• Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2

  Mushtaq, Sohail; Camara, Johanna E.; Wise, Stephen A.; Sempos, Christopher T.; University of Chester; National Institute of Standards and Technology (NIST); National Institutes of Health (NIH); Vitamin D Standardization Program LLC (Springer, 2021-06-28)

  An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.
• 

  Leading brand products and their supermarket economy line equivalents, is there a difference in nutritional content?

  Mushtaq, Sohail; Jackson, Emma; University of Chester

  Since the introduction of supermarket economy lines (SELs) in the early 1990s, their popularity has been established nationwide(1).However, these economical alternatives are commonly perceived to be of lower nutritional quality than their leading brand (LB)equivalents(2,3,4). The present study aimed to determine if there is a significant difference in nutritional content between the UKtop-selling LBs and their SEL equivalents. Additionally, the study aimed to investigate if on average, LBs or SELs provide better‘value for money’.The LBs of 38 most popular food categories were identified from UK market research, and equivalent SEL products were identifiedfrom each of the retailers with the top-five majority UK market share: Tesco, Sainsbury’s, Asda, Morrisons and Aldi. In each foodcategory, differences between LBs and SELs in: energy, fat, saturated fatty acid, carbohydrate, sugar,fibre, protein and salt content,per 100 g of food product were determined using a one-sample T-test. The nutritional quality of each product was also determined bya nutrient profiling system. Cost was analysed in relation to shopping baskets containing 33 equivalent products. Six shopping basketswere analysed, one containing LB products and one from each SEL retailer. The cost of each shopping basket was calculated usingpack price and price per 100 g or 100mL of food product.Data was collected for 219 products; 38 LBs and 181 SELs. 86 significant differences were identified in specific nutrients across thefood categories, but the direction of the differences was inconsistent. Based on pack price, the total LB shopping basket cost was£61·91 whereas average SEL basket cost £28·62, a difference of £33·29 or 54 % (P = 0·001). However, there was no difference betweenthe nutrient profile of LBs and SELs.Although significant differences were identified between nutrients in some food categories, overall, there appeared to be no differ-ence in nutritional content between LBs and SEL equivalents. This association is consistent with previous studies and is contrary tothe common perception that SELs are of lower nutritional quality than LBs(2,3,4,5,6). Pertinent to public health, the present studyfound that SEL breakfast cereals contained a significantly higher amount of salt than the LB (P = 0·035)(4,6). Additionally, althoughthe majority of food categories did not show a significant difference in energy content per 100 g of food product (29 of 38) LB pastahad significantly higher energy content per 100 g of food product than SEL equivalents (P = 0·017)(6).In conclusion, there appears to be no difference in nutritional content between the LB and SEL equivalents in 38 popular foodcategories, however, there appears to be twofold difference in price The cost analysis demonstrates that consumers can purchasethe same quantity of foodstuff for significantly less when opting for SEL products. Low income households may therefore be encour-aged to purchase SEL products to reduce weekly household expenditure and enable a greater proportion of the budget to be availablefor the purchase fresh produce such as fruit, vegetables and meat
• 

  Estimates of fibre intake and percentage of the population with intake below the dietary reference values (DRVs) in England (1991–2015)

  Mushtaq, Sohail; Farzad, Amirabdollahian; Buczkowski, Bartek; Davies, Ian; University of Chester

  In 1991, the Committee on Medical Aspects of Foods (COMA) defined dietary fibre as non-starch polysaccharides (NSP) and set the DRV as the population average intake of 18 g/day 1 , determined using the Englyst method of analysis 2 . The latest publication of the Scientific Advisory Committee on Nutrition (SACN) 3 broadened the definition of dietary fibre beyond NSP to broader definition of Association of Analytical Communities (AOAC) fibre, recommending the DRV to be 30 g/day based on AOAC method. The COMA 1991, DRV of 18 g/day of NSP corresponds to around 24 g/day of AOAC fibre 3 and therefore the new DRV of fibre would represent a higher recommendation (around 22·5 g fibre as per the Englyst method) for the average population. The purpose of this study was to investigate variation in fibre intake of English population by age and gender, in comparison with the COMA and SACN DRVs. Data on the core sample of the National Diet and Nutrition Survey rolling programme from 2008–2012 was reanalysed. Children aged below 16 years were excluded in consideration of their different DRVs. The data on dietary fibre was extracted from fully productive individuals (i.e. participants who completed three/four diary days), as an average daily intake based on the NSP/Englyst fibre. Inferential statistics included the analysis of variance to discover if there were any significant variations in fibre intake of males and females in relation to their age groups. The statistical significance was set at 0·05. For all age groups, the average fibre intake is below the DRVs. The average daily fibre intake slightly increased with age for both genders until 64 years. When differences in energy intake were taken into account, the average daily fibre density (g/1000 kcal) still increased with the age of participants. Overall, less than a third of populations had an intake above the COMA DRV 1 . More than 90 % of the population had intake below the SACN DRV 3 , demonstrating a challenge for future policies to meet the nutritional guidelines, particularly amongst females and younger adults. The findings should be treated with caution considering the definition of AOAC fibre used as the basis for the SACN DRV includes non-digestible oligosaccharides, resistant starch and polydextrose, going beyond NSP/Englyst variables analysed.
• 

  Full fat cheese intake and cardiovascular health: a randomised control trial

  Mushtaq, Sohail; Butler, Thomas; Davies, Ian; University of Chester

  Milk and milk products contribute approximately 22 % of the nation's saturated fat (SFA) intake. Recently, the role of dairy and its SFA composition and link to cardiovascular disease (CVD) has been analysed( 1 ), suggesting a beneficial action of this food group on reducing cardiovascular risk in high-risk groups( 2 , 3 ). The aim of this study was to examine the effects of 4 weeks full-fat cheese on circulating lipoprotein fractions, blood pressure and arterial stiffness in healthy adults. Participants were recruited in the city of Chester, UK. Those meeting entry criteria of: 18–65 years of age, not taking antihypercholesterolaemic or antihypertensive medication took part in the study. Participants were randomised to receive either 50 g of a full-fat Red Leicester (FFC) or placebo (virtually zero fat Cheddar cheese, ZFC) per day for 4 weeks. Anthropometry, blood pressure, brachial and aortic augmentation index (BAIX and AAIX, respectively), pulse-wave velocity (PWV) and a full lipid profile were determined at baseline and post-intervention. Participants were asked to keep a 3-day food diary prior to and for the last 3 days of the protocol. All procedures were approved by the Faculty of Medicine, Dentistry and Life Sciences Research Ethics Committee at the University of Chester. Eighty-six (86) individuals completed the study (43 per group). No significant changes were observed in any measured parameter (Table 1). Both ZFC and FFC groups showed a significant increase in calcium intake during the course of the study (1002·1 ± 639·1 mg to 1815·0 ± 1340·1 mg and 1219·6 ± 1169·1 mg 1845·8 ± 1463·2 mg, P < 0·001, respectively) showing good adherence to the protocol. In conclusion, these results suggest that inclusion of 50 g full fat cheese into the diet of a healthy population does not impact negatively on traditional CVD risk markers. Future strategies to reduce SFA intake should focus on – and acknowledge the importance of the source – of SFA in the diet.
• 

  Effect of a single serving of pecan nuts on blood lipids and weight: a single blind randomised control trial

  Mushtaq, Sohail; Butler, Thomas; Confue, Charlotte; Guild, Joanne; University of Chester

  Nuts are a common component of many traditional cardioprotective diets primarily due to their ability to lower blood lipids and reduce cardiovascular risk(1, 2). Studies consistently show nut intake is associated with favourable changes in energy balance(3). However there is a paucity of data examining the acute changes following nut consumption. We sought to examine the effect of a single serving of pecan nuts on plasma lipids and bodyweight. Participants were sampled from the University of Chester, UK. Individuals (n = 54) were screened for eligibility to participate. Those meeting entry criteria (n = 25) of being either male or female aged 30 years or more and with no previous history of CVD were randomised to either a control (CON) or pecan nut group (PECAN). Participants in the PECAN group received a single 50 g serving of pecan nuts. Capillary blood was taken for analysis of triacylglycerol, total-cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein-cholesterol and non-high density lipoprotein cholesterol (TAG, TC, LDL-C, HDL-C and non-HDL-C, respectively), and anthropometric measurements were performed. All measurements were repeated after 3 days. Participants were instructed to record all food and drink consumed, and not to change their habitual eating habits. Procedures were approved by the Faculty of Medicine, Dentistry and Clinical Sciences Research Ethics Committee, University of Chester. No significant effect on TC, HDL-C or TAG was observed during the study (Fig. 1A–C). LDL-C decreased by 0.09 ± 0.37 mmol/L and increased by 0.16 ± 0.40 mmol/L in CON and PECAN groups, respectively. Non-HDL-C showed a similar pattern with the CON group showing a decrease and PECAN group displaying an increase (−0.18 ± 0.36 mmol/L vs. 0.16 ± 0.40 mmol/L, respectively). Bodyweight significantly (P = 0.025) decreased in the PECAN group when compared to the CON group (−0.58 ± 0.56 kg vs. −0.05 ± 0.55 kg, respectively). In conclusion, a single serving of pecan nuts had no significant impact on lipid markers of cardiovascular risk. Bodyweight was significantly reduced consistent with recent literature showing a favourable relationship with nut intake and energy balance(3).
• 

  Acute and chronic effects of beetroot supplementation on blood pressure and arterial stiffness in humans

  Mushtaq, Sohail; Turner, Emma; University of Chester

  Dietary supplementation of beetroot juice, containing nitrate- a potent vasodilation agent, has been shown to be vasoprotective( 1 ), and dose dependent decreases in blood pressure (BP) have been previously demonstrated(2,3). To our knowledge there has been only one study investigating the effect of beetroot supplementation in humans on arterial stiffness, measured using pulsewave velocity (PWV) and, although there was no effect of supplementation on PWV, there was a significant reduction due to beetroot supplementation in acute diastolic BP (3hrs, P = 0·023)( 4 ). A double-blind, randomised, cross-over intervention trial was carried out in a cohort of 12 healthy male participants (mean age (SEM) = 43 (2·1) yrs, BMI = 27·8 (1·1) kg.m2) who underwent both beetroot juice and placebo supplementation for 14 days. The aim of the study was to assess the effect of 6·45 mmol of nitrate in a concentrated 70 ml beetroot drink (James White Ltd, Ipswich, UK) on systolic and diastolic BP, mean arterial pressure (MAP) and arterial stiffness (PWV, aortic augmentation index (Aix), brachial Aix) in humans. BP and arterial stiffness measurements weretaken using PWV (Arteriograph, TensioMed,Hungary). Measurements were taken intriplicate at baseline, 3 hours post-supplementation (either beetroot juice orplacebo) and post-intervention (day 15). This was followed by a 7-day washoutperiod before participants were transferred to the alternate supplement. Table 1 shows that there was no significant acute or short term effect of beetroot juice supplementation on the parameters measured when compared to placebo. However, there was a significant decrease in systolic BP (P = 0·009), diastolic BP (P = 0·035), MAP (P = 0·017), aortic and brachial AIX (P = 0·042 and 0·041 respectively), 3hours post beetroot supplementation. These results confirm previous findings( 4 ) that beetroot supplementation does not have an acute or short term effect on arterial stiffness measures. However, acute effects on arterial stiffness and BP within the beetroot juice supplementation group were observed. Further large scale studies on dietary nitrate supplementation and cardiovascular health are required to further assess efficacy.
• 

  Dietary supplementation with n-3 fatty acids (n-3 FA) for 4 weeks reduces post-exercise fatigue and delayed onset muscle soreness (DOMS) in trained male athletes

  Mushtaq, Sohail; Benson, Lindsay; University of Chester

  High intensity exercise in the form of eccentric contractions can lead to the formation of free radicals, stimulating an inflammatory response( 1 , 2 ). Consumption of n-3 FA may help modify inflammation and immune reactions beneficial to health by decreasing interleukin-6, tumour necrosis factor-alpha and C-reactive protein( 3 ). For trained athletes to improve athletic performance, recovery from training is important and DOMS is frequently experienced following eccentric exercise, impacting negatively on strength( 4 ). The Western diet is however, characterised by a high n-6 FA consumption relative to n-3 FA, formulating ratios often in excess of 16:1( 5 ). The use of non-steroidal anti-inflammatory drugs in the form of n-3 FA has been investigated by a number of clinical trials in untrained athletes, but whether this can be translated into attenuating exercise induced inflammation in trained athletes is still under investigation. A double-blind, randomised controlled trial was conducted in 22 trained male athletes who supplemented their diet with either 3000 mg/d of fish oil (gel capsules) consisting of 990 mg eicosapentaenoic acid (EPA) and 660 mg docosahexaenoic acid (DHA) (n = 11), or 3000 mg/d olive oil placebo (n = 11), for 28d. Participants underwent 3 sets of eccentric bicep curls in their dominant arm until failure and arm circumference, number of repetitions completed and DOMS/fatigue scores via visual analogue scale (VAS) were recorded at 0, 24 and 48 h after exercise, pre and post supplementation. No group performed better during the eccentric bicep test, pre and post supplementation, and at baseline, no differences were observed between groups for DOMS and fatigue. However, post supplementation, DOMS was significantly lower at 24 h (P = 0·005) and 48 h (P = 0·002) and fatigue was significantly lower at 24 h (P = 0·043) and 48 h post exercise (P < 0·001) in the n-3 FA group compared to the placebo group (Fig. 1). These findings indicate that n-3 FA supplementation has the potential to promote recovery and subsequently increase athletic performance in trained male athletes and may be a useful ergogenic aid. Possible anti-inflammatory mechanisms of n-3 FA should be further investigated using specific biomarkers of inflammation.
• 

  Estimation of fruit and vegetable consumption in a cohort of Ghanaian women and evaluation of knowledge, attitudes and practice

  Mushtaq, Sohail; Moss, Jennifer; University of Chester

  Non-communicable diseases (NCDs), previously confined to industrialised nations, are spreading through the developing world at unprecedented rates( 1 ). With communicable diseases still prevalent, this imposes a double-burden of disease in countries with limited resources and ill-equipped health systems( 2 ). An unhealthy diet, including insufficient consumption of fruit and vegetables is one of four main behavioural risk factors in the development of NCDs. However, consumption across the world, including Ghana, is below recommended levels( 3 ). Despite a wealth of research in developed countries, few studies have investigated barriers to fruit and vegetable consumption in Ghana. The aim of the present study was to assess fruit and vegetable consumption and evaluate knowledge, attitudes and practice in a cohort of Ghanaian women. A mixed-methods approach, incorporating a survey delivered in a guided interview format was utilised to investigate patterns and determinants of fruit and vegetable consumption in a cohort of Ghanaian women. Data were collected from female environmental health and nursing students aged 18–33 years (n=74, response rate 98.7%), residing in the Korle-Bu district of Accra, Ghana. A 24-hour recall questionnaire, local handy measures and a specially designed portion size assessment sheet allowed estimation of fruit and vegetable consumption, whilst a questionnaire comprising both qualitative and quantitative questions enabled investigation of barriers to consumption in Ghanaian society. A significantly greater level of low fruit and vegetable consumption was found in the present study (69%), than detailed in the World Health Survey 2002–03 (p⩽0.001). There was a significant positive association between meal frequency and consumption levels (p=0.025), however, no association was found between consumption and knowledge levels, income, home-production or perceived adequate consumption. Themes emerging from the research related to barriers affecting consumption included cost, availability, quality, and health and safety issues. Unhealthy snacking was found to be common, as were unhealthy substitutions during periods of scarcity. Numerous barriers causing the observed decrease in fruit and vegetable consumption were identified in the present study. The interrelating nature of the barriers identified suggests a multidirectional approach to address these issues would offer the greatest benefits for consumption levels. Increasing agricultural infrastructure is key, whilst educational initiatives should also play a major role in future strategies to increase fruit and vegetable consumption. Future investment and policy in Ghana should focus on prevention rather than cure, if the growing NCD burden is to be halted.
• 

  Dietary vitamin D supplementation improves haematological status following consumption of an iron-fortified cereal: an 8-week randomised controlled trial

  Mushtaq, Sohail; Ahmad Fuzi, Salma F.; University of Chester

  Vitamin D, a secosteroid, has recently been implicated in the stimulation of erythroid precursors and ultimately the rate of erythropoiesis. However, there are a paucity of randomised controlled trials (RCT), investigating the effect of vitamin D supplementation iron status, especially in populations at risk of iron deficiency. An eight-week, double-blind RCT was carried out in 50 female (mean age (± SD): 27 ± 9 years), iron-deficient (plasma ferritin concentration < 20 μg/L) participants, randomised to consume an iron-fortified cereal containing 9 mg of iron, with either a vitamin D supplement (1,500 international units (IU)/day, 38 μg/day) or placebo. The effect of dietary vitamin D supplementation on haematological indicators was investigated. Blood samples were collected at baseline, 4-weeks and 8-week timepoints for measurement of iron and vitamin D status biomarkers. The effect of intervention was analysed with a mixed-model repeated measures ANOVA using IBM SPSS statistical software (Version 21, IBM Corporation, New York, USA). Significant increases were observed in two haematological parameters: haemoglobin concentration and haematocrit level from baseline to post-intervention in the vitamin D group, but not in the placebo group. The increase from baseline to post-intervention in haemoglobin concentration in the vitamin D group (135 ± 11 to 138 ± 10 g/L) was significantly higher than in the placebo group (131 ± 15 to 128 ± 13 g/L) (P ≤ 0.05). The increase in haematocrit level from baseline to post-intervention was also significantly higher in the vitamin D group (42.0 ± 3.0 to 43.8 49 ± 3.4%) compared to the placebo group (41.2 ± 4.3 to 40.7 ± 3.6%) (P ≤ 0.05). Despite non-significant changes in plasma ferritin concentration, this study demonstrates that dietary supplementation with 1,500IU vitamin D, consumed daily with an iron-fortified cereal led to improvement in haemoglobin concentration and haematocrit levels in women with low iron stores. Further long-term studies are required, however, these findings suggest a potential role for improvement of vitamin D status as an adjunct therapy for recovery of iron status in iron-deficient populations.
• 

  Dietary vitamin D consumption, sunlight exposure, sunscreen use and parental knowledge of vitamin D sources in a cohort of children aged 1–6 years in North West England

  Mushtaq, Sohail; Aitken, Amanda; University of Chester

  Hospital admission for children with rickets in England has dramatically increased, from <1 child per 100,000 in the early 1990's to 4·78 (4·58–4·99) per 100 000 between 2007 and 2011( 1 ). The re-emergence of rickets thus suggests poor vitamin D status( 2 ). Additionally, there has been a plethora of publications associating low vitamin D status with many adverse health outcomes other than the classical role of vitamin D in the development, maintenance and function of a healthy skeleton( 3 ). Vitamin D is a fat lipophilic steroid pro hormone obtained from few foods in the diet. However, the majority (90–95%) of vitamin D is synthesised from exposure of bare skin to sunlight( 4 ), and casual sunlight exposure has been considered adequate for the majority of the population. Consequently, there is no reference nutrient intake (RNI) for ages 4–65 yrs( 5 ). With modern indoor lifestyles, cautious sun screen usage and changes in food habits, sunlight exposure may no longer be sufficient to maintain adequate vitamin D status. To avoid vitamin D deficiency, supplementation and fortification may need to play a more prominent role in everyday lives( 6 ). The aim of the present study was to investigate vitamin D dietary intake in children, parents’ knowledge of vitamin D sources, children's outdoor habits and sun screen application practices. A retrospective, cross sectional study approach was utilised. Parents of children (n = 42) aged between 1 and 6 yrs completed a semi-validated food frequency questionnaire, a sources of vitamin D knowledge questionnaire, and a sunlight exposure and sunscreen use questionnaire, in Adlington, N.W. England (latitude 55oN) during May 2013. Children's mean (±SD) dietary vitamin D intake was 4·4 ± 2·5 μg/d, significantly lower than 7μg/d (P = < 0·001, for comparison 7μg/d, the RNI for ages 3 months-4 yrs was used). As expected, children taking supplements had a significantly higher mean (±SD) vitamin D intake (8·49 ± 1·78 μg/d) compared to those that did not supplement (3·34 ± 1·23 μg/d, P < 0·001). The greatest contribution to dietary vitamin D intake from food was from butter and spreads (0·028μg/d), followed by cakes, biscuits & scones (0·023μg/d). Parents' knowledge of food sources was poor, with a mean (±SD) incorrect response of 76% ±11·2. Contrastingly, 93% correctly identified sunlight exposure as a potential source of vitamin D. Eighty nine percent of participants played outdoors daily for 1 hour or more, 81% used sunscreen with an SPF ≥30 and only 2% rarely applied sunscreen. This study revealed that children's diet in the NW England is lacking sufficient vitamin D content, in line with larger surveys( 7 , 8 ). Parents' knowledge regarding vitamin D dietary sources was poor but 93% of parents knew that sunlight was the non-dietary source of vitamin D. Outdoor play indicated sufficient exposure time to produce endogenous vitamin D but sunscreen usage may have potentially diminished epidermal UVB exposures. Further research is needed using biomarkers to confirm vitamin D insufficiency, and public health strategies should be implemented to promote existing recommendations regarding supplementation and consumption of vitamin D rich foods. Additionally, guidelines for safe sun exposure and sunscreen use are required.
• Nutrition knowledge and dietary intake of hurlers

  Murphy, John; orcid: 0000-0002-8337-722X; O’Reilly, James (SAGE Publications, 2020-11-26)

  The current study investigated the association between sports nutrition knowledge and dietary quality in a sample of adult Irish male hurling players. Nutrition knowledge was measured by the validated Sports Nutrition Knowledge Questionnaire (SNKQ). Diet quality was measured by the Australian Recommended Food Score (ARFS) calculated from food frequency questionnaire data. Analysis of variance and linear modelling were used to assess associations between variables. A total of 265 (129 elite, 136 sub-elite) players were recruited. No significant difference in nutrition knowledge (SNKQ) was found between groups. Results showed a significant difference (p = 0.02; d = 0.39 ± 0.25; small) in food score (ARFS) between groups. A weak, positive association (r = 0.3, p = 0.007) was found between nutrition knowledge and food score. Elite level players, aged 28–32, with college degrees, that have previously received nutritional guidance displayed the highest levels of both nutrition knowledge and food score. Higher levels of nutrition knowledge and food score were expected in elite players, however were only found in food score. Nutrition knowledge does contribute to dietary quality although future interventions should focus on specific gaps in knowledge such as how to meet total energy/carbohydrate requirements.
• 

  A call for action on the development and implementation of new methodologies for safety assessment of chemical-based products in the EU – A short communication

  Knight, Derek J.; Deluyker, Hubert; orcid: 0000-0002-3490-8661; Chaudhry, Qasim; Vidal, Jean-Marc; orcid: 0000-0002-4422-5880; de Boer, Alie

  Safety assessment of chemicals and products in the European Union (EU) is based on decades of practice using primarily animal toxicity studies to model hazardous effects in humans. Nevertheless, there has been a long-standing ethical concern about using experimental animals. In addition, animal models may fail to predict adverse effects in humans. This has provided a strong motivation to develop and use new approach methodologies and other alternative sources of evidence. A key challenge for this is integration of evidence from different sources. This paper is a call for action with regard to development, validation, and implementation of modern safety assessment approaches for human health assessment by means of focused applied research and development with three strands: (a) to improve screening and priority setting, (b) to enhance and partially replace animal studies under the current regulatory schemes and eventually (c) to fully replace animal studies, while achieving at least the same level of protection. For this gradual but systematic replacement of animal studies, a long-term concerted and coordinated effort with clear goals is needed at EU level, as a societal and political choice, to plan and motivate research and innovation in regulatory safety assessment.
• 

  A validated food frequency questionnaire to determine dietary intake of vitamin D

  Watkins, Stephanie; Mushtaq, Sohail; Freeborn, Ellen; University of Chester

  Objective The aim of the present study was to develop and validate a vitamin D food frequency questionnaire (FFQ) for assessment of dietary vitamin D intake in healthy adults in England, UK. Design: The current study assessed the agreement between a four-day food diary (4d-FD) and a new vitamin D FFQ to measure dietary intake of vitamin D. Dietary intake was estimated using Nutritics dietary analysis software and Spearman’s and Bland-Altman tests were utilised to assess correlation and agreement, respectively. Participants also provided a blood sample for plasma analysis of vitamin D concentrations. Setting: Home setting. Participants: Fifty participants were recruited to the study from the University of Chester and vicinity. Results: Results showed a strong correlation between vitamin D intake recorded by the FFQ and the 4d-FD (r = 0.609; P < 0.0001) within 95% limits of agreement. Furthermore, a significant correlation between plasma 25(OH)D concentrations and vitamin D intake measured by the FFQ (r = 0.290, P = 0.041) and the 4d-FD (r = 0.360, P = 0.01) was observed. Conclusion: Our analysis suggests this FFQ is a useful and rapid tool for researchers and health professionals to assess vitamin D dietary intakes in healthy adults in the UK.
• 

  Bioavailability of Orally Administered Active Lipid Compounds from four Different Greenshell™ Mussel Formats

  Miller, Matthew R.; Kruger, Marlena C.; Wynne, Chris; Waaka, Devonie; Li, Weili; Frampton, Chris; Wolber, Fran M.; Eason, Charles; Cawthron Institute; Massey University; Christchurch Clinical Studies Trust (CSST); University of Chester; University of Otago; (Emerald Publishing Limited, 2018-02-02)

  Abstract: Greenshell™ mussel (GSM, Perna canaliculus) is New Zealand’s most important aquaculture species. They are a good source of long chain-polyunsaturated fatty acids (n-3 LC PUFA). Beyond a traditional food product, GSMs are also sold as mussel powders and oil extract formats in the nutraceutical markets. In this study, a four-sequence, single dose, randomized crossover human trial with eight evaluable healthy male participants was undertaken to determine the bioavailability of the n-3 LC PUFA in four different GSM formats (oil, powder, food ingredient and half-shell unprocessed whole mussel) by measuring area under the curve (AUC) and maximal concentration (CMax). Blood samples were collected at baseline and up to 48 h after initiation of product consumption in each administration period. There were minor differences between the bioavailability of FA (fatty acid) between the different GSM formats. Eicosapentaenoic acid (EPA) peak concentrations and plasma exposures were significantly lower with GSM oil compared to GSM half-shell and GSM powder formats, which resulted in AUC0–48 for the intake of GSM half-shell mussel and GSM powder being significantly higher than that for GSM oil (p = 0.013, f= 4.84). This equated to a 20.6% and 24.3% increase in the amount of EPA present in the plasma after consumption of half-shell mussels and mussel powder respectively compared to GSM oil. GSM oil produced the shortest median time to maximal plasma n-3 LC PUFA concentration of all evaluated products demonstrated by a shorter maximum measured plasma concentration (TMax = 5 h). Docosahexaenoic acid (DHA) and n-3 LC PUFA plasma exposure parameters were statistically comparable across the four GSM products evaluated.
• 

  Ecotoxicological and regulatory aspects of environmental sustainability of nanopesticides.

  Grillo, Renato; email: renato.grillo@unesp.br; Fraceto, Leonardo F; Amorim, Mónica J B; Scott-Fordsmand, Janeck James; Schoonjans, Reinhilde; Chaudhry, Qasim (2020-10-02)

  Recent years have seen the development of various colloidal formulations of pesticides and other agrochemicals aimed at use in sustainable agriculture. These formulations include inorganic, organic or hybrid particulates, or nanocarriers composed of biodegradable polymers, that can provide a better control of the release of active ingredients. The very small particle sizes and high surface areas of nanopesticides may however also lead to some unintended (eco)toxicological effects due to the way in which they interact with the target and non-target species and the environment. The current level of knowledge on ecotoxicological effects of nanopesticides is scarce, especially in regard to the fate and behaviour of such formulations in the environment. Nanopesticides will however have to cross a stringent regulatory scrutiny before marketing in most countries for health and environmental risks under a range of regulatory frameworks that require pre-market notification, risk assessment and approval, followed by labelling, post-market monitoring and surveillance. This review provides an overview of the key regulatory and ecotoxicological aspects relating to nanopesticides that will need to be considered for environmentally-sustainable use in agriculture. [Abstract copyright: Copyright © 2020 Elsevier B.V. All rights reserved.]
• 

  Sarcopenia during COVID-19 lockdown restrictions: long-term health effects of short-term muscle loss.

  Kirwan, Richard; orcid: 0000-0003-4645-0077; email: r.p.kirwan@2018.ljmu.ac.uk; McCullough, Deaglan; orcid: 0000-0002-9882-9639; Butler, Tom; orcid: 0000-0003-0818-1566; email: t.butler@chester.ac.uk; Perez de Heredia, Fatima; orcid: 0000-0002-2537-3327; Davies, Ian G; orcid: 0000-0003-3722-8466; Stewart, Claire; orcid: 0000-0002-8104-4819 (2020-10-01)

  The COVID-19 pandemic is an extraordinary global emergency that has led to the implementation of unprecedented measures in order to stem the spread of the infection. Internationally, governments are enforcing measures such as travel bans, quarantine, isolation, and social distancing leading to an extended period of time at home. This has resulted in reductions in physical activity and changes in dietary intakes that have the potential to accelerate sarcopenia, a deterioration of muscle mass and function (more likely in older populations), as well as increases in body fat. These changes in body composition are associated with a number of chronic, lifestyle diseases including cardiovascular disease (CVD), diabetes, osteoporosis, frailty, cognitive decline, and depression. Furthermore, CVD, diabetes, and elevated body fat are associated with greater risk of COVID-19 infection and more severe symptomology, underscoring the importance of avoiding the development of such morbidities. Here we review mechanisms of sarcopenia and their relation to the current data on the effects of COVID-19 confinement on physical activity, dietary habits, sleep, and stress as well as extended bed rest due to COVID-19 hospitalization. The potential of these factors to lead to an increased likelihood of muscle loss and chronic disease will be discussed. By offering a number of home-based strategies including resistance exercise, higher protein intakes and supplementation, we can potentially guide public health authorities to avoid a lifestyle disease and rehabilitation crisis post-COVID-19. Such strategies may also serve as useful preventative measures for reducing the likelihood of sarcopenia in general and in the event of future periods of isolation.
• Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

  Isigonis, Panagiotis; orcid: 0000-0002-8404-7708; Afantitis, Antreas; Antunes, Dalila; Bartonova, Alena; Beitollahi, Ali; Bohmer, Nils; Bouman, Evert; Chaudhry, Qasim; Cimpan, Mihaela Roxana; Cimpan, Emil; et al. (2020-07-23)

  Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally. [Abstract copyright: © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.]
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  Response to invited commentary : Vitamin D3 supplementation for 8 weeks leads to improved haematological status following the consumption of an iron-fortified breakfast cereal: a double-blind randomised controlled trial in iron-deficient women

  Ahmad Fuzi, Salma F.; Mushtaq, Sohail; orcid: 0000-0001-7623-4661; University of Chester

  An abstract is not available for this article.
• Ketogenic diets for drug-resistant epilepsy

  Martin-McGill, Kirsty J; Bresnahan, Rebecca; Levy, Robert G; Cooper, Paul N; University of Chester; University of Liverpool; The CroD ShiDa Health Centre, Rochdale; Salford Royal Hospitals NHS Trust (Cochrane Database of Systematic Reviews, 2020-06-26)

  Background Ketogenic diets (KDs) are high in fat and low in carbohydrates and have been suggested to reduce seizure frequency in people with epilepsy. Such diets may be beneficial for children with drug-resistant epilepsy. This is an update of a review first published in 2003, and last updated in 2018. Objectives To assess the effects of ketogenic diets for people with drug-resistant epilepsy. Search methods For this update, we searched the Cochrane Register of Studies (CRS Web) and MEDLINE (Ovid, 1946 to 26 April 2019) on 29 April 2019. The Cochrane Register of Studies includes the Cochrane Epilepsy Group Specialized Register, the Cochrane Central Register of Controlled Trials (CENTRAL), and randomised controlled trials (RCTs) from Embase, ClinicalTrials.gov and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). We imposed no language restrictions. We checked the reference lists of retrieved studies for additional relevant studies. Selection criteria RCTs or quasi-RCTs of KDs for people of any age with drug-resistant epilepsy. Data collection and analysis Two review authors independently applied predefined criteria to extract data and evaluated study quality. We assessed the outcomes: seizure freedom, seizure reduction (50% or greater reduction in seizure frequency), adverse effects, cognition and behaviour, quality of life, and attrition rate. We incorporated a meta-analysis. We utilised an intention-to-treat (ITT) population for all primary analyses. We presented the results as risk ratios (RRs) with 95% confidence intervals (CIs). Main results We identified 13 studies with 932 participants; 711 children (4 months to 18 years) and 221 adults (16 years and over). We assessed all 13 studies to be at high risk of performance and detection bias, due to lack of blinding. Assessments varied from low to high risk of bias for all other domains. We rated the evidence for all outcomes as low to very low certainty. Ketogenic diets versus usual care for children Seizure freedom (RR 3.16, 95% CI 1.20 to 8.35; P = 0.02; 4 studies, 385 participants; very low-certainty evidence) and seizure reduction(RR 5.80, 95% CI 3.48 to 9.65; P < 0.001; 4 studies, 385 participants; low-certainty evidence) favoured KDs (including: classic KD, medium-chain triglyceride (MCT) KD combined, MCT KD only, simplified modified Atkins diet (MAD) compared to usual care for children. We are not confident that these estimated effects are accurate. The most commonly reported adverse effects were vomiting, constipation and diarrhoea for both the intervention and usual care group, but the true effect could be substantially different (low-certainty evidence). Ketogenic diet versus usual care for adults In adults, no participants experienced seizure freedom. Seizure reduction favoured KDs (MAD only) over usual care but, again, we are not confident that the effect estimated is accurate (RR 5.03, 95% CI 0.26 to 97.68; P = 0.29; 2 studies, 141 participants; very low-certainty evidence). Adults receiving MAD most commonly reported vomiting, constipation and diarrhoea (very low-certainty evidence). One study reported a reduction in body mass index (BMI) plus increased cholesterol in the MAD group. The other reported weight loss. The true effect could be substantially different to that reported. Ketogenic diet versus ketogenic diet for children Up to 55% of children achieved seizure freedom with a classical 4:1 KD aDer three months whilst up to 85% of children achieved seizure reduction (very low-certainty evidence). One trial reported a greater incidence of seizure reduction with gradual-onset KD, as opposed to fasting-onset KD. Up to 25% of children were seizure free with MAD and up to 60% achieved seizure reduction.Up to 25% of children became seizure free with MAD and up to 60% experienced seizure reduction. One study used a simplified MAD (sMAD)and reported that 15% of children gained seizure freedom rates and 56% achieved seizure reduction. We judged all the evidence described as very low certainty, thus we are very unsure whether the results are accurate.The most commonly reported adverse effects were vomiting, constipation and diarrhoea (5 studies, very low-certainty evidence). Two studies reported weight loss. One stated that weight loss and gastrointestinal disturbances were more frequent, with 4:1 versus 3:1 KD,whilst one reported no difference in weight loss with 20 mg/d versus 10 mg/d carbohydrates. In one study, there was a higher incidence of hypercalcuria amongst children receiving classic KD compared to MAD. All effects described are unlikely to be accurate. Ketogenic diet versus ketogenic diet for adults One study randomised 80 adults (aged 18 years and over) to either MAD plus KetoCal during the first month with MAD alone for the second month, or MAD alone for the first month followed by MAD plus KetoCal for the second month. No adults achieved seizure freedom. More adults achieved seizure reduction at one month with MAD alone (42.5%) compared to MAD plus KetoCal (32.5%), however, by three months only 10% of adults in both groups maintained seizure reduction. The evidence for both outcomes was of very low certainty; we are very uncertain whether the effects are accurate.Constipation was more frequently reported in the MAD plus KetoCal group (17.5%) compared to the MAD only group (5%) (1 study, very low-certainty evidence). Diarrhoea and increase/change in seizure pattern/semiology were also commonly reported (17.5% to 20% of participants). The true effects of the diets could be substantially different to that reported. Authors' conclusions The evidence suggests that KDs could demonstrate effectiveness in children with drug-resistant epilepsy, however, the evidence for the use of KDs in adults remains uncertain. We identified a limited number of studies which all had small sample sizes. Due to the associatedr isk of bias and imprecision caused by small study populations, the evidence for the use of KDs was of low to very low certainty.More palatable but related diets, such as the MAD, may have a similar effect on seizure control as the classical KD, but could be associated with fewer adverse effects. This assumption requires more investigation. For people who have drug-resistant epilepsy or who are unsuitable for surgical intervention, KDs remain a valid option. Further research is required, particularly for adults with drug-resistant epilepsy.
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  Gestational diabetes and progression to type two diabetes mellitus: missed opportunities of follow up and prevention?

  Walker, Emma; Flannery, Orla; Mackillop, Lucy; University of Chester; Manchester Metropolitan University; Nuffield Department of Reproductive Health, Oxford University Hospitals NHS Foundation Trust;

  Abstract Background: The incidence of type 2 diabetes (T2DM) is increasing. Having a pregnancy complicated by gestational diabetes mellitus (GDM) is a potent risk factor for the later development of T2DM. Aims: To explore the characteristics of women diagnosed with GDM in a single centre and their follow up for progression to T2DM. Methods: A retrospective cohort study using anonymised data of one hundred and fifty four (154) women with GDM receiving maternity care at the Oxford University Hospitals NHS Foundation Trust (OUHFT) in 2010 and their follow up until 2018. Results: The prevalence of GDM in women delivering in Oxfordshire in 2010 was 3.4%. 70% of pregnant women were overweight or obese (with 51% being obese) at booking. Gestational weight gain (GWG) was excessive in 29% of women, when compared to Institute of Medicine (IOM) guidelines. Almost a quarter of women (23.4%) had no follow up after delivery. Over a median follow up of 3.5 years (range 0-8 years) nearly one in six (16.9%) of the total cohort (22% of those tested) went on to develop T2DM. 74% of women with GDM were multiparous, and 65% of nulliparous women were tested compared to 81% of multiparous women. There was a significant difference between multiparous women (53.8%) compared to nulliparous women (46.2%) developing T2DM (p=0.01). There was no significant difference in BMI (p=0.866) or GWG (p=0.83) in women who progressed to T2DM versus those who did not. Conclusion: The risk of T2DM after GDM is substantial however, follow up rates of this population is poor. Subsequent screening of women with GDM and their management crosses secondary and primary care with scope for improvement in counselling of women of the importance of annual reviews, in data collection and follow up in both obstetrics and general practice. The implementation of a recall system, an education programme for general practitioners and/or a registry of women diagnosed with GDM could be useful to identify those at high risk of developing T2DM as well as providing a platform for the potential development of interventions to prevent progression to T2DM after GDM.

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