation into the remainder of the extrahepatic fatty acids was not different from that found for controls. Mice given a diet containing 20% of corn oil for 6hr./day adapted to the limited feeding regimen quicker and in 4 weeks did attain the weight and carcass fat content of controls. Incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads and the remainder of the extrahepatic fatty acids was respectively fivefold and threefold higher than in controls fed ad libitum. 4. The elevation in liver lipogenesis during re-feeding was greatest on a diet containing 1% of corn oil, whereas in extrahepatic tissues the increase in lipogenesis was greater when the mice were re-fed or were allowed limited access to a diet containing 20% of corn oil. These results suggest that the causes of the increased rate of incorporation of (14)C from [U-(14)C]glucose into fatty acids during re-feeding may be different in liver from that in extrahepatic tissues.     

Effect of bolus fluid intake on energy expenditure values as determined by the doubly labeled water method.

The doubly labeled water (DLW, 2H(2)18O) method is a highly accurate method for measuring energy expenditure (EE). A possible source of error is bolus fluid intake before body water sampling. If there is bolus fluid intake immediately before body water sampling, the saliva may reflect the ingested water disproportionately, because the ingested water may not have had time to mix fully with the body water pool. To ascertain the magnitude of this problem, EE was measured over a 5-day period by the DLW method. Six subjects were dosed with 2H2(18)O. After the reference salivas for the two-point determination were obtained, subjects drank water (700-1,000 ml), and serial saliva samples were collected for the next 3 h. Expressing the postbolus saliva enrichments as a percentage of the prebolus value, we found 1) a minimum in the saliva isotopic enrichments were reached at approximately 30 min with the minimum for 2H (95.48 +/- 0.43%) being significantly lower than the minimum for 18O (97.55 +/- 0.44, P less than 0.05) and 2) EE values calculated using the postbolus isotopic enrichments are appreciably higher (19.9 +/- 7.5%) than the prebolus reference values. In conclusion, it is not advisable to collect saliva samples for DLW measurements within approximately 1 h of bolus fluid intake.     

The expression, activity and localisation of the secretory pathway Ca-ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca²⁺-ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney.SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca²⁺ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca²⁺ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

Reliability in estimates of body composition of birds: oxygen-18 versus deuterium dilution

Body composition in birds was evaluated indirectly by 18O and 2H dilution. Body composition was determined by whole-body chemical analysis of eight adult roosters (Gallus gallus). In vivo measurements of total body water (TBW) were carried out using doubly labeled water (2H2 18O). Estimated dilution spaces using both the plateau and intercept approaches were compared with the results obtained by carcass lyophilization. Both 18O and 2H slightly overestimated TBW compared with the results obtained by lyophilization, by 2.2%+/-1.9% and 5.7%+/-0.2%, respectively; both differences were statistically significant (P<0.01). The difference between these isotope estimations was significant (P<0.001). However, isotope dilution spaces and TBW were highly correlated. There was a strong inverse relationship between total body fat and TBW percentages (r2=0.98, P<0.0001). The relation between TBW and body protein was significant. Water content in lean body mass (72.8%) obtained in our study was very close to that reported in mammals, demonstrating no fundamental difference in tissue water content between birds and mammals. Estimated body fat and protein values from isotopic dilution did not significantly differ from values obtained by direct chemical analysis (P>0.05), except for body fat in the Pace and Rathbun approach (Table 3). Although estimation of TBW and body composition by isotope dilution is time consuming and expensive, deuterium offers a reliable and low-cost alternative compared with 18O. The advantage of in vivo estimation of TBW with isotopic dilution in combination with the regression approach is that it permits repeated measurements of body composition on the same birds under laboratory and free-living conditions.