Marika Ferrari

Sampling and processing of fresh blood samples within a European multicenter nutritional study: Evaluation of biomarker stability during transport and storage

Introduction:Analysis of several biological markers improves the quality and physiologic comprehension of data obtained in epidemiological nutritional studies.Aim:To develop a methodology that guarantees the centralized analysis and quality assurance of the most relevant blood parameters from fresh blood samples in adolescents in a European multicenter study.Materials and methods:Stability of selected nutrients and biomarkers (vitamins, fatty acids, iron metabolism and immunological parameters) chosen with respect to time and temperature of sample transport and storage was evaluated as part of the pilot study of the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) project.Results:Routine biochemistry and iron status parameters included in the HELENA Cross-Sectional Study (CSS) protocol could be analyzed within 24 h from fresh blood samples without any stability problems (coefficient of variation (CV)<5%, P<0.05). However, stability tests for lymphocyte subpopulations, vitamin C and fatty acids showed that they are very unstable at room temperature without any treatment. Therefore, a special handling for these samples was developed. Vitamin C was stabilized with metaphosphoric acid and transported under cooled conditions (CV 4.4%, recovery rate >93%, P>0.05). According to the results, a specific methodology and transport system were developed to collect blood samples at schools in 10 European cities and to send them to the centralized laboratory (IEL, Bonn, Germany). To guarantee good clinical practice, the field workers were instructed in a training workshop and a manual of operation was developed.Conclusion:The handling and transport system for fresh blood samples developed for the European multicenter study HELENA is adequate for the final part of the HELENA-CSS and will provide, for the first time, reference values for several biological markers in European adolescents.

Objectively-measured and self-reported physical activity and fitness in relation to inflammatory markers in European adolescents: The HELENA Study

OBJECTIVE Atherogenesis involves an inflammatory process that occurs early in life even though clinical symptoms are not observed until adulthood. Two important protective factors for low-grade inflammation may be physical activity (PA) and fitness. We examined the independent associations of objective and subjective measurements of PA and fitness with low-grade inflammation in European adolescents. METHODS A total of 1045 adolescents, aged from 12.5 to 17.5 years old from 10 European cities, were selected from the HELENA-Cross-Sectional Study. Objectively-measured and self-reported PA variables were obtained by accelerometry and the International PA Questionnaire for Adolescents, respectively. Overall, cardiorespiratory, muscular and motor fitness variables were assessed by standardized field-based fitness tests and the International Fitness Scale. C-reactive protein (CRP), complement factors 3 (C3) and 4 (C4), interleukin-6 and TNF-α inflammatory markers were measured. RESULTS Objectively-measured vigorous PA was inversely associated with C3 (β=-0.094, P=0.021) but it did not remain significant after any objective fitness indicator was included in the model. Other objectively measured or self-reported assessments of PA were not significantly associated with inflammatory markers. All objective measures of fitness were inversely associated with CRP, C3 and C4, whereas only self-reported motor fitness remained significantly associated with C3, C4 and TNF-α. All these observations were independent of age, sex, city and body mass index or waist circumference. CONCLUSION High PA in adolescence may play an indirect role on lessening low-grade inflammation through improvements in fitness.

Evaluation of iron status in European adolescents through biochemical iron indicators: the HELENA Study

Background/Objectives:To assess the iron status among European adolescents through selected biochemical parameters in a cross-sectional study performed in 10 European cities.Subjects/Methods:Iron status was defined utilising biochemical indicators. Iron depletion was defined as low serum ferritin (SF<15 μg/l). Iron deficiency (ID) was defined as high-soluble transferrin receptor (sTfR>8.5 mg/l) plus iron depletion. Iron deficiency anaemia (IDA) was defined as ID with haemoglobin (Hb) below the WHO cutoff for age and sex: 12.0 g/dl for girls and for boys aged 12.5–14.99 years and 13.0 g/dl for boys aged ⩾15 years. Enzyme linked immunosorbent assay was used as analytical method for SF, sTfR and C-reactive protein (CRP). Subjects with indication of inflammation (CRP >5 mg/l) were excluded from the analyses. A total of 940 adolescents aged 12.5–17.49 years (438 boys and 502 girls) were involved.Results:The percentage of iron depletion was 17.6%, significantly higher in girls (21.0%) compared with boys (13.8%). The overall percentage of ID and IDA was 4.7 and 1.3%, respectively, with no significant differences between boys and girls. A correlation was observed between log (SF) and Hb (r=0.36, P<0.01), and between log (sTfR) and mean corpuscular haemoglobin (r=−0.30, P<0.01). Iron body stores were estimated on the basis of log (sTfR/SF). A higher percentage of negative values of body iron was recorded in girls (16.5%) with respect to boys (8.3%), and body iron values tended to increase with age in boys, whereas the values remained stable in girls.Conclusions:To ensure adequate iron stores, specific attention should be given to girls at European level to ensure that their dietary intake of iron is adequate.

The third National Food Consumption Survey, INRAN-SCAI 2005-06: major dietary sources of nutrients in Italy.

Abstract Introduction: To promote healthy food consumption patterns, information is required on the contribution of food groups to total nutrient intake. The objective of this paper is to identify the main dietary sources of nutrients in the diet of the population in Italy. Methods: Data collected through individual food records within the INRAN-SCAI 2005–06 survey were required. The final sample included 3323 subjects aged 0.1–97.7 years. Results: The percentage contributed by each food category to the intake of energy, dietary fibre and of 26 nutrients was calculated. Above 3 years of age, the main contributors to macro- and micro-nutrient intakes were similar among the various age-sex groupings with few exceptions. Conclusion: These data might be used to develop specific strategies for Italy in order to increase the intake of dietary fibre and to decrease that of total fats and of sugars in the population.

Cardiorespiratory fitness in males, and upper limbs muscular strength in females, are positively related with 25-hydroxyvitamin D plasma concentrations in European adolescents: the HELENA study

BACKGROUND High prevalence of vitamin D insufficiency (<75 nmol/l) has been previously reported in European adolescents. Vitamin D deficiency has been related to physical fitness and adiposity but it is not clearly known whether this relationship applies to growing children and adolescents. AIM To determine how body composition and physical fitness are related to 25-hydroxyvitamin D [25(OH)D] concentrations in European adolescents. DESIGN The HEalthy Lifestyle in Europe by Nutrition in Adolescence-CSS study was a multi-centre cross-sectional study. METHODS Plasma 25(OH)D, body composition and physical fitness measures were obtained in 1006 European adolescents (470 males) aged 12.5-17.5 years. Stepwise regression and ANCOVA were performed by gender using 25(OH)D as dependent variable, with body composition, physical fitness as independent variables controlling for age, seasonality and latitude. RESULTS For males, maximum oxygen consumption (VO2max) (B = 0.189) and body mass index (BMI) (B = -0.124) were independently associated with 25(OH)D concentrations (both P < 0.05). For females, handgrip strength (B = 0.168; P < 0.01) was independently associated with 25(OH)D concentrations. Those adolescents at lower BMI and high fitness score presented significant higher 25(OH)D concentrations than those at lower fitness score in the other BMI groups (P < 0.05). CONCLUSION Cardiorespiratory fitness and upper limbs muscular strength are positively associated with 25(OH)D concentrations in male and female adolescents, respectively. Adiposity in males and low fat free mass in females are related to hypovitaminosis D. The interaction between fitness and BMI has a positive effect on 25(OH)D concentrations. Therapeutic interventions to correct the high rates of vitamin D deficiency in adolescents should consider physical fitness.

Design and preparation of market baskets of European Union commercial baby foods for the assessment of infant exposure to food chemicals and to their effects

The assessment of acute and chronic dietary exposure to contaminants in baby foods is needed to ensure healthy infant growth. Monthly European Union market baskets of commercial baby foods were designed for the first 9 months of life by the ‘babyfood’ study group of the CASCADE Network of Excellence for the specific purpose of assessing exposure to potentially toxic substances in infants fed with such foods. The present paper reports the different steps that led to the preparation of monthly pooled samples of commercial baby foods (‘Infant formulae and follow-on formulae’ and ‘Other baby foods’) that may constitute the extreme case of the diet for an infant who would not be breast fed at all. Several market baskets were generated for an ‘average European Union infant’ and for infants of four selected countries (Italy, Sweden, Spain, and the Slovakia), fed with either milk infant formulae, soy infant formulae or hypoallergenic infant formulae and weaned (at the fifth month) with commercial baby foods and beverages available on the European Union market. Market share data for 2007 for baby foods were used to design the baskets. Holding companies and the name of all their products were identified. Monthly diets for European Union infants were elaborated in terms of food categories (e.g. infant cereals) of typologies of products (e.g. infant cereals without gluten) and of a specific product. The number of baskets generated was 30 for ‘Infant formulae and follow-on formulae’ (including 62 products) and 13 for ‘Other baby foods’ (including 35 products). These market baskets were designed to be used for the determination of certain contaminants and nutrients in the diet of European Union infants and for the assessment of their effects on infant health.

Combined influence of healthy diet and active lifestyle on cardiovascular disease risk factors in adolescents

To investigate the combined influence of diet quality and physical activity on cardiovascular disease (CVD) risk factors in adolescents, adolescents (n = 1513; 12.5–17.5 years) participating in the Healthy Lifestyle in Europe by Nutrition in Adolescence study were studied. Dietary intake was registered using a 24‐h recall and a diet quality index was calculated. Physical activity was assessed by accelerometry. Lifestyle groups were computed as: healthy diet and active, unhealthy diet but active, healthy diet but inactive, and unhealthy diet and inactive. CVD risk factor measurements included cardiorespiratory fitness, adiposity indicators, blood lipid profile, blood pressure, and insulin resistance. A CVD risk score was computed. The healthy diet and active group had a healthier cardiorespiratory profile, fat mass index (FMI), triglycerides, and high‐density lipoprotein cholesterol (HDL‐C) levels and total cholesterol (TC)/HDL‐C ratio (all P ≤ 0.05). Overall, active adolescents showed higher cardiorespiratory fitness, lower FMI, TC/HDL‐C ratio, and homeostasis model assessment index and healthier blood pressure than their inactive peers with either healthy or unhealthy diet (all P ≤ 0.05). Healthy diet and active group had healthier CVD risk score compared with the inactive groups (all P ≤ 0.02). Thus, a combination of healthy diet and active lifestyle is associated with decreased CVD risk in adolescents. Moreover, an active lifestyle may reduce the adverse consequences of an unhealthy diet.

Self-reported sleep duration, white blood cell counts and cytokine profiles in European adolescents: the HELENA study

BACKGROUND Sleep patterns face important changes during adolescence. This can have implications for the immune system, which is regulated by the sleep-wake cycle; however, most studies relating sleep and immune system have been conducted on adults. OBJECTIVE To study the relationships between sleep duration, immune cell counts, and cytokines in European adolescents participating in the HELENA Cross-Sectional Study. METHODS Adolescents (12.5-17.5 years; n = 933; 53.9% girls) were grouped according to self-reported sleep duration into <8, 8-8.9 and ≥9 h/night. Blood samples were collected in the morning after an overnight fast to analyze counts of white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, the lymphocyte subsets CD3(+), CD4(+), CD8(+), CD45RA(+), CD45RO(+), CD3(-)CD16(+)56(+) and CD19(+), and concentrations of cortisol, CRP, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α and IFN-γ. Pro-/anti-inflammatory and Th1/Th2 cytokine ratios were calculated. Immune parameters were correlated to sleep duration and compared between the three groups. RESULTS Sleep duration was negatively associated with cortisol levels and WBC, neutrophil, monocyte, CD4(+) and CD4(+)CD45RO(+) counts; in girls it is also negatively associated with IL-5 and IL-6 levels. The 8-8.9 h/night group presented the highest IL-4 values and the lowest pro-/anti-inflammatory and Th1/Th2 cytokine ratios. CONCLUSION A sleep duration of 8-8.9 h/night was associated with a healthier immune profile in our adolescents.

Inflammation profile in overweight/obese adolescents in Europe: an analysis in relation to iron status

Background/Objectives:The objectives of this study were to investigate the relationship between inflammatory parameters (CRP, c-reactive protein; AGP, α1-acid glycoprotein), iron status indicators (SF, serum ferritin; sTfR, soluble transferrin receptor) and body mass index (BMI) z-score, fat-free mass (FFM) and fat mass (FM) in European adolescents. Differences in intake for some nutrients (total iron, haem and non-haem iron, vitamin C, calcium, proteins) were assessed according to BMI categories, and the association of nutrient intakes with BMI z-score, FM and FFM was evaluated.Methods:A total of 876 adolescents participating in the Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study were included in the study sample.Results:Mean CRP values (standard error; s.e.) were significantly higher in overweight/obese adolescents (1.7±0.3 and 1.4±0.3 mg/l in boys and girls, respectively) than in thin/normal-weight adolescents (1.1±0.2 and 1.0±0.1 mg/l in boys and girls, respectively) (P<0.05). For boys, mean SF values (s.e.) were significantly higher in overweight/obese adolescents (46.9±2.7 μg/l) than in thin/normal-weight adolescents (35.7±1.7 μg/l) (P<0.001), whereas median sTfR values did not differ among BMI categories for both boys and girls. Multilevel regression analyses showed that BMI z-score and FM were significantly related to CRP and AGP (P<0.05). Dietary variables did not differ significantly among BMI categories, except for the intake of vegetable proteins, which, for boys, was higher in thin/normal-weight adolescents than in overweight/obese adolescents (P<0.05).Conclusions:The adiposity of the European adolescents was sufficient to cause chronic inflammation but not sufficient to impair iron status and cause iron deficiency.

Intake and dietary sources of haem and non-haem iron among European adolescents and their association with iron status and different lifestyle and socio-economic factors

Background/Objectives:Adolescents are at risk of iron deficiency because of their high iron requirements. The aims of this study were: (1) to assess iron intake, its determinants and its most important food sources and; (2) to evaluate the relation of iron intake and status in European adolescents.Subjects/Methods:Two non-consecutive 24-h recalls were completed by a computerised tool. The socio-demographic and socio-economic data were collected by a self-reported questionnaire. Weight and height were measured. A distinction was made between haem and non-haem iron.Results:The total iron intake was significantly higher among boys (13.8 mg/day; n=1077) than girls (11.0 mg/day; n=1253). About 97.3% of the boys and 87.8% of the girls met the estimated average requirement, and 72.4% of the boys and 13.7% of the girls met the recommendation for bio-available iron intake. The ratio of haem/non-haem iron intake was lower for girls than boys. Meat (19.2; 76%) and bread and rolls (12.6;3.9%) contributed most to total and haem iron intake. Bread and rolls (13.8%) and meat (10.8%) contributed most to non-haem iron intake. Age, sex and body mass index were associated with iron intake. Only red blood cell concentration was significantly negatively associated with total, haem and non-haem iron intake.Conclusion:Girls had lower iron intakes and ratio of haem/non-haem iron intake than boys. The main total iron and haem iron source was meat, while the main non-haem iron source was bread and rolls. Adolescent girls may be a group at risk for iron deficiency. Consequently, special attention and strategies are needed in order to improve iron intakes during adolescence.