A genome-wide association study implicates the APOE locus in nonpathological cognitive ageing.

Cognitive decline is a feared aspect of growing old. It is a major contributor to lower quality of life and loss of independence in old age. We investigated the genetic contribution to individual differences in nonpathological cognitive ageing in five cohorts of older adults. We undertook a genome-wide association analysis using 549 692 single-nucleotide polymorphisms (SNPs) in 3511 unrelated adults in the Cognitive Ageing Genetics in England and Scotland (CAGES) project. These individuals have detailed longitudinal cognitive data from which phenotypes measuring each individual's cognitive changes were constructed. One SNP--rs2075650, located in TOMM40 (translocase of the outer mitochondrial membrane 40 homolog)--had a genome-wide significant association with cognitive ageing (P=2.5 × 10(-8)). This result was replicated in a meta-analysis of three independent Swedish cohorts (P=2.41 × 10(-6)). An Apolipoprotein E (APOE) haplotype (adjacent to TOMM40), previously associated with cognitive ageing, had a significant effect on cognitive ageing in the CAGES sample (P=2.18 × 10(-8); females, P=1.66 × 10(-11); males, P=0.01). Fine SNP mapping of the TOMM40/APOE region identified both APOE (rs429358; P=3.66 × 10(-11)) and TOMM40 (rs11556505; P=2.45 × 10(-8)) as loci that were associated with cognitive ageing. Imputation and conditional analyses in the discovery and replication cohorts strongly suggest that this effect is due to APOE (rs429358). Functional genomic analysis indicated that SNPs in the TOMM40/APOE region have a functional, regulatory non-protein-coding effect. The APOE region is significantly associated with nonpathological cognitive ageing. The identity and mechanism of one or multiple causal variants remain unclear.

Genome-wide association studies establish that human intelligence is highly heritable and polygenic.

General intelligence is an important human quantitative trait that accounts for much of the variation in diverse cognitive abilities. Individual differences in intelligence are strongly associated with many important life outcomes, including educational and occupational attainments, income, health and lifespan. Data from twin and family studies are consistent with a high heritability of intelligence, but this inference has been controversial. We conducted a genome-wide analysis of 3511 unrelated adults with data on 549,692 single nucleotide polymorphisms (SNPs) and detailed phenotypes on cognitive traits. We estimate that 40% of the variation in crystallized-type intelligence and 51% of the variation in fluid-type intelligence between individuals is accounted for by linkage disequilibrium between genotyped common SNP markers and unknown causal variants. These estimates provide lower bounds for the narrow-sense heritability of the traits. We partitioned genetic variation on individual chromosomes and found that, on average, longer chromosomes explain more variation. Finally, using just SNP data we predicted ∼1% of the variance of crystallized and fluid cognitive phenotypes in an independent sample (P=0.009 and 0.028, respectively). Our results unequivocally confirm that a substantial proportion of individual differences in human intelligence is due to genetic variation, and are consistent with many genes of small effects underlying the additive genetic influences on intelligence.

Folic acid use in pregnancy and embryo selection.

OBJECTIVE: Folic acid supplement use is recommended in pregnancy to reduce the risk of neural tube defect but concerns have been raised that increasing folic acid intake may select for embryos with genotypes that increase disease risk in the offspring. Our aim was to test for this effect. DESIGN: Observational prospective cohort study. SETTING: Aberdeen Maternity Hospital. POPULATION OR SAMPLE: Women born before the introduction of folic acid advice (1970-80) and carrying singleton pregnancies (n = 1234) and their offspring (n = 1083) born after (2001-03). METHODS: We measured the genotype (MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TCN G776C) of mothers and their offspring, maternal supplement intake, intake of folate and vitamin B12 from natural foods and maternal blood folate and B12 status at 19 weeks of gestation. MAIN OUTCOME MEASURES: B vitamin related genotype of the offspring. RESULTS: There were no significant differences in any of the five genotype frequencies between mothers and their babies. There was no deviation from Hardy-Weinberg equilibrium in either generation and no change in the frequency of doubly homozygous MTHFR variants (677 TT/1298 CC). The genotype of the offspring was not related to maternal periconceptual supplement use, folate intake from foods or plasma and red cell folate measured at 19 weeks of gestation. CONCLUSIONS: We found no evidence to support the concern that folic acid fortification or supplement use in pregnancy results in selection of deleterious genotypes.

Effect of B vitamins and genetics on success of in-vitro fertilisation: prospective cohort study.

BACKGROUND: There is a need to understand what affects the success of in-vitro fertilisation (IVF) and the rate of resulting twin births so that pregnancy rates can be improved and multiple gestations avoided. Our aim was to assess the role of B vitamins and genetics. METHODS: We did a prospective cohort study of 602 women undergoing fertility treatment. We assessed intake of folate and vitamin B12 with a questionnaire and measured their plasma and red-blood-cell concentrations by radioimmunoassay. We measured five B-vitamin-related gene variants in women who received treatment and in 932 women who conceived naturally. FINDINGS: The likelihood of a twin birth after IVF rose with increased concentrations of plasma folate (1.52, 1.01-2.28; p=0.032) and red-cell folate (1.28, 1.00-1.65; p=0.039). There was no association between folate and vitamin B12 levels and likelihood of a successful pregnancy. Women homozygous for the 1298 CC variant of methylenetetrahydro-folate reductase (MTHFR), rather than the AA variant, were less likely to produce a livebirth after IVF (0.24, 0.08-0.71; p=0.003) or to have had a previous pregnancy (0.42, 0.21-0.81; p=0.008). INTERPRETATION: Our findings suggest that MTHFR genotype is linked to a woman's potential to produce healthy embryos (possibly through interaction with genes related to DNA methylation). In women likely to have a successful IVF pregnancy, high folate status increases the likelihood of twin birth after multiple embryo transfer. Proposals to fortify the UK diet with folic acid could lead to an increase in the number of twins born after IVF.

Energetic consequences of mild Giardia intestinalis infestation in Mexican children.

This study explored the effects of mild infestation with Giardia on energy intake and expenditure at rest and in activity in an urban Mexican population. Ten boys aged 6-10 y living in low-income sectors in northwest Mexico who had Giardia infestation were recruited. Energy intake, basal metabolic rate (BMR), and total free-living expenditure (TEE) measured by the doubly labeled water method were determined for 7 d during both infestation and after treatment. There was no significant difference in recorded energy intake between the two periods (7.76 and 7.70 MJ/d; P = 0.847). BMR showed no significant change in response to treatment; values were 4.79 and 4.86 MJ/d (P = 0.03). The mean TEE increased by almost 1 MJ/d in the Giardia-free period. This increase was observed in 8 of the 10 subjects; however, the overall change was not statistically significant (P = 0.08).

Long-chain polyunsaturated fatty acid transport across the perfused human placenta.

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 (P < 0.001) 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 (P < 0.001) 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 docosahexaenoic > alpha-linolenic > linoleic > oleic > arachidonic 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.

Leptin secretion to both the maternal and fetal circulation in the ex vivo perfused human term placenta.

The contribution of placental leptin, if any, to both the fetal and maternal circulation and its role in pregnancy remains to be determined. In an experiment to investigate this, 27 placentae from term pregnancies were perfused ex vivo (gestational age=39.5 s.d. 1.2; range=38-42 weeks: fetal weight=3285 s.d. 482; range=2480-4420; birthweight centile range=4th to the 98th) at both the maternal and fetal interface. Placental leptin was exported into both the maternal and fetal circulations. The log leptin production by the maternal side of the placenta was significantly greater (P=0.001) than that for the fetal side (5.193 s.d.1.049 versus 4.387 s.d. 0.768 ng/placenta/min). There was no significant relationship between maternal and fetal log leptin production and maternal body mass index, birthweight, birthweight centile, ponderal index or gestational age or with cord blood pO(2), pCO(2) and pH. There was however, a significant increase in the maternal log leptin production with increasing fetal to placental weight ratio (P=0.017; r(2)=20.7 per cent) but no corresponding relationship for fetal leptin production. It is proposed that such a mechanism would allow the placenta to modulate fat supply to the fetus in response to the fetal demand relative to placental supply.

Placental nutrient transfer capacity and fetal growth.

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.

Dietary intake and tissue concentration of fatty acids in omnivore, vegetarian and diabetic pregnancy.

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.

High omega-3:omega-6 fatty acid ratios in culture medium reduce endometrial-cell survival in combined endometrial gland and stromal cell cultures from women with and without endometriosis.

OBJECTIVE: To study the effects of omega-3 and omega-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 omega-3 PUFA and omega-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 omega-3:omega-6 PUFA ratios compared with cells incubated in the absence of fatty acids, in balanced omega-3:omega-6 PUFA ratios, and in high omega-6:omega-3 PUFA ratios. Endometrial cells from women with endometriosis secreted higher concentrations of IL-8, especially in the presence of high omega-3:omega-6 PUFA ratios. CONCLUSION(S): omega-3 PUFA may have a suppressive effect on the in vitro survival of endometrial cells and omega-3 PUFA be useful in the management of endometriosis by reducing the inflammatory response and modulating cytokine function.

Effect of placental function on fatty acid requirements during pregnancy.

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.

Basal metabolic rate and body fatness of adult men in northern Mexico.

To investigate the possibility that overprediction of basal metabolic rate (BMR) of tropical populations is related to differences in fatness between tropical and temperate populations, 32 Mexican men aged 18-40 years underwent measurements of BMR and body fat content. The men were divided into four body mass index (BMI) groups (< 20, 20-25, 25-30 and 30-40). The mean weight was 78 (range 52-119) kg and the mean height was 1.75 (range 1.61-1.89) m. BMR was measured in the subjects after an overnight stay in a residential unit by a Deltatrac ventilated hood indirect calorimetry system. Body fat content was determined by 2H2O dilution. The relationship between BMR and body weight was analysed by linear regression and the equations derived were compared to the values predicted using the FAO/WHO/UNU (1985) equations. The FAO/WHO/UNU equations overestimated measured BMR by 9.8%, 9.6%, 7.8% and 5.5% in the four groups. The overall difference was 8.2%, which was statistically significant (P < 0.001). The intercepts of the two equations were significantly different (P < 0.001) by 532 kJ/day, but there was no significant difference between the slopes of the two regression equations. There was no significant difference between BMR/kg fat-free mass in the four BMI groups. The results therefore do not support the possibility that the overprediction of BMR in tropical populations by international prediction equations based on body weight is accounted for by differences in fatness between tropical and temperate populations.

Validation of energy intake estimated from a food frequency questionnaire: a doubly labelled water study.

OBJECTIVE: The validation of dietary assessment methods is critical in the evaluation of the relation between dietary intake and health. The aim of this study was to assess the validity of a food frequency questionnaire by comparing energy intake with energy expenditure measured with the doubly labelled water method. DESIGN: Total energy expenditure was measured with the doubly labelled water (DLW) method during a 10 day period. Furthermore, the subjects filled in the food frequency questionnaire about 18-35 days after the DLW phase of the study was completed. SUBJECTS: Twenty-one healthy, non-pregnant females volunteered to participate in the study; only 17 subjects completed the study. RESULTS: The group energy intake was on average 10% lower than the energy expenditure, but the difference was not statistically significant. However, there was a wide range in reporting accuracy: seven subjects were identified as acceptable reporters, eight as under-reporters and two were identified as over-reporters. The width of the 95% confidence limits of agreement in a Bland and Altman plot for energy intake and energy expenditure varied from -5 to 3 MJ. CONCLUSION: The data showed that there was substantial variability in the accuracy of the food frequency questionnaire at the individual level. Furthermore, the results showed that the questionnaire was more accurate for groups than individuals.

The measurement of fetal liver T(*)(2) in utero before and after maternal oxygen breathing: progress towards a non-invasive measurement of fetal oxygenation and placental function.

Utero-placental insufficiency is thought to be a major cause of growth retardation in utero and an important risk factor in the perinatal period. The purpose of this study was to investigate whether MRI could detect changes of fetal oxygenation, based on the blood oxygenation level dependence (BOLD) of the MRI tissue signal. Nine third trimester women (34-38 weeks) with normal pregnancies underwent abdominal MRI examinations. Following localization of the fetal liver using T(2)-weighted single-shot HASTE scans, up to 7 breath-held transaxial single-slice gradient-echo image sets were obtained through the fetal liver. The mother then commenced oxygen breathing with the imaging procedure repeated after 20 minutes of O(2) breathing. For each image set, T(*)(2) values are calculated using linear regression of log (signal) versus TE for a region of interest within the fetal liver selected by the attending radiologist. Fetal liver T(*)(2) values were calculated before and after O(2) breathing for each multi-echo image acquisition set. A signed rank test was used to test for a significant change in fetal liver T(*)(2) between the pre-O(2) and post-O(2) image sets. A significant increase in T*(2) (alpha < 0.05) was seen in 5 of the 9 fetal livers, a smaller increase (of borderline statistical significance, alpha = 0.057) in 2 livers, and no significant change (alpha > 0.05) in 2 livers. Our study indicates that T(*)(2) measurement of the fetal liver may detect alteration in fetal oxygen level following maternal oxygenation using the BOLD effect. This technique may potentially be applied to the identification and understanding of placental dysfunction in intra-uterine growth retardation.

GWAS-based pathway analysis differentiates between fluid and crystallized intelligence.

Cognitive abilities vary among people. About 40-50% of this variability is due to general intelligence (g), which reflects the positive correlation among individuals' scores on diverse cognitive ability tests. g is positively correlated with many life outcomes, such as education, occupational status and health, motivating the investigation of its underlying biology. In psychometric research, a distinction is made between general fluid intelligence (gF) - the ability to reason in novel situations - and general crystallized intelligence (gC) - the ability to apply acquired knowledge. This distinction is supported by developmental and cognitive neuroscience studies. Classical epidemiological studies and recent genome-wide association studies (GWASs) have established that these cognitive traits have a large genetic component. However, no robust genetic associations have been published thus far due largely to the known polygenic nature of these traits and insufficient sample sizes. Here, using two GWAS datasets, in which the polygenicity of gF and gC traits was previously confirmed, a gene- and pathway-based approach was undertaken with the aim of characterizing and differentiating their genetic architecture. Pathway analysis, using genes selected on the basis of relaxed criteria, revealed notable differences between these two traits. gF appeared to be characterized by genes affecting the quantity and quality of neurons and therefore neuronal efficiency, whereas long-term depression (LTD) seemed to underlie gC. Thus, this study supports the gF-gC distinction at the genetic level and identifies functional annotations and pathways worthy of further investigation.

Fatty acid metabolism in human preimplantation embryos.

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 13C 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 13C 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 <8-cell stage, 4 at the 8-cell-morula stage and negligible at the blastocyst stage. For linoleic, there was little net accumulation at the 2-cell to <8-cell stage, 8 (8-cell-morula stage) and 17 pmol/embryo/24 h (blastocyst stage). CONCLUSION: Preimplantation human embryos actively take up individual fatty acids at different rates at different stages of development. The high unsaturated concentration at the later stages of development may be explained by preferential uptake of linoleic acid.

The doubly labeled water method: errors due to deuterium exchange and sequestration in ruminants.

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 effect of maternal smoking and ethanol on fatty acid transport by the human placenta.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas from non-smokers (n 5), smokers (>ten cigarettes/d; n 5) and after addition of ethanol at 2 mg/ml (n 4). The maternal side was of the placenta was perfused ex vivo for 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: 2 n-6), alpha-linolenic (alphaLN, 18: 3 n-3), arachidonic (AA, 20: 4 n-6) or docosahexaenoic acid (DHA, 22: 6 n-3), expressed per perfused area (calculated from H2(18)O exchange). However, the presence of ethanol in the perfusate at a concentration of 2 mg/ml significantly reduced (P<0.01) the absolute rate of transfer of the two n-3 polyunsaturated fatty acids, alphaLN and DHA. This specific effect of ethanol on alphaLN 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 > alphaLN > 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 > alphaLN > LA > DHA. The presence of ethanol in the perfusate was also associated with a significant reduction (P<0.05) in the clearance of H2(18)O. These results suggest that the presence of ethanol at a concentration of 2mg/ml may reduce the availability of polyunsaturated fatty acids to the developing fetus.

Measurement of fractionated water loss and CO2 production using triply labelled water.

The doubly labelled water (DLW) method for measuring CO2 production has recently been the subject of much interest since no other technique gives integrated values for CO2 production over long periods by free-living subjects. The importance of evaporative water loss and fractionation factors to the calculation of CO2 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 CO2 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 CO2 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 CO2 production in free-living subjects.