Archives of Biochemistry and Biophysics 572 (2015) 73–80
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Evaluating the relationship between plasma and skin carotenoids and reported dietary intake in elementary school children to assess fruit and vegetable intake Lori M. Nguyen a,b,1, Rachel E. Scherr a,b,1,⇑, Jessica D. Linnell a,b,1, Igor V. Ermakov c,2, Werner Gellermann c,2, Lisa Jahns d,3, Carl L. Keen a,e,1, Sheridan Miyamoto f,4, Francene M. Steinberg a,1, Heather M. Young f,4, Sheri Zidenberg-Cherr a,b,g,1 a
University of California, Davis, Department of Nutrition, United States Center for Nutrition in Schools, United States c University of Utah, Department of Physics and Astronomy, United States d United States Department of Agriculture, Agricultural Research Service, Grand Forks, United States e University of California, Davis, Department of Internal Medicine, United States f University of California, Davis, Betty Irene Moore School of Nursing, United States g University of California Agriculture and Natural Resources, University of California Cooperative Extension, United States b
a r t i c l e
i n f o
Article history: Received 18 October 2014 and in revised form 9 February 2015 Available online 9 March 2015 Keywords: Carotenoids Raman spectroscopy Validation Dietary assessment Biomarkers
a b s t r a c t Accurate assessment of dietary intake of children can be challenging due to the limited reliability of current dietary assessment methods. Plasma carotenoid concentration has been used to assess fruit and vegetable intake, but this testing is rarely conducted in school settings in children. Resonance Raman spectroscopy (RRS) is emerging as a useful method to objectively assess fruit and vegetable intake. This methodology has been validated in adults, but limited work has been done in children, particularly in the school setting. The purpose of this research is to further validate the RRS methodology in children. Children (9–12 year) participating in a school-based intervention were recruited. Plasma carotenoids were quantified using HPLC, skin carotenoid status was measured using RRS, and dietary intake of carotenoids was measured with the Block Food Frequency Questionnaire Ages 8–17. Total plasma carotenoid concentrations and skin carotenoid intensities were strongly correlated (r = 0.62, p < 0.001, n = 38). Reported total carotenoid intake correlated with skin carotenoids (r = 0.40, p < 0.0001, n = 128). Skin carotenoid status as measured by RRS can be a strong predictor of plasma carotenoid status and dietary intake of carotenoids in children. RRS may be used as a valid, non-invasive, and useful method to assess fruit and vegetable intakes in this population. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author at: University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. Fax: +1 (530) 752 8905. E-mail addresses: [email protected] (L.M. Nguyen), [email protected] (R.E. Scherr), [email protected] (I.V. Ermakov), [email protected] (W. Gellermann), [email protected] (L. Jahns), [email protected] (C.L. Keen), [email protected] (S. Miyamoto), [email protected] (F.M. Steinberg), [email protected] (H.M. Young), sazidenbergcherr@ ucdavis.edu (S. Zidenberg-Cherr). 1 University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. 2 University of Utah, Department of Physics and Astronomy, 115 S 1400 E, Salt Lake City, UT 84112, United States. 3 USDA-ARS Grand Forks Human Nutrition Research Center, 2420 2nd Avenue North, Grand Forks, ND 58203, United States. 4 Betty Irene Moore School of Nursing at UC Davis, UC Davis Health System, 4610 X Street, Suite 4202, Sacramento, CA 95817, United States. http://dx.doi.org/10.1016/j.abb.2015.02.015 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
Introduction Most children in the U.S. are not meeting the Dietary Guidelines for Americans recommendations for fruit and vegetable intake [1]. Fruits and vegetables are an important source of carotenoids, high intakes of which have been associated with a reduced risk for many chronic diseases [2,3]. While numerous intervention studies aimed at increasing fruit and vegetable intakes in children have been conducted [4], the success of these interventions has been difficult to evaluate due in part to a lack of validated methodologies for the
74
L.M. Nguyen et al. / Archives of Biochemistry and Biophysics 572 (2015) 73–80
assessment of changes in dietary behavior in children [5,6]. A commonly used assessment method is self-reported dietary intake, which includes methods such as 24-h recalls and food frequency questionnaires (FFQ).5 One drawback of self-reported methods is that they are dependent on the participant’s memory, and a second drawback is their inability to take into account the variability of food intake from day to day and throughout the year [6]. To improve recall of foods in children, parents or caretakers often assist in reporting their child’s dietary intake, but this can introduce errors as many of the child’s meals can be unobserved, for example during lunch time at school. Additional reporting biases and errors are problematic, therefore limiting the validity of these methods [5]. Additionally, many of these methods are labor and time intensive, making them less ideal for use in large community studies. One means to compensate for the problems outlined above is to use nutritional biomarkers, which can be metabolite concentrations in human tissues that indicate the consumption or metabolism of select nutrients, as an objective and independent measure of dietary intake [7]. Blood carotenoid concentration, as measured by High Performance Liquid Chromatography (HPLC), is currently considered an excellent biomarker of fruit and vegetable intake as carotenoids are concentrated in these foods, they can be absorbed, and they cannot be synthesized by humans [8]. Thus, plasma concentrations of carotenoids can reflect the overall intake of fruits and vegetables [9,10]. While the potential value of blood biomarkers, such as carotenoids, are well recognized, these types of assessments are rarely performed in school settings in children due to parental and child concerns regarding the invasive nature of blood draws and complicated plasma analysis methodologies. To minimize these barriers, a noninvasive measurement of carotenoids could provide a useful means to measure the effectiveness of school-based studies with children. Resonance Raman spectroscopy (RRS) is emerging as a useful method to assess fruit and vegetable intake [11–14]. This method is a quick, noninvasive, objective optical method that is capable of measuring dermal carotenoid status [15,16]. A direct relation between skin carotenoid RRS intensity levels in photon counts and skin carotenoid concentration levels via HPLC in micrograms per gram of tissue was previously reported [12]. In healthy adults, skin levels measured via RRS correlated with HPLCassessed tissue carotenoids (r = 0.66–0.95) [11,12], plasma carotenoids (r = 0.62–0.81) [9,11,15,17], and reported dietary intakes (r = 0.52) [11]. There are limited studies involving RRS in children. One study, using research-grade instrumentation [13], that helped to establish the feasibility of using RRS in preschool children, reported a near-normal distribution of inter-individual variability in dermal carotenoid status for the large sample (n = 381), and identified parent-reported fruit/vegetable intake as a major factor associated with the biomarker in this population [18]. Another study by Aguilar et al. involving children, ages 10–17 (n = 45), involved the use of a commercial RRS scanner, the ‘‘BioPhotonic Scanner’’ (NuSkinInc., Provo, UT); skin carotenoids were measured at three different time points using a different scanner unit each time, and at each time point the investigators observed a correlation between skin and serum carotenoids (R2 = 0.49, R2 = 0.51, R2 = 0.53) [19]. Aguilar et al. also reported a correlation between skin carotenoids and reported food intake (R2 = 0.32) [19]. To date, we are aware of three studies that have found positive associations between fruit and vegetable intake and RRS measurements in children [18–20], and a limited number of studies have found positive associations between intake 5 Abbreviations used: FFQ, food frequency questionnaires; HPLC, High Performance Liquid Chromatography; RRS, Resonance Raman spectroscopy; SHCP, Shaping Healthy Choices Program; CCD, charge coupled device array.
and plasma [18,21,22], but aside from the aforementioned study [19], no other studies have investigated the relationship of carotenoids in the skin and plasma of school aged children. Skin carotenoid status has also been reported to change in response to dietary intake in adults, making it possible to use this methodology in intervention studies where the aim is to detect changes in dietary behavior [15]. Having a valid, objective, and noninvasive assessment of fruit and vegetable intake that is feasible for use in community-based settings with children would be valuable for assessing the efficacy of programs that aim to change dietary behaviors in this population. The first objective of the current study was to validate the RRS methodology using a research-grade RRS instrument against plasma carotenoid concentrations and reported dietary intake for subsequent use as a tool for objective measurement of fruit and vegetable intakes in 4th and 5th-grade children. The second objective was to assess the feasibility of using and validating the RRS instrument in a school setting. Materials and methods The Shaping Healthy Choices Program intervention A subset of 128 4th and 5th-grade students participating in the Shaping Healthy Choices Program (SHCP) participated in the validation study. The SHCP, initiated in California in 2012, is a multi-component randomized-control nutrition intervention being implemented among 4th and 5th-grade students, ages 9–12 years (n = 490) [23]. Goals of the SHCP include: (1) increasing nutrition knowledge and use of critical thinking skills; (2) promoting the availability, consumption, and enjoyment of fruits and vegetables; (3) improving dietary patterns and level of physical activity; (4) fostering positive changes in the school environment; and (5) facilitating the development of an infrastructure to sustain the program. Resonance Raman spectroscopy was to be used as a means to assess changes in fruit and vegetable intake. Before dietary changes resulting from the intervention could be assessed, the current validity study of the RRS method compared to blood carotenoid concentrations measured by HPLC and to self-reported dietary intakes was conducted at baseline at a participating school and the possibility of using the RRS instrument in a school setting was assessed. The UC Davis Institutional Review Board approved all protocols and researchers obtained parent consent and child assent. Children participating in the study received a $20 gift card for participating in the blood draw. Data collection As part of the overall intervention, the SHCP research team collected anthropometric data from the children in the study. Students’ height, weight, and waist circumference were measured in light clothing after the removal of shoes. Height was measured to the nearest 0.1 cm using a transportable stadiometer (Seca; Chino, CA), body weight was measured to the nearest 0.1 kg using an electronic scale (Seca; Chino, CA); and waist circumference was measured to the nearest 0.1 cm using a body circumference measuring tape (AccuFitness, Greenwood Village, CO). Parents reported participant characteristics at home using a parent questionnaire developed by the SHCP team. Dietary intake was self-reported by all children with the help of their parents. A SHCP educator provided classroom instruction on how to complete the 2004 Block Food Frequency Questionnaire for Ages 8–17 (FFQ), which is a pre-coded form containing a large list of specific food items that assesses frequency and portion consumed over the past seven days. Children were given three bowls
L.M. Nguyen et al. / Archives of Biochemistry and Biophysics 572 (2015) 73–80
and a plate matching the sizes of the dishes in the diagram provided with the FFQ to use as a reference [23]. Completed FFQs were sent to NutritionQuest (Berkeley, CA) for analysis and quantification of nutrients and the amounts of each MyPlate Food Groups reported. Based on the Dietary Guidelines for Americans, MyPlate is a consumer-friendly icon that was designed to help build a healthy plate at mealtime [24]. MyPlate Food Groups describe a collection of foods that are nutritionally and biologically similar and make up a healthy diet. MyPlate divides the foods into the following Food Groups: fruits, vegetables, grains, protein, and dairy. Within each group are subgroups, for example, in the Vegetables Food Group there are dark-green vegetables, starchy vegetables, red and orange vegetables, beans and peas, and other vegetables. One week after the FFQs were completed, children were recruited for participation in the validation study. Due to the aforementioned difficulties in conducting blood draws in children, three different approaches were taken to recruit volunteers for the blood draw. During the first SHCP implementation year, the blood draw events took place on a Saturday morning at a school wide Health Fair organized by the SHCP team. The Health Fair was designed to promote health and wellness in order to inspire students to want to learn more about their health. Students needed to attend the fair to participate in the blood draw and skin scan, both of which occurred on the same day. In the second year with a new set of 4th-grade students, a one-day health screening was organized on a weekday morning before school started in hopes to recruit more participants since students would already be present in the classroom. A final blood draw event was held at a two-day, back-to-back, health screening to capture students who did not attend on the first day. In the second year, RRS data were collected on a school day during classroom time, increasing the number of scans. RRS data were collected either on the same day as the blood draw, or the following day. Children who volunteered to participate in the blood draw were asked to fast for 8 h before arrival. At each blood draw event, registered nurses drew blood samples in a private room from the antecubital vein into light-sensitive EDTA-coated tubes (10 mL) (BD; Franklin Lanes, NJ). Data analysis Samples were placed on ice and transported within 2 h of collection to a laboratory where samples were separated by centrifugation (1800 rpm for 15 min) and plasma was aliquoted and stored at 80 °C until analysis. Plasma carotenoids were analyzed via HPLC with a modified method that was previously published [25]. Briefly, carotenoids were quantified on an Agilent 1100 system with a mobile phase consisting of acetonitrile/methanol (80/ 20) at a flow rate of 1.1 mL/min with a column temperature at 25 °C. Separation was performed isocratically on a Luna C18(2) column, 150 4.6 mm, 3 lm particle size with guard cartridge system (Phenomenex, Torrance, CA). A photodiode array detector was used and carotenoids were detected at a wavelength of 450 nm. Each sample was analyzed for a-carotene, b-carotene, lutein, zeaxanthin, and lycopene. Lutein and zeaxanthin were not separated with this method and were reported together. Total carotenoids were calculated by taking the sum of the four measured carotenoids. Inter sample variation from samples prepared in parallel was 90th waist circumference percentile and the rest of the percentiles, (p = 0.04) (Table 2). Additionally, significant differences were observed in reported dietary intake of carotenoids in different quartiles of energy intake, p =
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Evaluating the relationship between plasma and skin carotenoids and reported dietary intake in elementary school children to assess fruit and vegetable intake Lori M. Nguyen a,b,1, Rachel E. Scherr a,b,1,⇑, Jessica D. Linnell a,b,1, Igor V. Ermakov c,2, Werner Gellermann c,2, Lisa Jahns d,3, Carl L. Keen a,e,1, Sheridan Miyamoto f,4, Francene M. Steinberg a,1, Heather M. Young f,4, Sheri Zidenberg-Cherr a,b,g,1 a
University of California, Davis, Department of Nutrition, United States Center for Nutrition in Schools, United States c University of Utah, Department of Physics and Astronomy, United States d United States Department of Agriculture, Agricultural Research Service, Grand Forks, United States e University of California, Davis, Department of Internal Medicine, United States f University of California, Davis, Betty Irene Moore School of Nursing, United States g University of California Agriculture and Natural Resources, University of California Cooperative Extension, United States b
a r t i c l e
i n f o
Article history: Received 18 October 2014 and in revised form 9 February 2015 Available online 9 March 2015 Keywords: Carotenoids Raman spectroscopy Validation Dietary assessment Biomarkers
a b s t r a c t Accurate assessment of dietary intake of children can be challenging due to the limited reliability of current dietary assessment methods. Plasma carotenoid concentration has been used to assess fruit and vegetable intake, but this testing is rarely conducted in school settings in children. Resonance Raman spectroscopy (RRS) is emerging as a useful method to objectively assess fruit and vegetable intake. This methodology has been validated in adults, but limited work has been done in children, particularly in the school setting. The purpose of this research is to further validate the RRS methodology in children. Children (9–12 year) participating in a school-based intervention were recruited. Plasma carotenoids were quantified using HPLC, skin carotenoid status was measured using RRS, and dietary intake of carotenoids was measured with the Block Food Frequency Questionnaire Ages 8–17. Total plasma carotenoid concentrations and skin carotenoid intensities were strongly correlated (r = 0.62, p < 0.001, n = 38). Reported total carotenoid intake correlated with skin carotenoids (r = 0.40, p < 0.0001, n = 128). Skin carotenoid status as measured by RRS can be a strong predictor of plasma carotenoid status and dietary intake of carotenoids in children. RRS may be used as a valid, non-invasive, and useful method to assess fruit and vegetable intakes in this population. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author at: University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. Fax: +1 (530) 752 8905. E-mail addresses: [email protected] (L.M. Nguyen), [email protected] (R.E. Scherr), [email protected] (I.V. Ermakov), [email protected] (W. Gellermann), [email protected] (L. Jahns), [email protected] (C.L. Keen), [email protected] (S. Miyamoto), [email protected] (F.M. Steinberg), [email protected] (H.M. Young), sazidenbergcherr@ ucdavis.edu (S. Zidenberg-Cherr). 1 University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. 2 University of Utah, Department of Physics and Astronomy, 115 S 1400 E, Salt Lake City, UT 84112, United States. 3 USDA-ARS Grand Forks Human Nutrition Research Center, 2420 2nd Avenue North, Grand Forks, ND 58203, United States. 4 Betty Irene Moore School of Nursing at UC Davis, UC Davis Health System, 4610 X Street, Suite 4202, Sacramento, CA 95817, United States. http://dx.doi.org/10.1016/j.abb.2015.02.015 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
Introduction Most children in the U.S. are not meeting the Dietary Guidelines for Americans recommendations for fruit and vegetable intake [1]. Fruits and vegetables are an important source of carotenoids, high intakes of which have been associated with a reduced risk for many chronic diseases [2,3]. While numerous intervention studies aimed at increasing fruit and vegetable intakes in children have been conducted [4], the success of these interventions has been difficult to evaluate due in part to a lack of validated methodologies for the
74
L.M. Nguyen et al. / Archives of Biochemistry and Biophysics 572 (2015) 73–80
assessment of changes in dietary behavior in children [5,6]. A commonly used assessment method is self-reported dietary intake, which includes methods such as 24-h recalls and food frequency questionnaires (FFQ).5 One drawback of self-reported methods is that they are dependent on the participant’s memory, and a second drawback is their inability to take into account the variability of food intake from day to day and throughout the year [6]. To improve recall of foods in children, parents or caretakers often assist in reporting their child’s dietary intake, but this can introduce errors as many of the child’s meals can be unobserved, for example during lunch time at school. Additional reporting biases and errors are problematic, therefore limiting the validity of these methods [5]. Additionally, many of these methods are labor and time intensive, making them less ideal for use in large community studies. One means to compensate for the problems outlined above is to use nutritional biomarkers, which can be metabolite concentrations in human tissues that indicate the consumption or metabolism of select nutrients, as an objective and independent measure of dietary intake [7]. Blood carotenoid concentration, as measured by High Performance Liquid Chromatography (HPLC), is currently considered an excellent biomarker of fruit and vegetable intake as carotenoids are concentrated in these foods, they can be absorbed, and they cannot be synthesized by humans [8]. Thus, plasma concentrations of carotenoids can reflect the overall intake of fruits and vegetables [9,10]. While the potential value of blood biomarkers, such as carotenoids, are well recognized, these types of assessments are rarely performed in school settings in children due to parental and child concerns regarding the invasive nature of blood draws and complicated plasma analysis methodologies. To minimize these barriers, a noninvasive measurement of carotenoids could provide a useful means to measure the effectiveness of school-based studies with children. Resonance Raman spectroscopy (RRS) is emerging as a useful method to assess fruit and vegetable intake [11–14]. This method is a quick, noninvasive, objective optical method that is capable of measuring dermal carotenoid status [15,16]. A direct relation between skin carotenoid RRS intensity levels in photon counts and skin carotenoid concentration levels via HPLC in micrograms per gram of tissue was previously reported [12]. In healthy adults, skin levels measured via RRS correlated with HPLCassessed tissue carotenoids (r = 0.66–0.95) [11,12], plasma carotenoids (r = 0.62–0.81) [9,11,15,17], and reported dietary intakes (r = 0.52) [11]. There are limited studies involving RRS in children. One study, using research-grade instrumentation [13], that helped to establish the feasibility of using RRS in preschool children, reported a near-normal distribution of inter-individual variability in dermal carotenoid status for the large sample (n = 381), and identified parent-reported fruit/vegetable intake as a major factor associated with the biomarker in this population [18]. Another study by Aguilar et al. involving children, ages 10–17 (n = 45), involved the use of a commercial RRS scanner, the ‘‘BioPhotonic Scanner’’ (NuSkinInc., Provo, UT); skin carotenoids were measured at three different time points using a different scanner unit each time, and at each time point the investigators observed a correlation between skin and serum carotenoids (R2 = 0.49, R2 = 0.51, R2 = 0.53) [19]. Aguilar et al. also reported a correlation between skin carotenoids and reported food intake (R2 = 0.32) [19]. To date, we are aware of three studies that have found positive associations between fruit and vegetable intake and RRS measurements in children [18–20], and a limited number of studies have found positive associations between intake 5 Abbreviations used: FFQ, food frequency questionnaires; HPLC, High Performance Liquid Chromatography; RRS, Resonance Raman spectroscopy; SHCP, Shaping Healthy Choices Program; CCD, charge coupled device array.
and plasma [18,21,22], but aside from the aforementioned study [19], no other studies have investigated the relationship of carotenoids in the skin and plasma of school aged children. Skin carotenoid status has also been reported to change in response to dietary intake in adults, making it possible to use this methodology in intervention studies where the aim is to detect changes in dietary behavior [15]. Having a valid, objective, and noninvasive assessment of fruit and vegetable intake that is feasible for use in community-based settings with children would be valuable for assessing the efficacy of programs that aim to change dietary behaviors in this population. The first objective of the current study was to validate the RRS methodology using a research-grade RRS instrument against plasma carotenoid concentrations and reported dietary intake for subsequent use as a tool for objective measurement of fruit and vegetable intakes in 4th and 5th-grade children. The second objective was to assess the feasibility of using and validating the RRS instrument in a school setting. Materials and methods The Shaping Healthy Choices Program intervention A subset of 128 4th and 5th-grade students participating in the Shaping Healthy Choices Program (SHCP) participated in the validation study. The SHCP, initiated in California in 2012, is a multi-component randomized-control nutrition intervention being implemented among 4th and 5th-grade students, ages 9–12 years (n = 490) [23]. Goals of the SHCP include: (1) increasing nutrition knowledge and use of critical thinking skills; (2) promoting the availability, consumption, and enjoyment of fruits and vegetables; (3) improving dietary patterns and level of physical activity; (4) fostering positive changes in the school environment; and (5) facilitating the development of an infrastructure to sustain the program. Resonance Raman spectroscopy was to be used as a means to assess changes in fruit and vegetable intake. Before dietary changes resulting from the intervention could be assessed, the current validity study of the RRS method compared to blood carotenoid concentrations measured by HPLC and to self-reported dietary intakes was conducted at baseline at a participating school and the possibility of using the RRS instrument in a school setting was assessed. The UC Davis Institutional Review Board approved all protocols and researchers obtained parent consent and child assent. Children participating in the study received a $20 gift card for participating in the blood draw. Data collection As part of the overall intervention, the SHCP research team collected anthropometric data from the children in the study. Students’ height, weight, and waist circumference were measured in light clothing after the removal of shoes. Height was measured to the nearest 0.1 cm using a transportable stadiometer (Seca; Chino, CA), body weight was measured to the nearest 0.1 kg using an electronic scale (Seca; Chino, CA); and waist circumference was measured to the nearest 0.1 cm using a body circumference measuring tape (AccuFitness, Greenwood Village, CO). Parents reported participant characteristics at home using a parent questionnaire developed by the SHCP team. Dietary intake was self-reported by all children with the help of their parents. A SHCP educator provided classroom instruction on how to complete the 2004 Block Food Frequency Questionnaire for Ages 8–17 (FFQ), which is a pre-coded form containing a large list of specific food items that assesses frequency and portion consumed over the past seven days. Children were given three bowls
L.M. Nguyen et al. / Archives of Biochemistry and Biophysics 572 (2015) 73–80
and a plate matching the sizes of the dishes in the diagram provided with the FFQ to use as a reference [23]. Completed FFQs were sent to NutritionQuest (Berkeley, CA) for analysis and quantification of nutrients and the amounts of each MyPlate Food Groups reported. Based on the Dietary Guidelines for Americans, MyPlate is a consumer-friendly icon that was designed to help build a healthy plate at mealtime [24]. MyPlate Food Groups describe a collection of foods that are nutritionally and biologically similar and make up a healthy diet. MyPlate divides the foods into the following Food Groups: fruits, vegetables, grains, protein, and dairy. Within each group are subgroups, for example, in the Vegetables Food Group there are dark-green vegetables, starchy vegetables, red and orange vegetables, beans and peas, and other vegetables. One week after the FFQs were completed, children were recruited for participation in the validation study. Due to the aforementioned difficulties in conducting blood draws in children, three different approaches were taken to recruit volunteers for the blood draw. During the first SHCP implementation year, the blood draw events took place on a Saturday morning at a school wide Health Fair organized by the SHCP team. The Health Fair was designed to promote health and wellness in order to inspire students to want to learn more about their health. Students needed to attend the fair to participate in the blood draw and skin scan, both of which occurred on the same day. In the second year with a new set of 4th-grade students, a one-day health screening was organized on a weekday morning before school started in hopes to recruit more participants since students would already be present in the classroom. A final blood draw event was held at a two-day, back-to-back, health screening to capture students who did not attend on the first day. In the second year, RRS data were collected on a school day during classroom time, increasing the number of scans. RRS data were collected either on the same day as the blood draw, or the following day. Children who volunteered to participate in the blood draw were asked to fast for 8 h before arrival. At each blood draw event, registered nurses drew blood samples in a private room from the antecubital vein into light-sensitive EDTA-coated tubes (10 mL) (BD; Franklin Lanes, NJ). Data analysis Samples were placed on ice and transported within 2 h of collection to a laboratory where samples were separated by centrifugation (1800 rpm for 15 min) and plasma was aliquoted and stored at 80 °C until analysis. Plasma carotenoids were analyzed via HPLC with a modified method that was previously published [25]. Briefly, carotenoids were quantified on an Agilent 1100 system with a mobile phase consisting of acetonitrile/methanol (80/ 20) at a flow rate of 1.1 mL/min with a column temperature at 25 °C. Separation was performed isocratically on a Luna C18(2) column, 150 4.6 mm, 3 lm particle size with guard cartridge system (Phenomenex, Torrance, CA). A photodiode array detector was used and carotenoids were detected at a wavelength of 450 nm. Each sample was analyzed for a-carotene, b-carotene, lutein, zeaxanthin, and lycopene. Lutein and zeaxanthin were not separated with this method and were reported together. Total carotenoids were calculated by taking the sum of the four measured carotenoids. Inter sample variation from samples prepared in parallel was 90th waist circumference percentile and the rest of the percentiles, (p = 0.04) (Table 2). Additionally, significant differences were observed in reported dietary intake of carotenoids in different quartiles of energy intake, p =
