Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin.

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins.

Cerebrolysin enhances the expression of the synaptogenic protein LRRTM4 in the hippocampus and improves learning and memory in senescent rats.

Aging reduces the efficiency of the organs and systems, including the cognitive functions. Brain aging is related to a decrease in the vascularity, neurogenesis, and synaptic plasticity. Cerebrolysin, a peptide and amino acid preparation, has been shown to improve the cognitive performance in animal models of Alzheimer's disease. Similarly, the leucine-rich repeat transmembrane 4 protein exhibits a strong synaptogenic activity in the hippocampal synapses. The aim of this study was to evaluate the effect of the cerebrolysin treatment on the learning and memory abilities, sensorimotor functions, and the leucine-rich repeat transmembrane 4 protein expression in the brain of 15-month-old rats. Cerebrolysin (1076 mg/kg) or vehicle was administered to Wistar rats intraperitoneally for 4 weeks. After the treatments, learning and memory were tested using the Barnes maze test, and the acoustic startle response, and its pre-pulse inhibition and habituation were measured. Finally, the leucine-rich repeat transmembrane 4 expression was measured in the brainstem, striatum, and hippocampus using a Western-blot assay. The 15-month-old vehicle-treated rats showed impairments in the habituation of the acoustic startle response and in learning and memory when compared to 3-month-old rats. These impairments were attenuated by the subchronic cerebrolysin treatment. The leucine-rich repeat transmembrane 4 protein expression was lower in the old vehicle-treated rats than in the young rats; the cerebrolysin treatment attenuated that decrease in the old rats. The leucine-rich repeat transmembrane 4 protein was not expressed in striatum or brainstem. These results suggest that the subchronic cerebrolysin treatment enhances the learning and memory abilities in aging by increasing the expression of the leucine-rich repeat transmembrane 4 protein in the hippocampus.

Effects of ALAD genotype on the relationship between lead exposure and anthropometry in a Cohort of Mexican children.

OBJECTIVES: Lead exposure is associated with children's growth, but this relationship may depend on the presence of susceptibility factors, including genetic variation. Blood lead levels (BLL) differ by ALAD (aminolevulinic acid dehydratase) genotype. We investigated the association between BLL and growth in Mexican first-graders with different ALAD genotypes. METHODS: Children between the ages of 6-8 years (n = 602) attending first grade in schools within the vicinity of a metal foundry in Torreón, Mexico were enrolled into a randomized controlled trial (RCT) testing the efficacy of iron and/or zinc supplementation on blood lead levels (BLL) and cognition. BLL and anthropometry were assessed at baseline (height, height-for-age z-score (HAZ), knee height, head circumference), after 6 (head circumference) and 12 months (height, HAZ, knee height). Children with ALAD1-1 and ALAD1-2/2-2 were compared. The study sample included 538 and 470 participants who had complete data at baseline and follow-up, respectively. Separate multivariable linear regression models adjusted for covariates were used to test the association between BLL at baseline and each anthropometric measure. Covariates included age, sex, hemoglobin, crowding, and maternal education. BLL x ALAD genotype interaction term was tested. RESULTS: Median BLL (10.1 µg/dL) did not differ by ALAD genotype. After covariate adjustment, baseline BLL was inversely associated with baseline height, HAZ, and knee height. The association (ß [95% CI]) between BLL and baseline height (-0.38[-0.68, -0.09]), HAZ (-0.07[-0.12, -0.02]) and knee height (-0.14[-0.25, -0.02]), was somewhat stronger in children with ALAD1-2/2-2 than ALAD1-1 (-0.09[-0.16, -0.02], -0.02[-0.03, -0.004] and -0.04[-0.06, -0.01], respectively). No associations between BLL and growth at 6 or 12 months were detected irrespective of ALAD genotype. CONCLUSIONS: BLL was adversely associated with anthropometric measures among Mexican children. ALAD genotype may be a susceptibility factor for the effects of lead on child growth.

Association between obesity and depressive symptoms in Mexican population.

PURPOSE: Obesity and depression are among the leading causes of disability in Mexico, but their association has not been explored yet. The aim of the current study was to investigate the association between obesity and depression in Mexican population. METHODS: We used data from the health and nutrition survey (ENSANUT 2012), which is representative of the Mexican population. Obesity was determined using the body mass index (BMI) and abdominal obesity by measuring waist circumference. Depressive symptoms were reported using the Center for Epidemiological Studies Depression Scale Short-Form (CES-D-SF, scale 0-21). Regression analyses were performed between obesity and depression, adjusting for gender, age, living with a partner, education, and diabetes history. RESULTS: Obese women had 1.28 (95% CI 1.07-1.53) times the odds of having depression in comparison with normal-weight women, whereas no association was found for men (OR 0.94; 95% CI 0.74-1.19). A significant association between BMI and depressive symptoms score (ß = 0.05, 95% CI 0.02-0.07) was present in women, but no association was found for men (ß = - 0.02, 95% CI - 0.05 to 0.00). There was a statistically significant association between waist circumference and depression scores again for women (ß = 0.03, 95% CI 0.01-0.04) but not for men (ß = 0.00, 95% CI - 0.01 to 0.01). No associations were found between abdominal obesity and depression for both genders. No association was found between different obesity severity levels and depression for both genders. CONCLUSION: Obesity was associated with depression in Mexican women, whereas no association was found between obesity and depression in men.

Use of urea-polyacrylamide electrophoresis for discrimination of A1 and A2 beta casein variants in raw cow's milk.

Beta-casein (BC) in cow's milk occurs in several genetic variants, where BC A1 (BCA1) and BC A2 (BCA2) are the most frequent. This work deals with a method based on modified polyacrylamide gel electrophoresis using urea PAGE to discriminate BCA1 and BCA2 variants from Holstein Friesian (HF) and genetically selected Jersey A2/A2 (JA2) cow's milk. Two well defined bands were obtained from BC fraction of HF milk, while that of JA2 showed a single band. Proteins from these bands were sequenced by HPLC-quadrupole linear ion trap/mass spectrometry, resulting in BCA1 and BCA2 separation from the BC fraction of HF milk, whereas BCA2 was the only constituent of JA2 fraction. This method represents a feasible and useful tool to on site phenotyping of BC fraction of cow's milk for pharmaceutical and food industries applications.

Iron and Zinc Supplementation Does Not Impact Urinary Arsenic Excretion in Mexican School Children.

OBJECTIVE: To examine the role of iron and zinc in arsenic excretion and metabolism in children. STUDY DESIGN: An analysis of urinary arsenic (UAs) concentrations from a double-blind randomized trial originally testing the efficacy of iron and zinc for lowering blood lead levels in children. A 2 × 2 factorial design was used, with children randomized individually, stratified by sex and classroom, to receive 30?mg ferrous fumarate (n?=?148), 30?mg zinc oxide (n?=?144), iron and zinc together (n?=?148), or placebo (n?=?151). Of the 602 children enrolled, 527 completed the 6-month treatment, and 485 had both baseline and final UAs values. The baseline total UAs concentration ranged from 3.2 to 215.9?µg/L. RESULTS: At baseline, children in the highest tertile of serum ferritin concentration had higher excretion of dimethylarsinic acid (DMA; 1.93?±?0.86%; P?

Fat mass obesity-associated (FTO) (rs9939609) and melanocortin 4 receptor (MC4R) (rs17782313) SNP are positively associated with obesity and blood pressure in Mexican school-aged children.

Childhood overweight and obesity are worldwide public health problems and risk factors for chronic diseases. The presence of SNP in several genes has been associated with the presence of obesity. A total of 580 children (8-13 years old) from Queretaro, Mexico, participated in this cross-sectional study, which evaluated the associations of rs9939609 (fat mass obesity-associated (FTO)), rs17782313 (melanocortin 4 receptor (MC4R)) and rs6548238 (transmembrane protein 18 (TMEM18)) SNP with obesity and metabolic risk factors. Overweight and obesity prevalence was 19·8 and 19·1 %, respectively. FTO, MC4R and TMEM18 risk allele frequency was 17, 9·8 and 89·5 %, respectively. A significant association between FTO homozygous and MC4R heterozygous risk alleles and obesity was found (OR 3·9; 95 % CI 1·46, 10·22, and OR 2·1; 95 % CI 1·22, 3·71; respectively). The FTO heterozygous subjects showed higher systolic and diastolic blood pressures, compared with the homozygous for the ancestral allele subjects. These results remain significant after considering adiposity as a covariate. The FTO and MC4R genotypes were not significantly associated with total cholesterol, HDL-cholesterol and insulin concentration. No association was found between TMEM18 risk allele and obesity and/or metabolic alterations. Our results show that, in addition to a higher BMI, there is also an association of the risk genotype with blood pressure in the presence of the FTO risk genotype. The possible presence of a risk genotype in obese children must be considered to offer a more comprehensive therapeutic approach in order to delay and/or prevent the development of chronic diseases.

Sex differences in the reduction of arsenic methylation capacity as a function of urinary total and inorganic arsenic in Mexican children.

Chronic arsenic (As) exposure decreases adult and children's ability to methylate inorganic As (iAs); however, few studies have examined children's sex differences. We measured urinary concentrations of iAs, monomethylarsonic (MMA), and dimethylarsinic (DMA) acids, and calculated the primary (PMI: MMA/iAs) and secondary (SMI: DMA/MMA) methylation capacity indexes in 591 children 6-8 years in Torreón, Mexico. We determined iAs, MMA, and DMA by hydride generation cryotrapping AAS. Lineal regression models estimated associations between methylation capacity and total As (TAs) or iAs. Interactions with sex were tested at p<0.10. Boys had significantly higher TAs levels, (58.4µg/L) than girls (46.2µg/L). We observed negative associations between TAs and PMI (ß=-0.039; p<0.18) and SMI (ß=-0.08; p=0.002) with significant sex differences; PMI reduction was significant in boys (ß=-0.09; p=0.02) but not in girls (ß=0.021; p=0.63), p for interaction=0.06. In contrast, SMI reduction was significantly more pronounced in girls. Furthermore, negative associations PMI (ß=-0.19; p<0.001) and SMI (ß=-0.35; p<0.001) were a function of urinary iAs levels, independently of TAs; however, the reduction in PMI was more pronounced in boys (ß=-0.24; p<0.001; girls ß=-0.15; p<0.001), p for interaction=0.04. A significant negative association was observed between SMI and iAs levels without significant sex differences. TAs and iAs associations with metabolite percentages were in good agreement with those observed with methylation indexes. Our results suggest that iAs plays an important role in reducing As methylation ability and that significant sex differences are present in As metabolism. These differences merit further investigation to confirm our findings and their potential implications for arsenic toxicity in children.

Beliefs and motives related to eating and body size: a comparison of high-BMI and normal-weight young adult women from rural and urban areas in Mexico.

BACKGROUND: Effective treatment and prevention of obesity and its co-morbidities requires the recognition and understanding of cultural and social aspects of eating practices. The objective of the present study was to identify social factors and beliefs that may explain undesirable eating practices among women with high body mass index (HBMI) compared with normal-weight (NW) women from rural and urban areas classified as middle-low socioeconomic status (SES) in the State of Querétaro, Mexico. METHODS: A qualitative technique with individual in-depth interviews was used. Fifty-five women with either NW or HBMI from rural and urban areas participated in the study. The responses were analyzed by coding and grouping text fragments into categories in a data matrix, in order to make comparisons between BMI groups and between rural and urban women. RESULTS: The habit of skipping breakfast prevailed among women with HBMI who also reported childhood food deprivation. Feelings related to eating seemed to be more important than losing weight among women with HBMI from urban and rural areas. Thus, overweight might be interpreted as a social symbol of the enjoyment of a good life, primarily in rural areas. Overweight was socially accepted when it occurred in children and in married woman, mainly because it is a symbol of the good life that the head of the household provides, and also because women may feel more relaxed about their weight when they already have a partner. The study also revealed that women with HBMI were not sufficiently motivated to lose weight unless they experience a physical indication of poor health. CONCLUSION: The findings from this study are helpful in the understanding of the reasons why strategies for the prevention and treatment of obesity may not be as effective as expected. The belief system of particular social groups within different SESs should be considered in order to understand the etiology of obesity and develop effective strategies.

4-Hydroxyisoleucine from Fenugreek (Trigonella foenum-graecum): Effects on Insulin Resistance Associated with Obesity.

Obesity and insulin resistance (IR) are interdependent multifactorial processes that cannot be understood separately. Obesity leads to systemic inflammation and increased levels of free fatty acids that provoke IR and lipotoxicity. At the same time, IR exacerbates adipose cell dysfunction, resulting in chronic inflammation and major lipotoxic effects on nonadipose tissues. 4-Hydroxyisoleucine (4-OHIle), a peculiar nonprotein amino acid isolated from fenugreek (Trigonella foenum-graecum) seeds, exhibits interesting effects on IR related to obesity. 4-OHIle increases glucose-induced insulin release, and the insulin response mediated by 4-OHIle depends on glucose concentration. The beneficial effects observed are related to the regulation of blood glucose, plasma triglycerides, total cholesterol, free fatty acid levels, and the improvement of liver function. The mechanism of action is related to increased Akt phosphorylation and reduced activation of Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB. Here, we present a review of the research regarding the insulinotropic and insulin-sensitising activity of 4-OHIle in in vitro and in vivo models.

Neuroprotective effect of Arthrospira (Spirulina) platensis against kainic acid-neuronal death.

Context Arthrospira (Spirulina) platensis (SP) is a cyanobacterium which has attracted attention because of its nutritional value and pharmacological properties. It was previously reported that SP reduces oxidative stress in the hippocampus and protects against damaging neurobehavioural effects of systemic kainic acid (KA). It is widely known that the systemic administration of KA induces neuronal damage, specifically in the CA3 hippocampal region. Objective The present study determines if the SP sub-chronic treatment has neuroprotective properties against KA. Materials and methods Male SW mice were treated with SP during 24 d, at doses of 0, 200, and 800 mg/kg, once daily, and with KA (35 mg/kg, ip) as a single dose on day 14. After the treatment, a histological analysis was performed and the number of atrophic neuronal cells in CA3 hippocampal region was quantified. Results Pretreatment with SP does not protect against seizures induced by KA. However, mortality in the SP 200 and the SP 800 groups was of 20%, while for the KA group, it was of 60%. A single KA ip administration produced a considerable neuronal damage, whereas both doses of SP sub-chronic treatment reduced the number of atrophic neurons in CA3 hippocampal region with respect to the KA group. Discussion The SP neurobehaviour improvement after KA systemic administration correlates with the capacity of SP to reduce KA-neuronal death in CA3 hippocampal cells. This neuroprotection may be related to the antioxidant properties of SP. Conclusion SP reduces KA-neuronal death in CA3 hippocampal cells.

[Lactose intolerance]. / Intolerancia a la lactosa.

The most common problem limiting milk consumption worldwide is lactose intolerance (LI), which is defined as the experience of gastrointestinal symptoms due to the intake of lactose-containing food. When symptoms ensue the intake of milk, the condition is referred as milk intolerance, and it may or may not be due to LI. The most common cause of LI is primary lactase deficiency which occurs in 30% of Mexican adults when one glass of milk is consumed (12-18 g of lactose). LI occurs in less than 15% of adults after the intake of this dose of lactose. Another cause of lactose intolerance is due to secondary lactase deficiency, which occurs because lactase is reduced due to diseases that affect the intestinal mucosa. Lactose intolerance can be eliminated or significantly reduced by elimination or reduction of the intake of milk and milk containing products. Recent studies demonstrate that when ß-casein-A1 contained in milk is hydrolyzed it produces ß-casomorphine-7 which is an opioid associated with milk intolerance.

Effect of a ß-Hydroxyphosphonate Analogue of ʟ-Carnitine on Insulin-Sensitive and Insulin-Resistant 3T3-L1 Adipocytes.

This study investigated the effect of a ß-x200B;hydroxyphosphonate analog of ʟ-carnitine (L-CA) (CAS number: 1220955-x200B;20-3, Component: 1221068-91-2, C12H29NO4PI), (3-Hexanaminium, 1-(dimethoxyphosphinyl)-2-hydroxy-N,N,N,5-x200B;tetramethy-iodide (1:1), (2R, 3S)) on parameters related with type-2 diabetes in an in vitro model. Nontoxic concentrations of L-CA were assayed and compared to commercial ʟ-carnitine effects. L-CA did not affect adipogenesis in normal cells, but an increment of TG accumulation was observed on insulin-resistant adipocytes (80%) when compared with resistant control. L-CA also stimulated glucose analog 2-NBDG uptakes on insulin-resistant adipocytes in a similar way as insulin when compared to insulin-resistant cells. Our results show that the L-CA promoted insulin-like responses on insulin-resistant adipocytes without appreciable pro-adipogenic effect in sensitive adipocytes.

Effectiveness of intra-articular injections of sodium bicarbonate and calcium gluconate in the treatment of osteoarthritis of the knee: a randomized double-blind clinical trial.

BACKGROUND: A novel therapeutic management of osteoarthritis (OA) of the knee was assessed. The study aimed to evaluate the effect of monthly sodium bicarbonate with a single (SBCG1) or double dose (SBCG2) of calcium gluconate injections on OA of the knee; as well as the efficacy and safety of both SBCG interventions in the long term. METHODS: A double-blind parallel-group clinical trial with 74 knee OA patients was performed during 12 months, both SBCG interventions were followed-up for another 6mo after intervention. The outcome variables were the Western Ontario-McMaster University Osteoarthritis Index (WOMAC), the Lequesne's functional index and joint-space width changes from serial radiographs. RESULTS: After 12 months, group SBCG1 decreased -14.8 (95% CI:-14.2, -17.0) and group SBCG2 decreased -14.6 (-16.9, -12.4) in the global WOMAC score, the mean changes represent 80% and 82% lessened pain, respectively. In the Lequesne Functional Index scale, SBCG1 decreased -11.9 (-10.4, -14.2) and SBCG2 decreased -11.9 (-13.8, -10.0), representing 66 and 69% of improvement. Both mean scores were maintained after intervention discontinued. SBCG2 improved the knees' joint space width more than SBCG1 at 3 and 18 months. Both SBCG interventions were well tolerated after 12 months of treatment CONCLUSION: A solution of sodium bicarbonate and calcium gluconate is effective on reducing the symptoms associated with OA. Its beneficial effect is maintained for one year of continuous monthly administration and at least for 6 months after the administration is discontinued. When the dose of calcium gluconate is increased, it prevents further narrowing of joint-space. TRIAL REGISTRATION: Clinicaltrials.gov NCT00977444 September 11, 2009.

Novelty exposure overcomes foot shock-induced spatial-memory impairment by processes of synaptic-tagging in rats.

Novelty processing can transform short-term into long-term memory. We propose that this memory-reinforcing effect of novelty could be explained by mechanisms outlined in the "synaptic tagging hypothesis." Initial short-term memory is sustained by a transient plasticity change at activated synapses and sets synaptic tags. These tags are later able to capture and process the plasticity-related proteins (PRPs), which are required to transform a short-term synaptic change into a long-term one. Novelty is involved in inducing the synthesis of PRPs [Moncada D, et al. (2011) Proc Natl Acad Sci USA 108:12937-12936], which are then captured by the tagged synapses, consolidating memory. In contrast to novelty, stress can impair learning, memory, and synaptic plasticity. Here, we address questions as to whether novelty-induced PRPs are able to prevent the loss of memory caused by stress and if the latter would not interact with the tag-setting process. We used water-maze (WM) training as a spatial learning paradigm to test our hypothesis. Stress was induced by a strong foot shock (FS; 5 × 1 mA, 2 s) applied 5 min after WM training. Our data show that FS reduced long-term but not short-term memory in the WM paradigm. This negative effect on memory consolidation was time- and training-dependent. Interestingly, novelty exposure prevented the stress-induced memory loss of the spatial task and increased BDNF and Arc expression. This rescuing effect was blocked by anisomycin, suggesting that WM-tagged synapses were not reset by FS and were thus able to capture the novelty-induced PRPs, re-establishing FS-impaired long-term memory.

Increased calorie intake at a specific mid-morning meal and increased intake of soft drinks are strongly associated with obesity in Mexican rural women.

The study investigated the dietary habits and foods that are associated with obesity in women from a rural area in Mexico. Anthropometry and body fat were measured in 580 women. Participants answered a socioeconomic and a food-frequency questionnaire; a subsample (n = 80) also answered three 24-hour-recall questionnaires. Results showed that obese women consumed more soft drinks and fat than did overweight and normal-weight women. Women who consumed more energy during a mid-morning meal had higher BMI. A strategy to decrease the prevalence of obesity in rural areas could be to encourage limiting the consumption of soft drinks and eliminating or reducing caloric intake at a mid-morning meal.

Pharmacokinetic and pharmacological effects of ß-hydroxyphosphocarnitine in animal models.

The purpose of this research was to describe the pharmacokinetic parameters of ß-hydroxyphosphocarnitine (ß-HPC; CAS No. 1220955-20-3) after a single oral dose in rats and rabbits as well as to assess the impact of 14 weeks of ß-HPC (100 mg/kg) treatment on the serum metabolites and liver enzymes, body weight, and hepatic steatosis of lean and obese Zucker fa/fa rats. In the case of the rat and rabbit study, the ß-HPC area under the curve, biological half-life, and clearance were 2,174.4 versus 3,128 µg ∙ h/ml, 23.7 versus 8.87 h, and 13.9 versus 151.1 ml/h in the rats versus the rabbits, respectively. The values for the time of maximal concentration were 0.58 versus 1.53 h, for the maximal concentration, they were 62.4 versus 221.4 µg/ml, and for the absorption rate constant 0.02 versus 2.40 h(-1), respectively. In the case of the Zucker fa/fa rat study, ß-HPC administered orally once a day reduced insulin, triglyceride, and cholesterol levels in the liver and serum; it also reduced weight gain and decreased liver steatosis in obese rats after 14 weeks. ß-HPC could therefore potentially be used in the treatment of metabolic syndrome.

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells.

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells.

Dietary Energy Density Is Associated with Biomarkers of Chronic Diseases-A Cross-Sectional Study of School-Aged Children in Rural Mexico.

Background: Dietary energy density (DED) is associated with chronic disease markers in adults. However, results in children are still controversial. Objective: To evaluate the DED of children and its association with obesity and biomarkers of chronic disease. Methods: In this cross-sectional study, we recruited 284 children (6-10 y) from rural Mexico. Dietary intake was assessed using three 24-h recalls. DED was calculated for "foods only" (DEDfo) and for "foods and beverages" (DEDfb). Weight, height, and body fat percent (dual-energy X-ray absorptiometry) were measured. Inflammatory cytokines, lipid profile, leptin, and insulin resistance were determined from a fasting blood sample. Results: DEDfo was 1.91 ± 0.36 kcal/g and DEDfb was 1.36 ± 0.31 kcal/g. Higher DEDfo and DEDfb were associated with higher risk to have insulin resistance [odds ratio (OR) = 3.92, 95% confidence interval (CI): 1.66, 9.22, P < 0.01; OR = 3.51, 95% CI: 1.25, 9.87, P = 0.02, respectively]. Higher DEDfo was associated with higher risk of higher leptin levels (OR = 3.17, 95% CI: 1.01, 10.23). Also, DEDfo and DEDfb were associated with higher concentrations of cholesterol (ß = 11.67, 95% CI: 1.81, 19.53, P = 0.03; and ß = 11.74, 95% CI: 2.69, 20.74 P = 0.01, respectively) and higher odds of having high insulin concentrations (OR = 2.52, 95% CI: 1.26, 5.06, P = 0.01; and OR = 2.95, 95% CI: 1.30, 6.70, P = 0.01). DEDfo and DEDfb were not associated with any measure of obesity and inflammatory cytokines in the adjusted models. Conclusions: DED was associated with higher leptin and cholesterol concentrations, and having insulin resistance, but not with any measure of obesity or inflammation. Reducing DED may reduce risk of cardiovascular disease and improve insulin sensitivity in school-aged children.