Plasma free fatty acids do not provide the link between obesity and insulin resistance or ß-cell dysfunction: results of the Reading, Imperial, Surrey, Cambridge, Kings (RISCK) study.

AIMS: To investigate the relationship between adiposity and plasma free fatty acid levels and the influence of total plasma free fatty acid level on insulin sensitivity and ß-cell function. METHODS: An insulin sensitivity index, acute insulin response to glucose and a disposition index, derived from i.v. glucose tolerance minimal model analysis and total fasting plasma free fatty acid levels were available for 533 participants in the Reading, Imperial, Surrey, Cambridge, Kings study. Bivariate correlations were made between insulin sensitivity index, acute insulin response to glucose and disposition index and both adiposity measures (BMI, waist circumference and body fat mass) and total plasma free fatty acid levels. Multivariate linear regression analysis was performed, controlling for age, sex, ethnicity and adiposity. RESULTS: After adjustment, all adiposity measures were inversely associated with insulin sensitivity index (BMI: ß = -0.357; waist circumference: ß = -0.380; body fat mass: ß = -0.375) and disposition index (BMI: ß = -0.215; waist circumference: ß = -0.248; body fat mass: ß = -0.221) and positively associated with acute insulin response to glucose [BMI: ß = 0.200; waist circumference: ß = 0.195; body fat mass ß = 0.209 (P values <0.001)]. Adiposity explained 13, 4 and 5% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. After adjustment, no adiposity measure was associated with free fatty acid level, but total plasma free fatty acid level was inversely associated with insulin sensitivity index (ß = -0.133), acute insulin response to glucose (ß = -0.148) and disposition index [ß = -0.218 (P values <0.01)]. Plasma free fatty acid concentration accounted for 1.5, 2 and 4% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. CONCLUSIONS: Plasma free fatty acid levels have a modest negative association with insulin sensitivity, ß-cell secretion and disposition index but no association with adiposity measures. It is unlikely that plasma free fatty acids are the primary mediators of obesity-related insulin resistance or ß-cell dysfunction.

An approach to quantifying abnormalities in energy expenditure and lean mass in metabolic disease.

BACKGROUND/OBJECTIVES: The objective of this study was to develop approaches to expressing resting energy expenditure (REE) and lean body mass (LM) phenotypes of metabolic disorders in terms of Z-scores relative to their predicted healthy values. SUBJECTS/METHODS: Body composition and REE were measured in 135 healthy participants. Prediction equations for LM and REE were obtained from linear regression and the range of normality by the standard deviation of residuals. Application is demonstrated in patients from three metabolic disorder groups (lipodystrophy, n=7; thyrotoxicosis, n=16; and resistance to thyroid hormone (RTH), n=46) in which altered REE and/or LM were characterised by departure from the predicted healthy values, expressed as a Z-score. RESULTS: REE (kJ/min) = -0.010 × age (years)+0.016 × FM (kg)+0.054 × fat-free mass (kg)+1.736 (R2 = 0.732, RSD = 0.36 kJ/min). LM (kg)=5.30 × bone mineral content (kg)+10.66 × height2 (m)+6.40 (male). LM (kg)=0.20 × fat (kg)+14.08 × height2 (m)-2.93 (female).(male R2=0.55, RSD = 3.90 kg; female R2 = 0.59, RSD=3.85 kg).We found average Z-scores for REE and LM of 1.77 kJ/min and -0.17 kg in the RTH group, 5.82 kJ/min and -1.23 kg in the thyrotoxic group and 2.97 kJ/min and 4.20 kg in the LD group. CONCLUSION: This approach enables comparison of data from individuals with metabolic disorders with those of healthy individuals, describing their departure from the healthy mean by a Z-score.

Effect of protein intake and weight gain velocity on body fat mass at 6 months of age: the EU Childhood Obesity Programme.

INTRODUCTION: Higher protein intake during the first year of life is associated with increased weight gain velocity and body mass index (BMI). However, the relationship of protein intake and weight gain velocity with body composition is unclear. OBJECTIVE: To assess if the increases in weight gain velocity and BMI induced by protein intake early in life are related to an increase in fat or fat-free mass. MATERIALS AND METHODS: In all, 41 infants randomized at birth to a higher or lower protein content formula (HP=17 and LP=24, respectively) and 25 breastfed infants were included. Anthropometric measures were assessed at baseline, 6, 12 and 24 months, and fat-free mass (FFM) and fat mass (FM) were assessed by isotope dilution at 6 months. RESULTS: Weight gain velocity (g per month) during the first 6 months of life was significantly higher among HP infants (807.8 (±93.8) vs 724.2 (±110.0) (P=0.015)). Weight gain velocity strongly correlated with FM z-score (r=0.564, P<0.001) but showed no association with FFM z-scores. FFM showed no association with BMI. Nevertheless, FM strongly correlated with BMI at 6, 12 and 24 months (r=0.475, P<0.001; r=0.332, P=0.007 and r=0.247, P=0.051, respectively). FFM and FM z-scores did not differ significantly between HP and LP infants (0.32±1.75 vs -0.31±1.17 and 0.54±2.81 vs -0.02±1.65, respectively). CONCLUSION: Our findings support the hypothesis that higher protein intakes early in life are associated with faster weight gain and in turn to higher adiposity. This mechanism could be a determinant factor for later obesity risk.

Measurement of in vivo nitric oxide synthesis in humans using stable isotopic methods: a systematic review.

Stable isotopic methods are considered the "gold standard" for the measurement of rates of in vivo NO production. However, values reported for healthy human individuals differ by more than 1 order of magnitude. The reason for the apparent variability in NO production is unclear. The primary aim of this review was to evaluate and compare the rates of in vivo NO production in health and disease using stable isotope methods. Articles were retrieved using the PubMed electronic database. Information on concentrations, isotopic enrichments of fluxes, and conversion rates of molecules involved in the NO metabolic pathway was extracted from selected articles; 35 articles were included in the final analysis. Three protocols were identified, including the arginine-citrulline, the arginine-nitrate, and the oxygen-nitrate protocols. The arginine-citrulline protocol showed a wider variability compared to the arginine-nitrate and oxygen-nitrate protocols. The direction of the association between disease state and rate of NO production was essentially determined by the etiopathogenesis of the disorder (inflammatory, metabolic, vascular). Considerable variation in methodologies used to assess whole-body NO synthesis in humans exists. The precision of several aspects of the techniques and the validity of some assumptions made remain unknown, and there is a paucity of information about physiological rates of NO production from childhood over adolescence to old age.

Post-challenge hyperglycaemia, nitric oxide production and endothelial dysfunction: the putative role of asymmetric dimethylarginine (ADMA).

The endothelium is a thin layer of cells at the internal surface of blood vessels in continuous contact with the circulating fluids. The endothelial cells represent the primary barrier for the transport of glucose from the vascular conduits into the interstitial space. Insulin and nitric oxide have an important role in the regulation of glucose transport and metabolism. Hyperglycaemia is the main criteria for the diagnosis of diabetes and is responsible for the micro- and macro-vascular pathology seen in diabetic patients. Recent evidence suggests that post-challenge hyperglycaemia is a better predictor of cardiovascular risk than fasting glucose. Acute glucose elevations have been associated with a reduced endothelial-dependent flow mediated dilation indicating a decrease in nitric oxide production. Post-prandial hyperglycaemic peaks have been directly associated with increased intima media thickness in type 2 diabetic patients indicative of an increased atherosclerotic risk. The increase in intra-cellular glucose concentrations in the endothelial cells induces a hyper-generation of reactive oxygen species via the activation of different pathways (polyol-sorbitol, hexosamine, advanced glycated end products, activation of PKC, asymmetric dimethylarginine (ADMA)). These mechanisms influence the expression of genes and release of signalling and structural molecules involved in several functions (inflammation, angiogenesis, coagulation, vascular tone and permeability, cellular migration, nutrient metabolism). ADMA is considered as a biomarker of endothelial dysfunction and it has been associated with an increased risk of atherosclerosis and cardiovascular diseases. The increased generation of ADMA and reactive oxygen species in subjects with persistent hyperglycaemia could lead to an impairment of nitric oxide synthesis.

Recent advances in the interpretation of the (13)C octanoate breath test for gastric emptying.

The (13)C octanoate breath test for gastric emptying has still not achieved its full potential in clinical practice, largely because of uncertainty in how to relate its results to those of more established techniques, such as gamma scintigraphy. Here we briefly review the test and then go on to discuss recent advances in its validation and interpretation.

A stable isotope method for the simultaneous measurement of vitamin K1 (phylloquinone) kinetics and absorption.

OBJECTIVES: To measure uptake and disposal kinetics and absolute absorption of vitamin K(1) using two stable isotope-labelled forms of vitamin K(1). SUBJECTS: Ten subjects (nine women and one man) aged between 22 and 31 years, with a mean (+/-standard deviation) body mass index of 22.5+/-2.4 kg/m(2). Subjects took capsules containing 3 microg of methyl-(13)C vitamin K(1), three times a day for six days to reach a steady state for plasma vitamin K(1) isotopic enrichment. On day seven, subjects were given an intravenous dose of Konakion MM to measure disposal kinetics and at the same time, a capsule containing 4 microg of ring-D(4) vitamin K(1) to measure absorption. Plasma vitamin K(1) concentration was measured by high-performance liquid chromatography and isotopic composition by gas chromatography mass spectrometry. RESULTS: The disposal kinetics of the intravenous dose of vitamin K(1) were resolved into two exponentials with half-times of 0.22 (+/-0.14) and 2.66 (+/-1.69) h. Absorption of oral, deuterated vitamin K(1) was 13 (+/-9)%. CONCLUSIONS: Two-compartmental kinetic parameters observed in this study are similar to those obtained previously using radioactive tracers, but there may be additional slow-turnover body pools acting as stores of vitamin K(1). The kinetic parameters determined from the intravenous dose allowed determination of the absolute absorption of vitamin K(1) from a bolus oral dose.

Frequent feeding delays the gastric emptying of a subsequent meal.

To assess the suitability of the 13C-octanoic acid breath test for measuring gastric emptying in circumstances other than the post-absorptive state, a preliminary study was performed where 6 hourly spaced isoenergetic meals preceded the determination of gastric emptying of a subsequent 2 MJ meal. Emptying was measured in three individuals on four separate occasions, with a reproducibility of 8%. A crossover study was then conducted to test the hypothesis that meal frequency can modulate the gastric emptying of a subsequent meal, with the potential to influence appetite regulation. Sixteen subjects were fed to energy balance, receiving food either as 2 isoenergetic meals 3 h apart or 6 isoenergetic meals fed hourly. Gastric emptying of a subsequent 2 MJ meal was investigated. Visual analogue scales were used throughout to assess appetite. The maximum rate of gastric emptying was unchanged but the onset of emptying was delayed by the more frequent feeding pattern. There was no significant difference in subjective appetite before or after the test meal. In conclusion, short-term increases in feeding frequency delayed the gastric emptying of a subsequent meal, but significant effects on post-meal appetite could not be demonstrated.

Delayed gastric emptying in the obese: an assessment using the non-invasive (13)C-octanoic acid breath test.

AIM: Much of the controversy surrounding the correlation between obesity and gastric emptying lies in the inconsistency of methodology and analysis. This study was designed to overcome some of the discrepancies encountered in previous studies and to test the hypothesis that obese individuals have altered gastric emptying compared to lean individuals. METHODS: Gastric emptying was measured using the (13)C-octanoic acid breath test in 16 lean and 16 obese women pair-matched for age. Following an overnight fast, subjects were given a standard 2 MJ egg meal labelled with 100 microl of [1-(13)C]-octanoic acid. Breath samples were collected at regular intervals over a 6-h period. (13)C-isotopic enrichment in the breath was analysed using isotope ratio mass spectrometry and the data fitted to the established gastric emptying model. The lag times (t(lag)), half excretion times (t(1/2)), latency phase (t(lat)) and ascension times (t(asc)) were calculated. RESULTS: The mean t(1/2)-values (+/-standard error of the mean) were 3.67 +/- 0.14 h and 4.23 +/- 0.18 h for lean and obese respectively, indicating significantly delayed gastric emptying in the obese (p = 0.019). The obese group also showed a significantly slower lag time (t(lag), p = 0.005) and latency phase (t(lat), p = 0.005), but no significant difference was found in the ascension time (t(asc), p = 0.154). Within groups, no correlation was found between half excretion times and body weight or half excretion times and body mass index. CONCLUSIONS: The present study demonstrated a prolonged lag phase and delayed gastric emptying in obese women when compared to lean women. This delay may be as a consequence of high-fat diets, a sedentary lifestyle and increased gastric distension associated with obesity, or a contributing factor in the pathogenesis of obesity resulting from the inactivation of gastrointestinal satiety signals and in an increase in food intake.

De novo lipogenesis during controlled overfeeding with sucrose or glucose in lean and obese women.

BACKGROUND: The results of previous studies suggest that de novo lipogenesis may play an important role in the etiology of obesity, particularly during overconsumption of different carbohydrates. OBJECTIVE: We hypothesized that de novo lipogenesis would increase during overfeeding, would vary depending on the type of carbohydrate consumed, and would be greater in obese than in lean women. DESIGN: De novo lipogenesis was measured during 96 h of overfeeding by 50% with either sucrose or glucose and during an energy balance treatment (control) in 8 lean and 5 obese women. De novo lipogenesis was determined by measuring the amount of deuterium incorporation into plasma triacylglycerols. Fat and carbohydrate balance were measured simultaneously by continuous whole-body calorimetry. RESULTS: De novo lipogenesis did not differ significantly between lean and obese subjects, except with the control treatment, for which de novo lipogenesis was greater in the obese subjects. De novo lipogenesis was 2- to 3-fold higher after overfeeding by 50% than after the control treatment in all subjects. The type of carbohydrate overfeeding (sucrose or glucose) had no significant effect on de novo lipogenesis in either subject group. Estimated amounts of absolute VLDL production ranged from a minimum of 2 g/d (control) to a maximum of 10 g/d after overfeeding. This compares with a mean fat balance of approximately 275 g after 96 h of overfeeding. Individual subjects showed characteristic amounts of de novo lipogenesis, suggesting constitutive (possibly genetic) differences. CONCLUSION: De novo lipogenesis increases after overfeeding with glucose and sucrose to the same extent in lean and obese women but does not contribute greatly to total fat balance.

Daidzein and genistein content of fruits and nuts.

Dietary phytoestrogens such as the isoflavones daidzein and genistein are thought to protect against chronic diseases that are common in Western societies, such as cancer, osteoporosis, and ischemic heart disease. In addition, there are concerns regarding the deleterious effects of hormone-like compounds, especially with respect to the development of infants. However, there is little information regarding the phytoestrogen content of foods, and therefore epidemiologic investigations of phytoestrogens are limited. As part of a study quantifying the consumption of phytoestrogens, the objective of this work was to assess the daidzein and genistein content of fruits and nuts commonly eaten in Europe. Eighty different fruits and nuts were sampled, prepared for eating, and freeze-dried. Daidzein and genistein were extracted from the dried foods, and the two isoflavones were quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 80 foods assayed, 43 contained no detectable daidzein or genistein, at a limit of quantification of 1 microg/kg dry weight of food. Nine foods contained more than 100 microg of the two isoflavones combined per kilogram wet weight, and 28 contained less than this amount. Currants and raisins were the richest sources of the isoflavones, containing 2,250 microg and 1,840 microg of the two isoflavones combined per kilogram of wet weight of food. Although fruits and nuts are not as rich in isoflavone phytoestrogens as are soy and other legumes, this is the first documentation of levels of daidzein and genistein occurring in these foods.

Daidzein and genistein contents of vegetables.

Food samples (n 114) were prepared from vegetables commonly eaten in Europe. The glycosidic forms of the phyto-oestrogens daidzein and genistein were extracted from the dried foods into aqueous methanol. The isoflavones were quantified by GC-MS after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 114 foods assayed, at a limit of quantification of 0.1 microg/kg dry weight, forty-eight contained no detectable daidzein or genistein, forty-one contained less than 100 microg/kg of the two isoflavones combined and the remaining twenty-five contained more than this amount. Soyabean products contained between 470 and 1420 mg (average of 960 mg) daidzein and genistein combined per kg wet weight of food, and legumes contained between 20 and 5750 microg/kg wet weight of food, with an average of 620 microg/kg. Cooking by boiling in water caused a decrease in the daidzein and genistein content of food in twenty-four of twenty-eight foods. The extent of the decrease was variable and warrants further investigation. The present paper comprises the first comprehensive description of the content of daidzein and genistein in vegetables.

The use of infrared spectrophotometry for measuring body water spaces.

BACKGROUND: The conventional method of measuring total body water by the deuterium isotope dilution method uses gas isotope ratio mass spectrometry (IRMS), which is both expensive and time-consuming. We investigated an alternative method, using Fourier transform infrared spectrophotometry (FTIR), which uses less expensive instrumentation and requires little sample preparation. METHOD: Total body water measurements in human subjects were made by obtaining plasma, saliva, and urine samples before and after oral dosing with 1.5 mol of deuterium oxide. The enrichments of the body fluids were determined from the FTIR spectra in the range 1800-2800 cm-1, using a novel algorithm for estimation of instrumental response, and by IRMS for comparison. RESULTS: The CV (n = 5) for repeat determinations of deuterium oxide in biological fluids and calibrator solutions (400-1000 micromol/mol) was found to be in the range 0.1-0.9%. The use of the novel algorithm instead of the integration routines supplied with the instrument gave at least a threefold increase in precision, and there was no significant difference between the results obtained with FTIR and those obtained with IRMS. CONCLUSION: This improved infrared method for measuring deuterium enrichment in plasma and saliva requires no sample preparation, is rapid, and has potential value to the clinician.

Extraction and quantification of daidzein and genistein in food.

A simple analytical method has been developed for routine quantification of a broad range of concentrations of the isoflavones daidzein and genistein in food. The synthetic glucosides daidzin and genistin were used as internal standards, combined with each food prior to extraction. The recovery of the aglycones daidzein and genistein from these internal standards were used to ensure the completeness of the extraction and aid quantification of isoflavones from the food. Hydrolytic enzymes from Aspergillus niger were used, in aqueous buffer, to liberate daidzein and genistein from their respective glycosides. The aglycone isoflavones were partitioned from the aqueous buffer into ethyl acetate. After evaporation of the ethyl acetate under nitrogen, the isoflavones were derivatized with N-tert-(butyldimethylsilyl)-N-methyltrifluoroacetamide and quantified by comparison with authentic synthetic standards using gas chromatography-mass spectrometry in selected ion mode. The isoflavone content of a stock soy flour was determined, using 36 separate assays, to be 1.05 mg daidzein and 1.11 mg genistein per gram of freeze-dried food, and the interassay coefficient of variation was 2.7 and 4.7, respectively.

Phyto-oestrogens: where are we now?

Phyto-oestrogens have emerged from their esoteric role in animal husbandry following the hypothesis that the human Western diet is relatively deficient in these substances compared with societies where large amounts of plant foods and legumes are eaten. Evidence is beginning to accrue that they may begin to offer protection against a wide range of human conditions, including breast, bowel, prostate and other cancers, cardiovascular disease, brain function, alcohol abuse, osteoporosis and menopausal symptoms. Of the two main classes of these weak oestrogens, the isoflavones are under intensive investigation due to their high levels in soyabean. Like the 'anti-oestrogen' Tamoxifen, these seem to have oestrogenic effects in human subjects in the cardiovascular system and bone. Although previously only available from food, isoflavones are now being marketed in health-food supplements or drinks, and tablets may soon be available over the counter as 'natural' hormone-replacement therapy. In cancer, anti-oestrogenic effects are thought to be important, although genistein especially has been shown to induce wide-ranging anti-cancer effects in cell lines independent of any hormone-related influence. There are few indications of harmful effects at present, although possible proliferative effects have been reported. In infants, the effects of high levels in soya milk formulas are uncertain. The second group, lignans, have been less investigated despite their known antioestrogenic effects and more widespread occurrence in foods. Investigation of the possible benefits of phyto-oestrogens is hampered by lack of analytical standards and, hence, inadequate methods for the measurement of low levels in most foods. This problem may prove to be a major dilemma for regulatory authorities, clinicians and others wishing to advise the general public on whether these compounds really do have the health benefits attributed to them.

Measurement of ascorbic acid kinetics in man using stable isotopes and gas chromatography/mass spectrometry.

The feasibility of studying ascorbic acid kinetics in man using stable isotope-labelled tracers and gas chromatographic (GC) separation followed by mass spectrometric (MS) quantitation was assessed. Preliminary studies with 13C-labelled material showed that although better precision at low levels could be achieved using the GC/combustion/MS technique, consideration of likely enrichments in a human study made the simpler GC/MS method just as suitable. On this basis, a small pilot study of the kinetics in man was carried out. The enrichment of the ascorbic acid in plasma was measured for a 24 h period after oral administration of 13C-labelled material. The results were fitted to a simple three-compartment model and rate constants and pool sizes were deduced. The results obtained are comparable to those obtained in other published studies, from which we conclude that the technique may be useful as a non-invasive method for the assessment of nutritional status in a variety of populations.