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The aim of this review is to summarize recent evidence on the importance of individual long chain polyunsaturated fatty acid (LCPUFA) to the developing fetus and the maternal dietary requirement for these.Large-scale randomized controlled trials and innovative genetic and stable isotope studies are providing new insights in this field.Large randomized controlled trials of LCPUFA supplementation in pregnancy suggest that higher n-3 LCPUFA intake reduces the risk of preterm birth and increases the length of gestation, with secondary effects on birth weight. There is little evidence of an effect on postnatal visual function and cognition, but interpretation is complicated by maternal metabolic adaptations and adipose tissue status in the newborn. The links between polymorphisms in the FADS genes and tissue fatty acid composition suggest that LCPUFA synthesis influences overall availability. Stable isotope studies have also demonstrated the capacity for LCPUFA synthesis in pregnancy, the fact that n-6 synthesis is greater than n-3, metabolic channeling of individual fatty acids to different fates, and selective placental transfer. Studies linking FADS genotype to cognition imply that n-3 LCPUFA synthesis could have an effect on infant cognition, but more large-scale genetic studies are needed.

Deposition of fat in the fetus increases exponentially with gestational age, reaching its maximal rate—around 7 g/day or 90% of energy deposition—at term. In late pregnancy, many women consuming contemporary Western diets may not be able to meet the fetal demand for n-3 long chain polyunsaturated fatty acids (LCPUFAs) from the diet alone. Numerous mechanisms have evolved to protect human offspring from extreme variation or deficiency in the maternal diet during pregnancy. Maternal adipose tissue is an important source of LCPUFA. Temporal changes in placental function are synchronized with maternal metabolic and physiological changes to ensure a continuous supply of n-3 and n-6 LCPUFA-enriched fat to the fetus. LCPUFA storage in fetal adipose tissue provides an important source of LCPUFA during the critical first months of postnatal life. An appreciation of these adaptations is important in any nutritional strategy designed to improve the availability of fatty acids to the fetus.

More than 90 per cent of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy during which it increases exponentially to reach a rate of accretion of around 7g/day close to term. All of the n -3 and n -6 fatty acid structure acquired by the fetus has to cross the placenta and fetal blood is enriched in long chain polyunsaturated fatty acids (LCPUFA) relative to the maternal supply. The placenta may regulate its own fatty acid substrate supply via the action of placental leptin on maternal adipose tissue. Fatty acids cross the microvillous and basal membranes by simple diffusion and via the action of membrane bound and cytosolic fatty acid binding proteins (FABPs). The direction and magnitude of fatty acid flux is mainly dictated by the relative abundance of available binding sites. The fatty acid mix delivered to the fetus is largely determined by the fatty acid composition of the maternal blood although the placenta is able to preferentially transfer the important PUFA to the fetus as a result of selective uptake by the syncytiotrophoblast, intracellular metabolic channelling of individual fatty acids, and selective export to the fetal circulation. Placental FABP polymorphisms may affect these processes. There is little evidence to suggest that placental delivery of fatty acids limits normal fetal growth although the importance of the in utero supply may be to support post-natal development as most of the LCPUFA accumulated by the fetus is stored in the adipose tissue for use in early post-natal life.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas from non-smokers (n5), smokers (>ten cigarettes/d;n5) and after addition of ethanol at 2 mg/ml (n4). The maternal side was of the placenta was perfusedex vivofor 90 min with a physiological mixture of fatty acids and fatty acid:human albumin ratio. There was no effect of smoking on the transfer of linoleic (LA, 18 : 2n-6), α-linolenic (αLN, 18 : 3n-3), arachidonic (AA, 20 : 4n-6) or docosahexaenoic acid (DHA, 22 : 6n-3), expressed per perfused area (calculated from H218O exchange). However, the presence of ethanol in the perfusate at a concentration of 2 mg/ml significantly reduced (Pn-3 polyunsaturated fatty acids, αLN and DHA. This specific effect of ethanol on αLN and DHA also resulted in an altered selectivity for transfer of individual fatty acids. In the non-smoking control group the placenta selectively transferred polyunsaturated fatty acids to the fetus in the order DHA>AA>αLN>LA. The order of selectivity was unaltered in placentas from smokers, but the addition of ethanol to the perfusion medium altered the order of selectivity to AA>αLN>LA>DHA. The presence of ethanol in the perfusate was also associated with a significant reduction (P218O. These results suggest that the presence of ethanol at a concentration of 2 mg/ml may reduce the availability of polyunsaturated fatty acids to the developing fetus.

The objective of this study was to determine whether the ability of the human placenta to transfer glucose and fatty acids is related to normal fetal growth. The intrinsic nutrient transport capacity of the placenta was measured under standardized conditions during in vitro perfusion of 30 human term placentas and related to birth weight (range 2640–4640g), birth weight centile (8th–99th), ponderal index (2.43–3.69), placental weight (418–1030g) and placental:fetal weight (0.14–0.31). There was no statistically significant change in the rate of nutrient transfer per placenta or per kg fetal weight, with birth weight, birth weight centile, ponderal index, placental weight and placental:fetal weight. There was a weak but significant relationship ( P =0.020, r 2 =9 per cent) between the ratio of glucose to fatty acid transport and birth weight centile, largely due to the high ratio found in the lowest birth weight quartile where the babies are thinnest. This study provides no evidence that placental nutrient transport capacity limits fetal growth across a wide range of birth weights in normal pregnancies. It is proposed that the fetus itself may regulate placental nutrient transport in vivo via the fetal cardiac output and the rate of fetal nutrient utilization.

The purpose of the present study was to estimate whole-body fatty acid and cholesterol synthesis in weight-stable adults and to determine the likely effect on the doubly-labelled water (DLW) method for measuring energy expenditure. Synthesis was measured by2H incorporation over 14 d in six adult males in approximate energy balance following noradrenaline infusion to maximize mobilization of free fatty acid from adipose tissue. The inter-individual variation in synthesis rates was large and in one subject the proportion of free fatty acid synthesized was ten times that of the mean of the rest of the group; the fasting concentration of esterified fatty acid in this subject was five times that of the rest of the group indicating likely violation of the assumptions underlying the calculation of whole-body synthesis. After 14 d of labelling in the other five subjects, 0·9 (SEM 0·3) % OF THE CIRCULATING FREE FATTY ACID, 9·3 (sem 3·0) % of the esterified fatty acid, 14·6 (sem 2·4) % of the free cholesterol and 28·3 (sem 3·7) % of esterified cholesterol had been synthesizedde novo. A high rate of synthesis correlated with a low pre-dose2H abundance both within and between lipid classes suggesting that natural2H abundance variations in some lipid classes may be used to determine their metabolic origin. Whole-body synthetic rates were 8 g/d for fatty acid and 0·3–0·5 g/d for cholesterol. These values correspond to very small errors on DLW-derived estimates of CO2production; -2·5 litres/d for fatty acid and -0·1 to -0·2 litres/d for cholesterol. These results, obtained in subjects typically consuming a diet with a lower fat and cholesterol content that the typical Western diet, suggest that the DLW method is unlikely to be affected by fatty acid and cholesterol synthesis in subjects in energy balance consuming a typical Western diet.

The aim of this study was to determine the effect of fatty acid intake and insulin dependent diabetes on the fatty acid composition of maternal erythrocytes, the placenta and cord. Fatty acid intake (from food frequency questionnaire) and the fatty acid composition of maternal erythrocytes, the placenta and cord from pregnant vegetarians (n = 4) and insulin dependent diabetics (n = 5) was compared with pregnant omnivores (n = 10). There was a significantly lower intake of n-6 long chain polyunsaturated fatty acid (LCPUFA) (-75% P < 0.01) and n-3 LCPUFA (-92% P < 0.01) and increased ratio of n-6/n-3 LCPUFA in the vegetarians (103%; P < 0.001). The concentrations of 22:4 n-6 (+28%; P < 0.05) and 22:5 n-3 (+40%; P < 0.05) were higher in vegetarian erythrocytes. Placental 18:2 n-6 (+26.9%; P < 0.05) 18:3 n-3 (+139%; P < 0.05) and 22:5 n-3 (+24%; P < 0.05) were increased while 20:5 n-3 (-36%; P < 0.05), 22:6 n-3 (-16%; P = 0.059), and the ratios of 20:4 n-6/18:2 n-6 (P < 0.01) and 22:6 n-3/18:3 n-3 were reduced. 22:6 n-6 (-49%; P < 0.05) and total n-3 LCPUFA (-11%; P < 0.01) were reduced in vegetarian cord. For the diabetic mothers, all of the n-6 LCPUFA and n-3 LCPUFA were reduced in the maternal erythrocytes; 22:4 n-6 (-42%; P < 0.05), 22:5 n-6 (-46%; P < 0.05) and 22:6 n-3 (-41%; P < 0.05). For the diabetic placenta and cord the general pattern of n-3 LCPUFA was the same as that in the vegetarians. In the vegetarian mothers, the PUFA profiles in the maternal erythrocytes, placenta and cord are consistent with an elevation in the rate of LCPUFA synthesis in order to make up the relative deficit in LCPUFA intake. However, it may be that the higher level of desaturase activity is not able to overcome the dietary deficit of 22-6 n-3 and 22:6 n-6. Despite the fact that the dietary LCPUFA intake in the pregnant diabetic was comparable with that in the pregnant 'normal' omnivore mothers, the pattern of PUFA in the tissues resembled that of the vegetarian mothers.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentae (n = 9) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids and ratio of fatty acid to human albumin. There was no evidence of chain elongation and desaturation of the essential fatty acids. Relative to the value for oleic acid, the rate of transfer to the fetal circulation was: 1.30 +/- 0.02 (P0.001) for linoleic acid, 1.61 +/- 0.09 (P = 0.002) for alpha-linolenic acid, 0.67 +/- 0.10 (P = 0.033) for arachidonic acid and 2.10 +/- 0.16 (P = 0.003) for docosahexaenoic acid. For tissue accumulation the values were 1.47 +/- 0.39 (P0.001) for linoleic acid, 2.24 +/- 0.37 (P = 0.027) for alpha-linolenic acid, 9.84 +/- 1.03 (P = 0.001) for arachidonic acid, and 3.01 +/- 0.79 (P = 0.064) for docosahexaenoic acid. The order of selectivity for transfer from the maternal to the fetal circulation was docosahexaenoicalpha-linoleniclinoleicoleicarachidonic acid. Such a mechanism would allow the preferential transfer of docosahexaenoic acid and the essential fatty acids to the fetal circulation, thereby protecting the polyunsaturated fatty acid supply to the fetus during a critical period of development.

The CO2 production (rCO2) of eight growing pigs was determined by continuous collection of CO2 over 21 days and simultaneously estimated using the doubly labeled water (DLW) method. The aim was to assess the accuracy of the method before and after correction for known sources of error and to test for any residual discrepancy arising from as yet unidentified sources of error. Mass spectrometer accuracy was verified by analyzing serial dilutions of the dose material in the form of an artificial decay curve; no significant bias was detected. The physiological errors were linearly dependent on weight gain. DLW-derived rCO2 (corrected only for fractionated water loss) underestimated the true value by 0.270 l CO2/g wt gain or -8% in the restricted (group R) and -16% in the ad libitum-fed (group AL) groups. Known sources of error accounted for -0.006 (methane), -0.032 (fecal 2H losses), -0.108 (fat synthesis), and -0.146 (changing pool size) l CO2/g wt gain. After correction for these sources of error the DLW-derived rCO2 differed from the true value by -2 +/- 3% in group R and 0 +/- 3% in group AL. Thus there was no significant bias in the DLW method after correction for known sources of error, even during rapid weight gain or at weight stability with or without correction. The precision estimates include both dose and background errors and uncertainty in the correction factors used. Strategies for optimizing precision are presented.

Carbon dioxide production (rCO2) was estimated in four sheep over a period of 10 days using doubly labeled water (2H and 18O) and was compared with simultaneous respiration chamber measurements of CO2. The excess 2H and 18O measurements were corrected for the empirically determined effects of isotope rebreathing within the confines of the chambers. A weighted monoexponential curve was then fitted to the data from which isotope flux rates and ultimately rCO2 and water turnover (rH2O) estimates were made. The curve fits were weighted assuming a Poisson model. Selection of this weighting policy did not bias the results, and curvature in the data also appeared to have little effect on the rCO2 estimates. Fractionated evaporative water loss expressed as a fraction of rH2O (X) was estimated from water balance and breath water production estimates; the mean X was 0.145 and ranged from 0.108 to 0.183. Corrections for 2H loss in fecal solids reduced the mean rH2O (4,746 g/day) by 35.5 g/day and increased the mean rCO2 (332.3 l/day) by 21.2 l/day. Further corrections to account for 2H loss in methane (mean production rate 27.2 l/day) reduced rH2O by 33.8 g/day and increased rCO2 by 20.3 l/day. The final isotopic estimates of rH2O were 14.6 +/- 6.59% (n = 4) lower than direct measurements and the mean rCO2 was 3.5 +/- 14.48% (n = 4) lower than the chamber measured rCO2. However, in one of the animals studied the rCO2 deviated markedly from the chamber-derived value, and this discrepancy has yet to be explained. When this animal was excluded from the comparisons, the standard deviation was greatly reduced (+/- 3.6, n = 3) and the mean overall error on rCO2 was +3.6%.

Increasing use of 18O-enriched water in biological studies such as those involving doubly labeled water has led to a requirement for a simple cost effective method for analyzing large numbers of 18O-enriched biological samples. We evaluated the influence of various factors on the precision and accuracy of an equilibration technique in which aqueous samples are equilibrated with CO2 in evacuated blood collection tubes, Vacutainers™. Using 10 mL Vacutainers with degassed rubber septa, 1 mL water samples equilibrated in an atmosphere of CO2 after 16 h on an orbital shaker. δ18O values became more variable when the sample volume was reduced to 0.25 mL. Varying the evacuation time for the tubes prior to filling with CO2 between 15 and 75 s had no marked influence on the results. Similarly, sonicating the water samples during this process to remove dissolved gases also had little effect. Bubbling air or CO2 through the samples prior to analysis led to a reduction in the 18O content of enriched water samples due to the introduction of 16O; however, N2 had no effect on the δ18O values. Memory effects between samples and instrumental drift were detected with large changes in enrichment (up to 140‰) during mass spectrometric analysis. An analytical procedure and calculations are presented, which in conjunction with two reference waters, may be used to remove these drift effects. This procedure coupled with suggested sample analysis sequences minimizes the influence of memory and allows precision to be optimized. Even in the absence of these precautions, analytical precision on water with δ18OV-SMOW content of +30‰ was ca ±0.1‰, and at +175‰ was ±0.37‰, comparable to other 18O equilibration methods.

BACKGROUND: Little is known of fatty acid metabolism in human embryos. This information would be useful in developing metabolic tests of embryo quality and improving embryo culture media. METHODS: The fatty acid composition of human embryos and their ability to accumulate 13 C labelled fatty acids was assessed in relation to the stage of development using gas-chromatography and combustion-isotope-ratio-mass spectrometry. RESULTS: Compared with embryos which did not develop beyond the 4-cell stage, those that did had significantly higher concentrations of the unsaturates, linoleic (12% versus 3%; P = 0.02) and oleic (14% versus 7%; P = 0.02), and a lower concentration of total saturates (62% versus 77%; P = 0.04). There was uptake of both 13 C linoleic and palmitic, but the developmental pattern was different for each fatty acid. The net accumulation in pmol/embryo/24h for palmitic was 1 at the 2-cell to

The fetus has an absolute requirement for the n-3/n-6 fatty acids and docosahexaenoic acid (22:6 n-3; DHA) in particular is essential for the development of the brain and retina. Most of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy. The likely rate of DHA utilisation during late pregnancy cannot be met from dietary sources alone in a significant proportion of mothers. De novo synthesis makes up some of the shortfall but the available evidence suggests that the maternal adipose tissue makes a significant contribution to placental transport to the fetus. The placenta plays a crucial role in mobilising the maternal adipose tissue and actively concentrating and channelling the important n-3/n-6 fatty acids to the fetus via multiple mechanisms including selective uptake by the syncytiotrophoblast, intracellular metabolic channelling, and selective export to the fetal circulation. These mechanisms protect the fetus against low long-chain polyunsaturated fatty acid (LCPUFA) intakes in the last trimester of pregnancy and have the effect of reducing the maternal dietary requirement for preformed DHA at this time. As a result of these adaptations, small changes in the composition of the habitual maternal diet before pregnancy are likely to be more effective in improving LCPUFA delivery to the fetus than large dietary changes in late pregnancy. There is little evidence that DHA intake/status in the second half of pregnancy affects visual and cognitive function in the offspring, but more studies are needed, particularly in children born to vegetarian and vegan and mothers who may have very low intakes of DHA.

Objective: To study the effects of ω-3 and ω-6 polyunsaturated fatty acid (PUFA) on in vitro proliferation of endometrial cells and their production of the cytokine interleukin-8 (IL-8). Design: In vitro study. Setting: Obstetrics and gynecology department, University of Aberdeen. Patient(s): Women attending an infertility clinic. Intervention(s): In vitro cell cultures using culture mediums supplemented with normal and high ratios of ω-3 PUFA and ω-6 PUFA. Main Outcome Measure(s): In vitro survival and production of IL-8 by dispersed endometrial cells. Result(s): In vitro survival of endometrial cells from women with and without endometriosis was significantly reduced in the presence of high ω-3:ω-6 PUFA ratios compared with cells incubated in the absence of fatty acids, in balanced ω-3:ω-6 PUFA ratios, and in high ω-6:ω-3 PUFA ratios. Endometrial cells from women with endometriosis secreted higher concentrations of IL-8, especially in the presence of high ω-3:ω-6 PUFA ratios. Conclusion(s): ω-3 PUFA may have a suppressive effect on the in vitro survival of endometrial cells and ω-3 PUFA be useful in the management of endometriosis by reducing the inflammatory response and modulating cytokine function.

An underlying assumption when estimating total energy expenditure (TEE) using doubly labelled water (DLW) is that the injected isotopes (lsO and 2H) leave the body only in the form of CO, and H20. However, both isotopes have additional routes of loss. We quantified the loss of 2H (i) attached to faecal solids and (ii) by sequestration into newly synthesised fat in reindeer (Rangifer tarandus tarandus). Estimates of the errors caused by these processes were applied to data from DLW studies with reindeer in summer and in winter. Given the net rate of faecal dry matter output and lipid synthesis in the present study, ignoring both sources of error caused the TEE of reindeer to be underestimated by approximately 5% in winter and approximately 9% in summer. The separate effect of each source of error was evaluated in summer. If ignored, loss of 2H through sequestration alone caused TEE to be underestimated by approximately 3.7%. Similarly, if ignored, loss of 2H attached to faecal solids alone caused TEE to be underestimated by approximately 5.9%.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas (n = 5) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids – designed to mimic the composition of non-esterified fatty acids (NEFA) measured in the last trimester of pregnancy (n = 10) – and ratio of fatty acid to human albumin. The selectivity for α-linolenic acid (αLN) transfer to the fetal circulation was not significantly different from that observed when using the triglyceride (TG) composition (1.21 ± 0.04), but significantly different for AA (1.43 ± 0.12; p < 0.001) and docosahexaenoic acid (DHA; 2.02 ± 0.09; p = 0.048). The absolute rate of transfer (nmol · ml–1) compared to that using the TG maternal perfusate composition was significantly different for LA (0.562 ± 0.038; p = 0.50), αLN (0.130 ± 0.009; p < 0.001), arachidonic acid (AA; 0.218 ± 0.022; p = 0.001) and DHA (0.383 ± 0.04; p < 0.001). Thus, placental selectivity for αLN and DHA appears to be relatively unresponsive to changes in the mixture of fatty acids in the maternal circulation but the selectivity for AA increased with the increase in the maternal AA concentration. For an 8-fold increase in the concentration of DHA in the maternal circulation there was a 13-fold increase in the transfer of DHA to the fetal circulation. For a 2-fold increase in the concentration of AA, transfer was increased 8-fold. For a 1.3-fold increase in the concentration of αLN, transfer was increased 2.1-fold. These results suggest that the maternal concentration of individual fatty acids, and hence the composition of the maternal diet, can have large effects on polyunsaturated/long-chain polyunsaturated fatty acids delivery to the fetus.

The reliance on samples of blood or urine to estimate isotopic abundance in studies of energy metabolism using the doubly labeled water method has restricted application of the technique to animals that are either tame or easy to catch. This is generally not the case with large, free-ranging wild mammals. The use of feces as a source of body water in which to measure the concentration of isotopic markers was investigated in four female reindeer in summer and in winter.2H2O and H2 18O were injected to ∼160 parts per million excess. Samples of plasma and feces were then collected simultaneously for up to 456 h. Both isotopes were equilibrated with body water at 8 h postdose. There were no significant differences by animal between dilution spaces, rate constants, rates of CO2production, and total energy expenditure (TEE) calculated based on samples of plasma or feces in any trial. Mean TEE was 3.557 W/kg (SD 0.907, n = 4) in summer and 1.865 W/kg (SD 0.166, n = 4) in winter.

The doubly labeled water (DLW) technique allows the CO2 production (rCO2) of free living animals to be estimated from the difference between the turnover of 2H2O and H218O in the body water. A fundamental assumption of this technique is that neither of the isotopes used are lost in products other than CO2 and H2O. We found, however, that 2H was lost in both exchangeable and nonexchangeable positions in the feces of sheep. Negligible amounts of 18O were lost in exchangeable positions. 2H losses led to a 0.75% (SE 0.06, n = 4) overestimation of the measured 2H2O flux, leading to an average error in rCO2 estimates of 20.3 l/day. For a typical rCO2 rate of 370 l/day, this would amount to an error of approximately 5% (range -7.0 to -4.3%, n = 4). Correction factors to account for this loss were presented. The error in rCO2 due to 2H sequestration into fat was calculated to be at most 2.1 l/day or about -0.66% in lambs with a rCO2 of 320 l/day. In a triply labeled water (TLW) study the maximum error in the estimation of fractionated evaporative water loss (X) would lead to a 0.81% underestimation of rCO2. We recommend that during a DLW study involving ruminant animals the correction factors presented here be used to compensate for 2H loss in feces. This may be particularly important where the diet has a high roughage content leading to a significant fecal dry matter production.

The doubly labelled water (DLW) method for measuring CO 2 production has recently been the subject of much interest since no other technique gives integrated values for CO 2 production over long periods by free-living subjects. The importance of evaporative water loss and fractionation factors to the calculation of CO 2 production using this technique is described. Present methods of estimating evaporative water loss and the use of fractionation factors are summarized together with a discussion of their limitations. A novel technique is proposed whereby water labelled with three isotopes can be used to measure evaporative water loss and CO 2 production in completely free-living subjects, and the feasibility of the method is tested in simulations using experimental data. This technique has three advantages over existing methods of estimating evaporative water loss: (1) it can be used in completely free-living subjects without any additional experimental procedures (e.g. water-balance studies or physical trapping of water vapour); (2) it gives a direct estimate of fractionated evaporative water loss in each subject, since non-fractionated water lost as vapour is automatically compensated for; and (3) the routes of water loss do not have to be known. The appropriate calculations are presented together with a discussion of the difficulties of measuring oxygen-17 by mass spectrometry. It is estimated that the maximum theoretical error on calculated CO 2 production is ±0·3%. Practical ways of achieving this theoretical level of accuracy are suggested. We conclude that the proposed technique will allow correction for evaporative water loss to be made more exactly, thereby increasing the accuracy of the heavy water technique for measuring CO 2 production in free-living subjects.

The doubly labeled water (DLW) technique for measuring CO2 production (rCO2) in free-living animals requires an assessment of the elimination of both 2H and 18O from the body over a long period of time. To calculate rCO2, it is necessary to calculate water turnover (rH2O) from the 2H flux rate. In ruminant animals, the accuracy of this calculation is affected by the loss of 2H in methane. We have quantified the effect of methane production (rCH4) on the 2H flux rate, determined in four sheep given 2H2O. The methane produced was depleted in 2H relative to the urine. A relationship between the enrichment of the methane and urine was established. The ratio of urine to methane enrichment was found on average to be 0.6536, and this value was unaffected by the level of rCH4 but showed some dependence on the absolute concentration of 2H in urine. For this reason, the ratio value obtained from four sheep not given 2H2O was different, a mean of 0.6886 was measured, this ratio was unaffected by changes in the diet supplied to the animals. Computer modeling was used to illustrate the dependence of the isotopically derived value for rCO2 on not only rCH4 but also the magnitude of rCO2 itself. The effect of rCH4 on the DLW method can be predicted from the observed ratio of rCO2 to rCH4 and the value of 0.6536 obtained for the ratio of methane to urine enrichment. With the use of data from several studies at this Institute, a limited range of 10 to 20 was found for rCO2/rCH4 in animals fed at or above maintenance.(ABSTRACT TRUNCATED AT 250 WORDS)