Nutritional and social contribution of meat in diets: Interplays among young urban and rural men.

Considering the recent increase in the demand for meat and its subsequent implications for health and food security, there is an increasing need to explore its nutritional and social importance among young men in settings experiencing nutrition transition. A better understanding of meat in the diets of this group could contribute to the design of socio-culturally appropriate interventions to improve healthy eating, as these men are key decision makers in family food choices. This mixed-methods study aimed to assess the nutritional and social contribution of meat in the diet of young adult men in urban and rural Zambia. A food frequency questionnaire, multiple pass 24-h dietary recall, anthropometric measurements and a socio-demographic questionnaire were utilized while qualitative interviews explored the socio-cultural importance of meat consumption. Rural and urban participants had an isocaloric diet. All macronutrient intakes except carbohydrates were significantly higher in the urban population than the rural population (p < 0.01). Zinc intake was significantly greater in the urban than the rural sample (χ2 (39) = 40, p-value = 0.04). Except for vitamin A, calcium and folate, participants met the recommendations for all micronutrients. Regardless of being rural or urban, the higher the participant's level of education, the weaker the socio-cultural importance of meat. In both settings, increased consumption of meat was associated with prosperity, authority and respect within society. There are strong social and cultural beliefs among participants about meat consumption, reflecting the symbolic meaning in their customs. These findings could help improve the design and implementation of dietary interventions, incorporating specific cultural beliefs and socio-economic factors in the targeted population, to achieve healthy eating practices.

A systematic review of iodine deficiency among women in the UK.

OBJECTIVE: The present study reviewed the literature on iodine status among women of childbearing age and pregnant women in the UK. Particular attention was given to study quality and methods used to assess iodine status. DESIGN: A systematic review was conducted to examine the literature and critically evaluate study design. SETTING: Studies were identified in PubMed, Web of Science, Scopus and Ovid MEDLINE databases, as well as from secondary references. PARTICIPANTS: Women of childbearing age or pregnant, living in the UK. RESULTS: Fifty-seven articles were identified and twelve articles were selected, including a total of 5283 women. Nine studies conducted urinary iodine assessments, three studies conducted dietary assessments only, and seven studies classified their target population as iodine deficient according to WHO criteria. CONCLUSIONS: No single study from the selected articles could produce nationally representative results regarding the prevalence of iodine deficiency among the female population in the UK. Consideration of the evidence as a whole suggests that women of childbearing age and pregnant women in the UK are generally iodine insufficient. Further large-scale research is required for more accurate and reliable evidence on iodine status in the UK.

Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype.

PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. RESULTS: HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. CONCLUSION: Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC.

Fortified breakfast cereal consumed daily for 12 wk leads to a significant improvement in micronutrient intake and micronutrient status in adolescent girls: a randomised controlled trial.

BACKGROUND: Poor micronutrient status is reported among adolescents across Europe and USA. This may be related to the well-documented decline in the regular consumption of breakfast by this group. The regular consumption of a breakfast cereal offers a possible means to improve micronutrient status; fortified cereal is likely to have enhanced benefit. A study was conducted to determine the efficacy of the regular consumption of a fortified cereal with milk, compared with unfortified cereal, consumed either as a breakfast or a supper, in improving micronutrient intake and micronutrient status of adolescent girls. METHODS: A randomised, double-blind, placebo-controlled intervention trial was conducted in girls recruited at ages 16-19 years, from schools and colleges in Sheffield, UK. Girls were randomised to receive 50 g fortified or unfortified cereal, with 150 ml semi-skimmed milk, daily, for 12 weeks, as a breakfast or as a supper. Dietary intake was estimated using a 4-d food diary and blood collected for the assessment of nutritional status. Within-group changes were tested using a paired sample t test; two-way ANOVA was used to analyse effects of the intervention, with cereal type and time of consumption as factors, correcting for baseline values. The analysis was conducted on 71 girls who completed the study. RESULTS: Consumption of unfortified cereal elicited an increase in the intake of vitamins B1, B2 and B6; consumption of fortified cereal elicited increases in vitamins B1, B2, B6, B12, folate and iron (P < 0.001) and of vitamin D (P = 0.007), all increases were significantly greater than for unfortified cereal. Consumption of the fortified cereal also led to a significant improvement in biomarkers of status for vitamins B2, B12, folate and of iron, compared with girls receiving the unfortified cereal, and maintained vitamin D status, in contrast with the girls receiving the unfortified cereal (P < 0.001). CONCLUSIONS: The daily consumption of cereal with milk for 12 weeks by adolescent girls, increased intakes of micronutrients. The consumption of fortified cereal elicited greater increases than for unfortified cereal and improved biomarkers of micronutrient status. The findings justify strategies to encourage the consumption of fortified cereal with milk by adolescents, either as a breakfast or a supper. TRIAL REGISTRATION: Registered with Current Controlled Trials (Registration: ISRCTN55141306 ).

Effects of an exercise and hypocaloric healthy eating intervention on indices of psychological health status, hypothalamic-pituitary-adrenal axis regulation and immune function after early-stage breast cancer: a randomised controlled trial.

INTRODUCTION: Many women experience emotional distress, depression and anxiety after a diagnosis of breast cancer. Psychological stress and depression have been associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation that may adversely affect immune system functioning and impact upon survival. This study investigated the effects of a lifestyle intervention on indices of psychological health status, HPA axis regulation and immune function in overweight women recovering from early-stage breast cancer treatment. METHODS: A total of 85 women treated for breast cancer 3 to 18 months previously were randomly allocated to a 6-month exercise and hypocaloric healthy eating program plus usual care or usual care alone (control group). Women in the intervention group received three supervised exercise sessions per week and individualized dietary advice, supplemented by weekly nutrition seminars. Depressive symptoms (Beck Depression Inventory version II: BDI-II), perceived stress (Perceived Stress Scale: PSS), salivary diurnal cortisol rhythms; inflammatory cytokines (IL-6 and Tumor necrosis factor-α), leukocyte phenotype counts, natural killer (NK) cell cytotoxicity and lymphocyte proliferation following mitogenic stimulation were assessed at baseline and 6-month follow up. RESULTS: Compared with the control group, the intervention group exhibited a reduction in depressive symptoms (adjusted mean difference, 95% confidence intervals (95% CI): -3.12, -1.03 to -5.26; P = 0.004) at the 6-month follow-up but no significant decrease in PSS scores (-2.07, -4.96 to 0.82; P = 0.16). The lifestyle intervention also had a significant impact on diurnal salivary cortisol rhythm compared with usual care alone, as evidenced by an increase in morning salivary cortisol at the 6-month follow-up (P <0.04), indicating a change in HPA axis regulation. Women in the control group had higher total leukocyte, neutrophil and lymphocyte counts in comparison to the intervention group at the 6-month follow-up (P ≤0.05), whereas there was no difference in NK cell counts (P = 0.46), NK cell cytotoxicity (P = 0.85) or lymphocyte proliferation responses (P = 0.11) between the two groups. CONCLUSION: Our results show that the lifestyle intervention resulted in a reduction in depressive symptoms and a normalisation of HPA axis regulation. Such changes could have important implications for long-term survival in women recovering from early-breast cancer treatment. TRIAL REGISTRATION: Current Controlled Trials: ISRCTN08045231.

Tumour suppressor gene methylation and cervical cell folate concentration are determinants of high-risk human papillomavirus persistence: a nested case control study.

BACKGROUND: Persistent infection with one or more high-risk human papillomavirus [HR-HPV] types increases the risk of intraepithelial neoplasia and cervical cancer. A nested case-control study was conducted to investigate the importance of cervical cell folate concentration and tumour suppressor gene methylation as risk factors for HR-HPV persistence. METHODS: Cervical cell samples from 955 women with HR-HPV infection and normal, borderline or mild dyskaryosis were retrieved from the archive of a population-based screening trial. Women were classified as cases or controls, reflecting the presence or absence [respectively] of any HR-HPV infection at a follow-up clinic at least 6 months from baseline. Cervical cell folate concentration and promoter methylation of five tumour suppressor genes were measured in independent samples from cases and controls. RESULTS: A higher cervical cell folate concentration [P = 0.015] was an independent predictor of infection at follow-up, together with infection with HPV-16 or infection with multiple HR-HPV types. Methylation of the tumour suppressor gene DAPK was associated with a 2.64-fold [95% CI, 1.35-5.17] increased likelihood of HPV infection whilst CDH1 methylation was associated with a 0.53-fold [95% CI, 0.331-0.844] likelihood of HR-HPV infection at follow-up. When considering women with normal or abnormal cytology, the predictive effect of higher cervical cell folate was only seen in women with mild cytology [P = 0.021]; similarly the effect of DAPK methylation was seen in women with mild or borderline cytology [P < 0.05]. CONCLUSIONS: Higher cervical cell folate concentration and promoter methylation of the tumour suppressor gene, DAPK, in women with cervical cell dyskaryosis, are associated with increased risk of HR-HPV persistence.

A vitamin B-12 supplement of 500 µg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 µg/d, 100 µg/d, and 10 µg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (µmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 µg/d, but even 500 µg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 µg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Riboflavin depletion of intestinal cells in vitro leads to impaired energy generation and enhanced oxidative stress.

BACKGROUND: Riboflavin is an essential component of the human diet, with an established role for its derivative cofactors in oxidative metabolism. Our previous in vivo data suggest that riboflavin may act as a signalling molecule in the intestinal lumen, regulating crypt development and cell turnover. Our in vitro studies in riboflavin-depleted intestinal cells in culture indicate that riboflavin depletion impairs normal mitosis. METHODS: The aim of the study was to establish an improved intestinal cell model of riboflavin depletion using the structural analogue of riboflavin, lumiflavin (7,8,10-trimethyl-isoalloxazine) and to determine effects on cell function. The study was conducted using three intestinal cell lines, Caco-2, HCT116 and HT29 cells. RESULTS: Cell growth was inhibited in all three cell lines, in a lumiflavin concentration-dependent manner. Riboflavin depletion was confirmed through a significant decrease in intracellular riboflavin concentrations in Caco-2 and HT29 cell lines and a significant increase in the activation coefficient for the flavin adenine dinucleotide-dependent enzyme glutathione reductase. Riboflavin depletion led to a significant reduction in intracellular ATP concentration, and an enhanced generation of reactive oxygen species was also observed in response to riboflavin depletion, in all cell lines; effects were at least fivefold greater in Caco-2 cells than other cells. Riboflavin-depleted Caco-2 and HCT116 cells also showed an irreversible loss of proliferative potential. CONCLUSIONS: A model system of intracellular riboflavin depletion in intestinal epithelial cells has been developed. Riboflavin depletion induced by lumiflavin results in oxidative stress and a disruption of energy generation, which may contribute to observed effects on cell proliferation.

Systemic folate status, rectal mucosal folate concentration and dietary intake in patients at differential risk of bowel cancer (The FAB2 Study).

BACKGROUND/OBJECTIVES: Folate has been strongly implicated in the aetiology of colorectal cancer. However, the relationship between dietary folate intake, rectal mucosal folate status and colorectal cancer risk is uncertain. The study aimed to estimate nutrient intakes and measure systemic folate status and rectal mucosal folate concentration in people at differential risk of developing colorectal cancer. METHODS: Two hundred and twenty-eight individuals were recruited from gastroenterology clinics and subdivided into three patient groups: untreated colorectal cancer (n = 43), adenomatous polyps (n = 90) or normal bowel (n = 95). Biopsies from macroscopically normal rectal mucosa and blood were collected and used for the measurement of rectal mucosal 5-methyltetrahydrofolate (5-MeTHF) and systemic markers of folate status, respectively. Nutrient intake was estimated using a validated food frequency questionnaire. RESULTS: Dietary intake variables, plasma 5-MeTHF and red cell folate and plasma homocysteine concentrations were similar in all three subject groups and 95% CI fell within normal range for each variable. Rectal mucosal 5-MeTHF concentration was higher in the normal mucosa of adenomatous polyp patients than in normal subjects (P = 0.055). Rectal mucosal 5-MeTHF was associated significantly with plasma folate (P < 0.001, r = 0.294), red cell folate (P = 0.014, r = 0.305), plasma homocysteine (P = 0.017, r = -0.163) and dietary folate intake (P = 0.036, r = 0.152). CONCLUSIONS: This study demonstrates adequate folate status of patients attending gastroenterology clinics for the investigation of bowel symptoms, with no significant difference in dietary intakes or systemic folate status indices according to diagnosis. Rectal mucosal 5-MeTHF concentrations were elevated in adenomatous polyp patients, but failed to reach significance. Further studies are required to determine the biological significance of this observation.

Riboflavin in development and cell fate.

Riboflavin (7,8-dimethyl-10-ribitylisoalloxazine; vitamin B2) is a water-soluble vitamin, cofactor derivatives of which (FAD, FMN) act as electron acceptors in the oxidative metabolism of carbohydrate, amino acids and fatty acids and which in the reduced state can donate electrons to complex II of the electron transport chain. This means that riboflavin is essential for energy generation in the aerobic cell, through oxidative phosphorylation. The classic effects of riboflavin deficiency on growth and development have generally been explained in terms of these functions. However, research also suggests that riboflavin may have specific functions associated with cell fate determination, which would have implications for growth and development. In particular, riboflavin depletion interferes with the normal progression of the cell cycle, probably through effects on the expression of regulatory genes, exerted at both the transcriptional and proteomic level.

Effects of mixed dietary supplements on total plasma homocysteine concentrations (tHcy): a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Although a number of studies have reported raised total plasma homocysteine (tHcy) concentrations in free-living older people, there are no data on homocysteine response to a mixed nutrient supplement in older patients. A raised plasma homocysteine concentration in older patients is partly a reflection of their co-morbidity, including impaired renal function, and there is uncertainty about the extent to which dietary interventions can improve plasma tHcy. AIM: To determine the plasma tHcy response to dietary supplements during acute illness. METHODS: Two-hundred and thirty-six hospitalized, acutely ill older patients, who were part of a randomized double-blind placebo-controlled trial, were assigned to receive a daily oral nutritional supplement drink containing 1.3 mg of vitamin B2, 1.4 mg of vitamin B6, 1.5 µg of B12, 200 µg of folic acid, or a placebo, for 6 weeks. Outcome measures were plasma tHcy concentration at baseline, 6 weeks, and 6 months. RESULTS: The mean plasma tHcy concentration fell among patients given the supplements (mean difference 4.1 µmol/L [95 % C.I, 0.14 to 8.03), p = 0.043], but tHcy concentration increased between 6 weeks and 6 months, after patients stopped taking the supplements [mean difference -2.0 µmol/L (95 % C.I, -03.9 to -0.18), p = 0.033]. About 46 % of patients in the placebo group and 55 % of patients in the supplement group had hyperhomocysteinemia (>14 µmol/L) at baseline compared with 45 % and 29 % at the end of the treatment period. CONCLUSIONS: A mixed nutrient supplement containing physiological amounts of B vitamins significantly reduced plasma tHcy concentrations in older patients recovering from acute illness.

Riboflavin depletion impairs cell proliferation in adult human duodenum: identification of potential effectors.

BACKGROUND AND AIMS: Riboflavin (vitamin B2) is an essential dietary component with a known function in oxidative metabolism. Our previous data using a rat model of riboflavin deficiency suggested that riboflavin also functions as a luminal signaling molecule regulating crypt development and cell turnover. Riboflavin deficiency is prevalent in both high- and low-income countries across the globe. This study aims to establish whether riboflavin deficiency has consequences for gastrointestinal (GI) morphology in adults and what the effects and effectors of any such alteration may be. METHODS: Duodenal biopsies and blood samples were collected from a cross-section of gastroscopy patients. Crypt morphology and cell division were studied by immunohistochemistry, and biochemical riboflavin status was determined. Additionally a cell culture model of riboflavin deficiency was developed and analyzed using a combination of flow cytometry, and microarray and clonogenic assays. RESULT: Duodenal crypts from subjects in the lowest quartile of riboflavin status were significantly shorter (P=0.023), less cellular (P=0.007), and had fewer cell divisions (P=0.034) than the crypts of subjects in the top quartile of riboflavin status. Following riboflavin depletion of colon cells in culture, cell cycle slowed. Microscopy revealed impaired mitosis and accumulation of aneuploid cells. Alterations in gene expression profiles reflected this alteration, with several mitosis-related genes altered, including AspM, cyclin B1, and Birc5 downregulated and Kif23 upregulated. Riboflavin depletion in vitro caused irreversible loss of proliferative potential of cells. CONCLUSIONS: Riboflavin depletion in adult humans impairs proliferation and proliferative potential of intestinal cells, which may have implications for gastrointestinal function.

Approaches to setting dietary reference values for micronutrients, and translation into recommendations.

Dietary reference values (DRV) are estimates of the daily amounts of nutrients or food energy that meet the needs of healthy people. In the UK, three terms are used to express these estimates, assuming a normal distribution of requirements in a population. These are the estimated average requirement, the lower reference nutrient intake and the reference nutrient intake. DRV are for use in a variety of settings, including the assessment of adequacy and safety of nutrient or energy intake in a population group, in the design of meal provision in care settings, in food labelling and in considering food fortification strategies. DRV, and other expressions of nutrient requirements, assume a relationship between the intake of a nutrient and some criterion of adequacy, the outcome. Estimates of requirements are based on a diverse range of measures of adequacy, according to available evidence. The Scientific Advisory Committee on Nutrition (SACN) is the body responsible for reviewing and setting DRV for the UK population. The work of SACN is guided by a framework of evidence that relates food and nutrients to health. There have been calls for the harmonisation of approaches used in the setting of nutrient requirements, globally, and an increased transparency in the decision-making process. Some progress has been made in this regard, but there is a great deal of work to be done.

Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation.

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

Comparison of (6 S)-5-methyltetrahydrofolic acid v. folic acid as the reference folate in longer-term human dietary intervention studies assessing the relative bioavailability of natural food folates: comparative changes in folate status following a 16-week placebo-controlled study in healthy adults.

Folic acid (pteroylmonoglutamic acid) has historically been used as the reference folate in human intervention studies assessing the relative bioavailability of dietary folate. Recent studies using labelled folates indicated different plasma response kinetics to folic acid than to natural (food) folates, thus obviously precluding its use in single-dose experiments. Since differences in tissue distribution and site of biotransformation were hypothesised, the question is whether folic acid remains suitable as a reference folate for longer-term intervention studies, where the relative bioavailability of natural (food) folate is assessed based on changes in folate status. Healthy adults aged 18-65 years (n 163) completed a 16-week placebo-controlled intervention study in which the relative bioavailability of increased folate intake (453 nmol/d) from folate-rich foods was assessed by comparing changes in plasma and erythrocyte folate concentration with changes induced by an equal reference dose of supplemental (6S)-5-methyltetrahydrofolic acid or folic acid. The relative increase in plasma folate concentration in the food group was 31 % when compared with that induced by folic acid, but 39 % when compared with (6S)-5-methyltetrahydrofolic acid. The relative increase in erythrocyte folate concentration in the food group when compared with that induced by folic acid was 43 %, and 40 % when compared with (6S)-5-methyltetrahydrofolic acid. When recent published observations were additionally taken into account it was concluded that, in principle, folic acid should not be used as the reference folate when attempting to estimate relative natural (food) folate bioavailability in longer-term human intervention studies. Using (6S)-5-methyltetrahydrofolic acid as the reference folate would avoid future results' validity being questioned.

Recruiting older people to a randomised controlled dietary intervention trial--how hard can it be?

BACKGROUND: The success of a human intervention trial depends upon the ability to recruit eligible volunteers. Many trials fail because of unrealistic recruitment targets and flawed recruitment strategies. In order to predict recruitment rates accurately, researchers need information on the relative success of various recruitment strategies. Few published trials include such information and the number of participants screened or approached is not always cited. METHODS: This paper will describe in detail the recruitment strategies employed to identify older adults for recruitment to a 6-month randomised controlled dietary intervention trial which aimed to explore the relationship between diet and immune function (The FIT study). The number of people approached and recruited, and the reasons for exclusion, will be discussed. RESULTS: Two hundred and seventeen participants were recruited to the trial. A total of 7,482 letters were sent to potential recruits using names and addresses that had been supplied by local Family (General) Practices. Eight hundred and forty three potential recruits replied to all methods of recruitment (528 from GP letters and 315 from other methods). The eligibility of those who replied was determined using a screening telephone interview, 217 of whom were found to be suitable and agreed to take part in the study. CONCLUSION: The study demonstrates the application of multiple recruitment methods to successfully recruit older people to a randomised controlled trial. The most successful recruitment method was by contacting potential recruits by letter on NHS headed note paper using contacts provided from General Practices. Ninety percent of recruitment was achieved using this method. Adequate recruitment is fundamental to the success of a research project, and appropriate strategies must therefore be adopted in order to identify eligible individuals and achieve recruitment targets.

Effects of methodological variation on assessment of riboflavin status using the erythrocyte glutathione reductase activation coefficient assay.

Riboflavin status is usually measured as the in vitro stimulation with flavin adenine dinucleotide of the erythrocyte enzyme glutathione reductase, and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). This method is used for the National Diet and Nutrition Surveys (NDNS) of the UK. In the period between the 1990 and 2003 surveys of UK adults, the estimated prevalence of riboflavin deficiency, expressed as an EGRAC value > or = 1.30, increased from 2 to 46 % in males and from 1 to 34 % in females. We hypothesised that subtle but important differences in the detail of the methodology between the two NDNS accounted for this difference. We carried out an evaluation of the performance of the methods used in the two NDNS and compared against an 'in-house' method, using blood samples collected from a riboflavin intervention study. Results indicated that the method used for the 1990 NDNS gave a significantly lower mean EGRAC value than both the 2003 NDNS method and the 'in-house' method (P < 0.0001). The key differences between the methods relate to the concentration of FAD used in the assay and the duration of the period of incubation of FAD with enzyme. The details of the EGRAC method should be standardised for use in different laboratories and over time. Additionally, it is proposed that consideration be given to re-evaluating the basis of the EGRAC threshold for riboflavin deficiency.

Study Protocol: randomised controlled trial to investigate the functional significance of marginal riboflavin status in young women in the UK (RIBOFEM).

BACKGROUND: The functional significance of moderate riboflavin deficiency as it is currently assessed is not well understood. Animal and human studies have suggested a role for riboflavin in the absorption and mobilisation of iron and as such may be important in maintaining haematological status. Recent National Diet and Nutrition Surveys in the United Kingdom have shown that young women in particular are at risk of moderate riboflavin deficiency and low iron status. METHODS/DESIGN: A randomised placebo controlled intervention trial was conducted to investigate the effect of riboflavin supplementation on various measures of haematological status in a group of moderately riboflavin deficient young women aged 19 to 25 years. Women who were low milk consumers were initially screened for riboflavin status as assessed by the erythrocyte glutathione reductase activation coefficient assay (EGRAC). One hundred and twenty three women with EGRAC values >1.40 were randomised to receive 2 mg, 4 mg riboflavin or placebo for 8 weeks. In addition 36 of these women were randomly allocated to an iron bioavailability study to investigate the effect of the intervention on the absorption or utilisation of iron using an established red cell incorporation technique. DISCUSSION: One hundred and nineteen women completed the intervention study, of whom 36 completed the bioavailability arm. Compliance was 96 +/- 6% (mean +/- SD). The most effective recruitment strategy for this gender and age group was e-communication (e-mail and website). The results of this study will clarify the functional significance of the current biochemical deficiency threshold for riboflavin status and will inform a re-evaluation of this biochemical threshold. TRIAL REGISTRATION: Current Controlled Trials Registration No. ISRCTN35811298.

Quantification of the bioavailability of riboflavin from foods by use of stable-isotope labels and kinetic modeling.

BACKGROUND: Discrepancies have been reported between estimates of the prevalence of riboflavin deficiency based on intakes of riboflavin and estimates based on measures of riboflavin status. One reason for this may be an overestimate of the bioavailability of riboflavin from foods, about which relatively little is known. OBJECTIVE: We aimed to quantify the bioavailability of riboflavin from milk and spinach by using stable-isotope labels and a urinary monitoring technique and by a plasma appearance method based on kinetic modeling. DESIGN: Twenty healthy women aged 18-65 y were recruited for a randomized crossover study performed with extrinsically labeled (13C) milk and intrinsically labeled (15N) spinach as sources of riboflavin. An intravenous bolus of labeled riboflavin was administered with each test meal to assess the apparent volume of distribution of riboflavin in plasma. RESULTS: No significant differences were noted in riboflavin absorption from the spinach meal and from the milk meal according to either the urinary monitoring technique (60 +/- 8.0% and 67 +/- 5.4%, respectively; P = 0.549) or the plasma appearance method (20 +/- 2.8% and 23 +/- 5.3%, respectively; P = 0.670). CONCLUSIONS: A large fraction of newly absorbed riboflavin is removed by the liver on "first pass." The plasma appearance method therefore underestimates riboflavin bioavailability and should not be used to estimate riboflavin bioavailability from foodstuffs. Urinary monitoring suggests that riboflavin from spinach is as bioavailable as is riboflavin from milk.

Responses of biomarkers of folate and riboflavin status to folate and riboflavin supplementation in healthy and colorectal polyp patients (the FAB2 Study).

Epidemiologic data suggest that increasing folate intake may protect against colorectal cancer. Riboflavin may interact with folate to modulate the effect. A double-blind randomized placebo-controlled intervention study (the FAB2 Study) was carried out in healthy controls and patients with colorectal polyps (adenomatous and hyperplastic) to examine effects of folic acid and riboflavin supplements on biomarkers of nutrient status and on putative biomarkers of colorectal cancer risk (DNA methylation and DNA damage; to be reported elsewhere). Ninety-eight healthy controls and 106 patients with colorectal polyps were stratified for the thermolabile variant of methylene tetrahydrofolate reductase, MTHFR C677T, and were randomized to receive 400 microg of folic acid, 1,200 microg of folic acid, or 400 microg of folic acid plus 5 mg of riboflavin or placebo for 6 to 8 weeks. Blood samples and colon biopsy samples were collected for the measurement of biomarkers of folate and riboflavin status. Supplementation with folic acid elicited a significant increase in mucosal 5-methyl tetrahydrofolate, and a marked increase in RBC and plasma, with a dose-response. Measures of riboflavin status improved in response to riboflavin supplementation. Riboflavin supplement enhanced the response to low-dose folate in people carrying at least one T allele and having polyps. The magnitude of the response in mucosal folate was positively related to the increase in plasma 5-methyl tetrahydrofolate but was not different between the healthy group and polyp patients. Colorectal mucosal folate concentration responds to folic acid supplementation to an extent comparable to that seen in plasma, but with a suggestion of an upper limit.