The effect of food preparation on the bioavailability of carotenoids from carrots using intrinsic labelling.

A strategy to reduce the incidence of vitamin A deficiency is to improve precursor bioavailability from meals. Since vitamin A precursors are fat-soluble, we noted that carotenoids are more easily absorbed from food if prepared in such a way that the food matrix containing provitamin A (ß-carotene) is sufficiently fat rich. To quantify this effect, we have developed a stable isotope methodology. By regular watering with 2H-labelled water, we were able to produce several kg of intrinsically labelled carrots, with carotenoids labelled to 0.63 % excess 2H. These were divided into 100 g portions and fed to a small group of healthy subjects both raw and stir-fried. To normalise for inter-individual variation in absorption and subsequent metabolism, small quantities of extrinsically 13C-labelled ß-carotene and 2H-labelled retinol acetate were also incorporated into the meal. After ingestion of the carrots, blood lipids were monitored for a period of 3 d in order to determine the kinetics of ß-carotene and retinol. From kinetic data, it was estimated that the bioavailability of carrot-derived ß-carotene compared with pure ß-carotene was about 11 % for raw carrots, but 75 % when the carrots were stir-fried. Conversely, there was a slight reduction in the bioconversion to retinol from ß-carotene when the latter was derived from the stir-fried meal compared with that from raw carrots. When these two factors are combined, the yield of retinol from the carotene in carrots was found to be enhanced by a factor of 6.5 by stir-frying.

The effect of different meals on the absorption of stable isotope-labelled phylloquinone.

Few studies have investigated the absorption of phylloquinone (vitamin K1). We recruited twelve healthy, non-obese adults. On each study day, fasted subjects took a capsule containing 20 microg of 13C-labelled phylloquinone with one of three meals, defined as convenience, cosmopolitan and animal-oriented, in a three-way crossover design. The meals were formulated from the characteristics of clusters identified in dietary pattern analysis of data from the National Diet and Nutrition Survey conducted in 2000-1. Plasma phylloquinone concentration and isotopic enrichment were measured over 8 h. Significantly more phylloquinone tracer was absorbed when consumed with the cosmopolitan and animal-oriented meals than with the convenience meal (P = 0.001 and 0.035, respectively). Estimates of the relative availability of phylloquinone from the meals were: convenience meal = 1.00; cosmopolitan meal = 0.31; animal-oriented meal = 0.23. Combining the tracer data with availability estimates for phylloquinone from the meals provides overall relative bioavailability values of convenience = 1.00, cosmopolitan = 0.46 and animal-oriented = 0.29. Stable isotopes provide a useful tool to investigate further the bioavailability of low doses of phylloquinone. Different meals can affect the absorption of free phylloquinone. The meal-based study design used in the present work provides an approach that reflects more closely the way foods are eaten in a free-living population.

Energy density of the diet and change in body fatness from childhood to adolescence; is there a relation?

BACKGROUND: The contribution of energy density (ED) of the total diet to increased risk of obesity from childhood into adolescence is unclear. OBJECTIVE: We assessed the relation between the ED of the diet in childhood, calculated in a number of ways, and change in adiposity from childhood to adolescence. DESIGN: In a prospective study, 48 children (30 boys, 18 girls) were initially studied at age 6-8 y (baseline) and followed up at age 13-17 y. Daily ED, energy intake, and food intake were assessed at baseline by 7-d weighed food records concurrent with estimates of total energy expenditure (TEE) by doubly labeled water. ED was calculated with the use of 5 published methods. Obesity risk was defined with the use of body fat from total body water by isotope dilution. Body fat was normalized for height and expressed as fat mass index (FMI). Change in adiposity was calculated as follow-up FMI minus baseline FMI. RESULTS: Misreporting of energy intake at the group level at baseline was low relative to the TEE. ED of the total diet at baseline by the 3 methods for calculating ED that excluded all or most beverages was prospectively associated with change in FMI. However, ED of the total diet by any of the methods was not associated with change in the percentage body fat, body mass index, or waist circumference z scores. CONCLUSION: The methods used to calculate ED and to assess obesity risk lead to different conclusions about the relation between the ED of the diet in childhood and gain in fat into adolescence.

Efficiency of autoregulatory homeostatic responses to imposed caloric excess in lean men.

Obesity implies a failure of autoregulatory homeostatic responses to caloric excess. We studied the mechanisms, effectiveness, and limits of such responses in six lean (21.9 +/- 1.3 kg/m(2)), healthy men based in a metabolic suite for 17 wk of progressive intermittent overfeeding (OF) (3 wk, baseline; 3 wk, 20% OF; 1 wk, ad libitum; 3 wk, 40% OF; 1 wk, ad libitum; 3 wk, 60% OF; 3 wk, ad libitum). Body composition was assessed by a four-compartment model using dual X-ray absorptiometry, deuterium dilution, and plethysmography. Magnetic resonance imaging assessed subcutaneous/visceral fat at abdominal level at baseline and at the end of 60% OF. Energy intake was assessed throughout, energy expenditure (EE) and substrate oxidation rates were measured repeatedly by whole body calorimetry (calEE), and free-living EE (TEE) was measured by doubly labeled water at baseline and after 60% OF. At the end of 60% OF, calEE and TEE had increased by just 11.4% (P = 0.001) and 16.2% (P = 0.001), respectively. Weight and body fat (fat mass) had increased by 5.98 kg (8.8%, P = 0.001) and 3.31 kg (22.6%, P = 0.01), respectively. The relative increase in visceral fat (32.6%, P = 0.02) exceeded that of subcutaneous fat (13.3%, P = 0.002) in the abdominal region. The computed energy cost of tissue accretion differed from the excess ingested by only 13.1% (using calEE) and 11.6% (using TEE), indicating an absence of effective dissipative mechanisms. We conclude that elevations in EE provide very limited autoregulatory capacity in body weight regulation, and that regulation must be dominated by hypothalamic modulation of energy intake. This result supports present conclusions from genetic studies in which all known causes of human obesity are related to defects in the regulation of appetite.

Use of stable-isotope techniques to validate infant feeding practices reported by Bangladeshi women receiving breastfeeding counseling.

BACKGROUND: The World Health Organization recommends exclusive breastfeeding until age 6 mo. Studies relying on mothers' self-reported behaviors have shown that lactation counseling increases both the rate and duration of exclusive breastfeeding. OBJECTIVE: We aimed to validate reported infant feeding practices in rural Bangladesh; intakes of breast milk and nonbreast-milk water were measured by the dose-given-to-the mother deuterium dilution technique. DESIGN: Subjects were drawn from the large-scale Maternal and Infant Nutrition Interventions, Matlab, study of combined interventions to improve maternal and infant health, in which women were randomly assigned to receive either exclusive breastfeeding counseling or standard health care messages. Data on infant feeding practices were collected by questionnaire at monthly visits. Intakes of breast milk and nonbreast-milk water were measured in a subsample of 98 mother-infant pairs (mean infant age: 14.3 wk) and compared with questionnaire data reporting feeding practices. RESULTS: Seventy-five of the 98 subjects reported exclusive breastfeeding. Mean (+/-SD) breast milk intake was 884 +/- 163 mL/d in that group and 791 +/- 180 mL/d in the group reported as nonexclusively breastfed (P = 0.0267). Intakes of nonbreast-milk water were 40 +/- 80.6 and 166 +/- 214 mL/d (P < 0.0001), respectively. Objective cross-validation using deuterium dilution data showed good accuracy in reporting of feeding practices, although apparent misreporting was widely present in both groups. CONCLUSIONS: The dose-given-to-the-mother deuterium dilution technique can be applied to validate reported feeding behaviors. Whereas this technique shows that the reports of feeding practices were accurate at the group level, it is not adequate to distinguish between feeding practices in individual infants.

Comparative dietary intake and sources of phylloquinone (vitamin K1) among British adults in 1986-7 and 2000-1.

Using data from 7 d weighed dietary records, dietary intake and sources of phylloquinone (vitamin K1) were examined by socio-demographic and lifestyle factors in 1916 participants aged 16-64 years from the 1986-7 Dietary and Nutritional Survey of British Adults, and 1423 participants aged 19-64 years from the 2000-1 National Diet and Nutrition Survey. Using UK-specific food content data, geometric mean phylloquinone intakes were estimated as 72 (95% CI 70, 74) and 67 (95% CI 65, 69) microg/d in 1986-7 and 2000-1 respectively (P<0.001). In 1986-7, 47% of participants had phylloquinone intakes below the UK guideline for adequacy (> or =1 microg/kg body weight per d), compared with 59% in 2000-1 (P<0.001). In both surveys, daily phylloquinone intake was higher among men than women and increased significantly with age. Participants of manual occupational social class, or who were smokers, had lower phylloquinone intake than their counterparts. Participants living in Scotland and northern England had lower phylloquinone intake than those living elsewhere in mainland Britain, particularly in 1986-7 when the contribution from vegetables was also lower than elsewhere. However, by 2000-1 this regional difference was no longer significant. Overall, vegetables contributed 63% of phylloquinone intake in 1986-7 and 60% in 2000-1, with cooked leafy green vegetables (LGV) providing 23 and 19% respectively. In both surveys, the contribution of vegetables (cooked LGV in particular) was directly associated with age. These data show a decrease in phylloquinone intake from 1986-7 to 2000-1, mainly owing to lower consumption of cooked LGV.

Plasma phylloquinone (vitamin K1) concentration and its relationship to intake in British adults aged 19-64 years.

Plasma phylloquinone (vitamin K1) concentration from non-fasted blood samples was examined by season, smoking status, socio-demographic factors and phylloquinone intake in a nationally representative sample of 1154 British individuals aged 19-64 years from the 2000-1 National Diet and Nutrition Survey. Geometric mean plasma phylloquinone concentration was 0.94 (95% CI 0.88, 1.00) nmol/l, with 95% of values in the range 0.10-8.72 nmol/l. Plasma phylloquinone concentrations of 530 men were significantly higher than those of 624 women (1.13 (95% CI 1.04, 1.22) v. 0.81 (95% CI 0.74, 0.88) nmol/l; P<0.001), independent of other factors. Women aged 19-34 years had significantly lower plasma phylloquinone concentration than their older counterparts. Women were also found to have lower plasma phylloquinone concentrations during summer compared with winter and spring (each P<0.01). In contrast, plasma phylloquinone concentration in men did not vary significantly by season or any of the socio-demographic or lifestyle factors. Plasma phylloquinone concentrations were positively correlated with phylloquinone intake in men and women (r 0.26 and 0.32 respectively; each P<0.001). Overall, forward stepwise multiple regression analysis revealed that 8% of the variation in plasma phylloquinone concentration was explained by phylloquinone intake, with a further 10% of its variation explained by plasma concentrations of gamma-tocopherol (6%) and retinyl palmitate (4%). After adjustment for age and corresponding nutrient intakes, plasma phylloquinone concentration was significantly associated (each P<0.01) with plasma concentrations of total and LDL-cholesterol, alpha- and gamma-tocopherols, retinyl palmitate, beta-carotene, lycopene and lutein plus zeaxanthin in men and women.

Analysis of isotope ratios in vitamin K1 (phylloquinone) from human plasma by gas chromatography/mass spectrometry.

Vitamin K(1) is a fat-soluble vitamin required for the gamma-carboxylation of vitamin K-dependent proteins. Recent work has suggested an important role for vitamin K(1) in bone health beyond its more established function in the control and regulation of blood coagulation. However, current UK recommended intakes do not reflect this recent evidence. The use of stable isotopes provides a powerful tool to investigate vitamin K kinetics, turnover and absorption in man, although published methods have reported difficulties in the extraction and analysis of isotope ratios of vitamin K in human plasma. In this paper, we report a new methodology for the extraction and measurement of isotope ratios in vitamin K(1). Sample clean-up is achieved with liquid-liquid extraction, enzyme hydrolysis with lipase and cholesterol esterase, and solid-phase extraction. Isotopic analysis of the pentafluoropropionyl derivative of vitamin K(1) is performed by gas chromatography/mass spectrometry (GC/MS). The limit of quantitation is equivalent to at least 0.3 nmol/L and the method is demonstrated to be linear over a range of enrichments. This method provides a robust alternative to previous work requiring the use of semi-preparative high-performance liquid chromatography (HPLC).

Measurement of gastric emptying by the 13C-octanoate breath test--rationalization with scintigraphy.

The (13)C-octanoate breath test has not achieved universal acceptance for the measurement of solid phase gastric emptying, largely because the results are not comparable with those from direct methods such as scintigraphy. To convert breath-test data to their scintigraphic equivalent scale corrections are applied which have been obtained from population studies, but there is no guarantee that these are applicable in all cases. We propose an alternative method applicable on an individual basis based upon a simple physiological model which does not require any change to the breath-test protocol. It is demonstrated by comparison with scintigraphy and the octanoate saliva test. Results from an existing dataset of simultaneous breath test, saliva test and scintigraphic determinations of solid phase gastric emptying from nine healthy subjects were re-analysed. The corrected breath tests gave gastric emptying curves which were shown to be not significantly different to those obtained from scintigraphy. The method provides a simple but effective way of generating gastric emptying curves from breath-test data that are directly comparable with direct measurement methods, which is advantageous since it allows the whole of the gastric emptying profile to be generated, not just values for the lag phase and half-emptying times.

A stable isotope minimal model protocol with oral glucose administration.

A protocol for investigating glucose metabolism whereby stable isotope tracer is given intravenously after an oral glucose challenge is described. Frequent sampling of plasma glucose and insulin allows the tracer disappearance to be interpreted on the basis of established minimal models. We have investigated the glucose effectiveness and insulin sensitivity parameters and their reproducibility in a group of six healthy adults, each studied twice. A mono-compartmental description of glucose distribution did not provide a physiological description of glucose kinetics, whereas a two-compartment model gave adequate results in every case. The estimates of glucose effectiveness and insulin sensitivity were 2-3 times higher than those obtained in similar populations using the conventional protocol of the frequently sampled intravenous glucose tolerance test, and this appeared to be related to the kinetics of transport of glucose from accessible to remote pools. The indices of insulin sensitivity obtained in this way were highly reproducible, with a between-test correlation of 93%.

13C- and 2H-labelled glucose compared for minimal model estimates of glucose metabolism in man.

In the present study, we have investigated the use of 1-[(13)C]glucose and GC/combustion/isotope-ratio MS as an alternative to 6,6-[(2)H(2)]glucose and GC/MS in the determination of parameters of glucose metabolism using the IVGTT (intravenous glucose tolerance test) interpreted by labelled (hot) minimal models. The study has been done in four populations, normoglycaemics (subdivided into lean and obese individuals), subjects with impaired glucose tolerance and those with diabetes mellitus. Although the use of carbon label may in some circumstances be compromised by substrate recycling, our hypothesis was that this would not be an issue under the condition of suppression of hepatic glucose production during the short timescale of an IVGTT. In all four groups, we found that the methodology employing the carbon label gave equivalent results to those obtained using the conventional deuterated material, but the sensitivity of the measurement technique in the new approach was sufficient to allow an approx. 15-fold reduction in the quantity of isotope administered. In addition to the clear cost advantages, this represents a significant scientific advance in that true tracer status is more nearly attained in these measurements with near-physiological tracee loads.

The 'anomalous' absorption of labelled and unlabelled vitamin C in man.

Previous studies of vitamin C absorption in man using stable isotope probes have given results which cannot easily be reconciled with those obtained using non-isotope measurement. In order to investigate some of the apparent paradoxes we have conducted a study using two consecutive doses of vitamin C, one labelled and one unlabelled, given 90 min apart. Compatibility of the experimental results with two feasible models was investigated. In Model 1, ingested vitamin C enters a pre-existing pool before absorption, which occurs only when a threshold is exceeded; in Model 2, ingested vitamin C is exchanged with a pre-existing flux before absorption. The key difference between these two models lies in the predicted profile of labelled material in plasma. Model 1 predicts that the second unlabelled dose will produce a secondary release of labelled vitamin C which will not be observed on the basis of Model 2. In all subjects Model 1 failed to predict the observed plasma concentration profiles for labelled and unlabelled vitamin C, but Model 2 fitted the experimental observations. We speculate on possible physiological explanations for this behaviour, but from the limited information available cannot unequivocally confirm the model structure by identifying the source of the supposed flux.

Glycogenesis and glucose oxidation during an intravenous glucose tolerance test in man.

The quantity of deuterated glucose customarily given in labelled IVGTTs (intravenous glucose tolerance tests) changes the isotopic composition of the subject's body water enough to be detected by mass spectrometric techniques. Glucose undergoing direct glycogenesis does not contribute label to the body water pool, and isotope incorporated into it must have come from glucose that has either been oxidized or undergone indirect glycogenesis. By subtracting the amount of label found in body water from the total amount of glucose utilized, as calculated from the minimal model of glucose disappearance, it should be possible to study the partitioning of the dose given between direct glycogenesis in skeletal muscle and other metabolic pathways. To establish these principles, we used isotope ratio MS to determine body water composition in groups of healthy ( n =7; mean weight, 76 kg; fasting plasma glucose and insulin, 5.1 mmol and 40 pmol respectively) and Type II diabetic ( n =5; mean weight, 84 kg; fasting plasma glucose and insulin, 6.2 mmol and 75 pmol respectively) subjects undergoing IVGTTs. It was found that, for healthy subjects, 31% of the dose given was utilized in direct glycogenesis and this was decreased to 15% in diabetes. Defects in muscle glycogen synthesis in diabetes of the same order are well known from magnetic resonance studies. We conclude that measurement of label incorporation into body water is potentially useful for investigation of the metabolism of a glucose load in vivo during an IVGTT.

Evaluación del consumo de leche humana por dilución con deuterio y detección por espectroscopia de infrarrojo / Assessment of human milk consumption by dilution with deuterium and detection by infrared spectroscopy

Objetivo. Adaptar la metodología de evaluación de ingestión de leche humana con isótopos estables utilizando un espectroscopio infrarrojo en lugar de un espectrómetro de masas. Material y métodos. Previo consentimiento informado, 12 mujeres que estaban amamantando recibieron 30 g de D2O por vía oral. a las 4 horas y 1, 3, 6, 9 y 14 días depués de la dosificación, se les tomó una muestra de saliva. Las muestras de saliva de los bebés se tomaron a los 1,2,5,6,13 y 14 días. Todas las muestras fueron preparadas por sublimación y centrifucación y la determinación de la concentración de D2O se determinó por espectroscopía de infrarrojo. Resultados. La recuperación del sistema de sublimación fue superior al 95 por ciento del volumen total tanto de agua sola como de saliva. Se encontró que con tres evaluaciones se alcanza la misma confiabilidad que con 5 evaluaciones. Los volúmenes de ingestión de leche humana fueron similares a los informados por otros autores