Greater bioavailability of xanthophylls compared to carotenes from orange juice (high-pressure processed, pulsed electric field treated, low-temperature pasteurised, and freshly squeezed) in a crossover study in healthy individuals.

This study examined the effect of the intake of orange juice provided freshly squeezed (FS) or processed using low-temperature pasteurisation (LP), high-pressure processing (HPP), or pulsed electric field (PEF) treatment on the serum carotenoid concentrations of 12 healthy individuals, aged 20-32 years, enrolled in a crossover study. Participants were instructed to consume 500 ml of orange juice/day for 14 days. Carotenoid concentrations in the orange juice as well as serum samples retrieved on days 7 and 14 were analysed via HPLC. A significant increase in serum xanthophyll concentrations, but not serum carotenes, was observed, with the highest increase in α- and ß-cryptoxanthin. The processing technologies applied appeared to affect serum carotenoid concentrations, with concentrations being similar in the HPP and FS orange juice types. As high variability in serum carotenoid concentrations was observed, the effect of different technologies on serum carotenoid concentration warrants further studies with larger sample sizes.

A positive impact on the serum lipid profile and cytokines after the consumption of a plant sterol-enriched beverage with a milk fat globule membrane: a clinical study.

The hypocholesterolemic effect and the modification of serum biomarkers of a dietary plant sterol (PS) intake, cholesterol precursors and cytokines after the consumption of milk-based fruit beverages with a milk fat globule membrane were evaluated by a randomized, double-blind, crossover, multiple dose bioavailability study. Postmenopausal women (n = 38) consumed daily 250 mL of a beverage with or without 2 g of PS added during 6 weeks in each of the study periods. With the intake of the PS-added beverage, significant decreases (mg dL-1) in serum total cholesterol (pre-treatment: 220.0 ± 27.8 vs. post-treatment: 212.9 ± 25.8; p < 0.05) and LDL-cholesterol (129.4 ± 28.5 vs. 121.7 ± 24.4; p < 0.05) were detected. The cholesterol precursor lathosterol (11.2%), markers of the dietary PS intake (campesterol 43.1% and ß-sitosterol 32.5%), and anti-inflammatory IL-10 cytokine (22.5%) increased significantly, with a concomitant significant reduction in pro-inflammatory IL-1ß (6.7%). No variations in HDL-cholesterol, other sterols (desmosterol and stigmasterol) or cytokines (IL-6, IL-8, IL-12p70 and TNF-α) were detected. These results indicated that this kind of PS-enriched milk-based fruit beverage is suitable during the period of clinical intervention, and its consumption may be an adequate way to improve PS functionality since a significant reduction in cholesterol levels has been observed. Therefore, the intake of this beverage could contribute to reduce the risk of cardiovascular disease also obtaining a beneficial effect on the serum inflammatory status in postmenopausal women.

Functional Foods and Health Effects: A Nutritional Biochemistry Perspective.

BACKGROUND: Increased consumer´s interest in health has driven the development of foods that offer specific beneficial effects. The list of foods and ingredients includes essential and non-essential nutrients, plant and marine components, whole foods, microorganisms, microalgae and technological approaches. Traditionally, health outcomes focussed on the prevention of chronic diseases but health targets have expanded to cover areas such as brain health, inflammation, eye health, women´s health, healthy ageing and beauty. OBJECTIVE: This review highlights, from a nutritional biochemistry perspective, differential aspects on designing and interpreting human studies to support the health effects of functional foods. RESULTS: Despite the available evidence from in vitro, animal and observational studies, welldesigned human studies are necessary to support the health effects of functional foods. Intervention trials with foods are complex as they imply limitations due to methodological, food-related and host-related factors. The use of responsive, validated and clinically relevant markers becomes essential even though there is a lack of reliable biomarkers of exposure for many bioactives. Furthermore, the effect of modulating factors such as subclinical inflammation, gut microbiota and genetic variability should be taken into account. Multiple indicators may provide a more reliable alternative to assess physiological processes while emerging biomarkers (microRNAs, epigenetic changes) constitute a promising approach. Additionally, the magnitude of the change is critical to support any health effect although interventions may have a limited clinical impact but be epidemiologically relevant. Also, based on the available data, the premise that bioactivescontaining foods are safe may be questionable. CONCLUSION: An integrated approach including multiple biomarkers, genetic variability, effect of gut microbiota and risk/benefit assessment should be used to support the potential health effects of functional foods.

Criteria of adequacy for vitamin D testing and prevalence of deficiency in clinical practice.

BACKGROUND: Vitamin D deficiency is an important concern in clinical settings although there is no consensus on who should undergo 25-OH-vitamin D testing. We studied the prevalence of vitamin D deficiency before and after introducing adequacy (clinical and biochemical) criteria for testing. METHODS: A total of 32,363 tests for 25-OH-vitamin D were retrospectively evaluated. Requests were unrestricted until December 2010 and justification criteria were applied from January 2011. During 6 years, 25,656 samples were analyzed (UHPLC) of which 12,315 were considered the first visit. The prevalence of deficiency was assessed for all the samples and according to the year, sex, season, age, origin of the requests, inclusion of adequacy criteria and consecutive visits. RESULTS: A significant proportion of the requests (25%) were unjustified and less than half of the clinically or biochemically-justified tests displayed serum concentrations indicative of deficiency. Application of adequacy criteria resulted in a non-significant increase in the prevalence of deficiency, both at the first visit (36.5 vs. 41.7, p=0.17) and for all the samples analyzed (32.0 vs. 35.5, p=0.14). The percentage of deficiency decreased in consecutive visits although 2/3 and 41% of the patients remained deficient on the second and third visit, respectively. Moreover, at least 1/5 of sufficient patients at the first test became deficient in subsequent evaluations. CONCLUSIONS: A significant proportion of the requests was unjustified by clinical or biochemical criteria. Our data also indicate that clinical and biochemical criteria may be necessary (to be present) to justify vitamin D testing but not sufficient (predictive) to indicate the presence of vitamin D deficiency.

ß-Cryptoxanthin Synergistically Enhances the Antitumoral Activity of Oxaliplatin through ΔNP73 Negative Regulation in Colon Cancer.

BACKGROUND: The acquired resistance to chemotherapy represents the major limitation in the treatment of cancer. New strategies to solve this failure and improve patients' outcomes are necessary. The cancer preventive effect of ß-cryptoxanthin has been widely described in population studies. Few reports support its putative use as an antitumoral compound. Here we focus on the therapeutic potential of ß-cryptoxanthin individually or in combination with oxaliplatin in colon cancer and try to decipher the molecular basis underlying its effect. METHODS: Apoptosis, viability and proliferation assays, mouse models, and an intervention study in 20 healthy subjects were performed. A PCR array was carried out to unravel the molecular putative basis of the ß-cryptoxanthin effect, and further signaling experiments were conducted. Comet Assay was completed to evaluate the genotoxicity of the treatments. RESULTS: ß-Cryptoxanthin differentially regulates the expression of the P73 variants in vitro, in vivo, and in a human intervention study. This carotenoid decreases the proliferation of cancer cells and cooperates with oxaliplatin to induce apoptosis through the negative regulation of ΔNP73. The antitumoral concentrations of oxaliplatin decrease in the presence of ß-cryptoxanthin to achieve same percentage of growth inhibition. The genotoxicity in peripheral blood mononuclear cells of mice decreased in the combined treatment. CONCLUSIONS: We propose a putative novel therapeutic strategy for the treatment of colon cancer based on the combination of ß-cryptoxanthin and oxaliplatin. The combined regimen produced more benefit than either individual modality without increasing side effects. In addition, the concentration-limiting toxicity of oxaliplatin is reduced in the presence of the carotenoid.

Critical evaluation of assays for vitamin D status.

PURPOSE OF REVIEW: The objective is to highlight new aspects and methodological developments in the assessment of vitamin D status in clinical settings. RECENT FINDINGS: Recent advances in the assessment of vitamin D status mostly concern two areas: the emerging clinical and analytical interest in other circulating vitamin D metabolites [i.e. 3-epimer and 24,25-(OH)2 D] that opens a new perspective for the vitamin D metabolite profiling in health and disease and methodological improvements including the development of reference measurement procedures, the certification of Standard Reference Materials, the development and application of Standardization protocols and the change of the Vitamin D External Quality Assurance Survey (DEQAS) to an accuracy-based proficiency testing scheme. SUMMARY: Despite the improvement in the analytical imprecision between methods and laboratories over the last years, the reported prevalence of vitamin D deficiency in a given population still depends on the method used. Improvements in quality control procedures, however, will improve the standardization, accuracy and comparability of methods. At present, the analytical evaluation of other circulating vitamin D metabolites (vitamin D profiling) and their clinical utility is, however, limited to liquid chromatography users.

Modulation of DNA-induced damage and repair capacity in humans after dietary intervention with lutein-enriched fermented milk.

Dietary factors provide protection against several forms of DNA damage. Additionally, consumer demand for natural products favours the development of bioactive food ingredients with health benefits. Lutein is a promising biologically active component in the food industry. The EFSA Panel on Dietetic Products, Nutrition and Allergies considers that protection from oxidative damage may be a beneficial physiological effect but that a cause and effect relationship has not been established. Thus, our aim was to evaluate the safety and potential functional effect of a lutein-enriched milk product using the Comet Assay in order to analyze the baseline, the induced DNA-damage and the repair capacity in the lymphocytes of 10 healthy donors before and after the intake of the mentioned product. Our data suggest that the regular consumption of lutein-enriched fermented milk results in a significant increase in serum lutein levels and this change is associated with an improvement in the resistance of DNA to damage and the capacity of DNA repair in lymphocytes. Our results also support the lack of a genotoxic effect at the doses supplied as well as the absence of interactions and side effects on other nutritional and biochemicals markers.

Iridophores and not carotenoids account for chromatic variation of carotenoid-based coloration in common lizards (Lacerta vivipara).

Abstract Carotenoids typically need reflective background components to shine. Such components, iridophores, leucophores, and keratin- and collagen-derived structures, are generally assumed to show no or little environmental variability. Here, we investigate the origin of environmentally induced variation in the carotenoid-based ventral coloration of male common lizards (Lacerta vivipara) by investigating the effects of dietary carotenoids and corticosterone on both carotenoid- and background-related reflectance. We observed a general negative chromatic change that was prevented by ß-carotene supplementation. However, chromatic changes did not result from changes in carotenoid-related reflectance or skin carotenoid content but from changes in background-related reflectance that may have been mediated by vitamin A1. An in vitro experiment showed that the encountered chromatic changes most likely resulted from changes in iridophore reflectance. Our findings demonstrate that chromatic variation in carotenoid-based ornaments may not exclusively reflect differences in integumentary carotenoid content and, hence, in qualities linked to carotenoid deposition (e.g., foraging ability, immune response, or antioxidant capacity). Moreover, skin carotenoid content and carotenoid-related reflectance were related to male color polymorphism, suggesting that carotenoid-based coloration of male common lizards is a multicomponent signal, with iridophores reflecting environmental conditions and carotenoids reflecting genetically based color morphs.

The antioxidant status response to low-fat and walnut paste-enriched meat differs in volunteers at high cardiovascular Risk carrying different PON-1 polymorphisms.

BACKGROUND: Cardiovascular risk largely depends on diet, antioxidant status, and gene polymorphisms. Low-fat meat (CM) and walnut-enriched meat (WM) products may exert potential beneficial health effects with respect to conventional meat products. OBJECTIVE: To compare the effects of consuming WM vs CM on reduced and oxidized glutathione, lipoperoxides, α- and γ-tocopherol levels, and paraoxonase (PON-1), catalase (CAT), and superoxide dismutase (SOD) activities in 22 volunteers (mean age 54.8 years and body mass index 29.6 kg/m(2)) at high cardiovascular risk carrying different PON-1 192/55 polymorphisms. DESIGN: The study was a 5-week nonblinded, randomized, crossover, controlled trial. RESULTS: In general term, WM vs CM improved the volunteers' antioxidant status, with several result modifications occurring after the WM period. CM consumption increased oxidized glutathione and decreased PON-1 activity (at least p < 0.05). When WM vs CM effects were compared, SOD, CAT, and PON-1 enzyme activities increased (at least p < 0.05) in PON-1 192QQ carriers. γ-tocopherol levels and SOD and PON-1 activities increased in PON-1 192QR+RR carriers besides the significant decrease of lipoperoxide levels. In PON-1 55LM+MM carriers, the intervention increased significantly all the investigated enzyme activities and glutathione levels, whereas PON-1 55LL carriers increased their PON-1 activities. CONCLUSIONS: WM consumption should be preferred to CM. The intake of WM vs CM increased PON-1 but the effect upon other antioxidant enzymes and substrates varied depending on the individual's PON-1 polymorphism. PON-1 192QR+RR carriers appear the targets for WM consumption as they increased enzyme activities and γ-tocopherol levels and decreased lipoperoxides.

The prevalence of vitamin deficiency in clinical practice is assay-dependent.

BACKGROUND & AIM: Vitamin D deficiency is an important concern in clinical settings and recently, international agencies have recognised the importance of 25-OHD assays in defining vitamin D status. Thus, our aim was to assess the consistency of different vitamin D assays in clinical practice. METHODS: 25-OH-vitamin D was measured in 332 patients by ultra-fast liquid chromatography (UHPLC) and two immunoassays (Liaison Total 25(OH) and ADVIA Centaur Vitamin D Total Assay). Samples from the Vitamin D External Quality Survey (DEQAS) and the Standard Reference Material SRM 972 were used for analytical quality control. RESULTS: All methods displayed an acceptable performance with DEQAS samples but immunoassays showed a significant bias against certified materials. Compared to UHPLC, differences were significant for both immunoassays in the deficiency interval but the systematic bias was higher for the ADVIA assay throughout the whole range of concentrations. CONCLUSION: The prevalence of vitamin D deficiency in clinical practice is assay-dependent and physicians should be aware of the uncertainty associated with vitamin D assessment.

Bioavailability of ß-cryptoxanthin in the presence of phytosterols: in vitro and in vivo studies.

Bioactive compounds are used in the design and development of new food products with potential health benefits, although little is known regarding their bioavailability and interactions. This study assessed the stability, in vitro bioaccessibility, and human bioavailability of ß-cryptoxanthin from ß-cryptoxanthin-rich drinks with and without added phytosterols developed for this purpose. The developed drinks showed no difference in the content of ß-cryptoxanthin, and they were stable over 6 months. In vitro, hydrolysis of ß-cryptoxanthin esters and the amount of free ß-cryptoxanthin at duodenal and micellar phases were similar regardless of the presence of phytosterols. In the human study, the daily intake provoked significant increments of ß-cryptoxanthin in serum regardless of the type of the drink. In conclusion, in vitro and in vivo human studies have shown that the bioavailability of ß-cryptoxanthin is not significantly affected by the presence of phytosterols when they are simultaneously supplied in a drink.

Depletion of serum carotenoid and other fat-soluble vitamin concentrations following obesity surgery.

BACKGROUND: Obesity constitutes a growing health problem, and surgical treatment of severe obesity is increasingly used. Nutrient deficiencies are common following bariatric surgery and the evidence indicates a progressive increase in the incidence and severity of the deficiency of certain vitamins and related clinical conditions. Because of the potential role of carotenoids in disease prevention, our aim was to assess the carotenoid status in candidates for obesity surgery and the time-course changes following two bariatric procedures. METHODS: Seventy-five candidates for bariatric surgery (17 men, 58 women; age 43 ± 10 years) and a total of 362 serum samples after obesity surgery (i.e., Roux-en-Y gastric bypass (n = 187) and biliopancreatic diversion (n = 175)) were consecutively collected and assessed. Retinol, α- and γ-tocopherol, 25-OH-vitamin D3, lutein, zeaxanthin, α- and ß-cryptoxanthin, lycopene (trans and cis), α- and ß-carotene (trans and cis) were analyzed by high-performance liquid chromatography. RESULTS: Mean serum levels of carotenoids in candidates for obesity surgery were within the reference values reported in controls and seasonal variations were present in several analytes. After surgery, and regardless of the type of intervention, all serum carotenoids dropped following first-order kinetics. Cis/trans ratio of lycopene and ß-carotene did not change after surgery, over the time or between surgical procedures. On a long-term, serum carotenoids were at or below fifth percentile of reference groups. CONCLUSIONS: The chronic low levels of carotenoids in these patients compromise their availability to tissues, constituting an additional risk factor for other clinical conditions. Dietary advice on carotenoid-rich, fortified foods or supplements should be also evaluated in these patients.

Time-course changes in bone turnover markers and fat-soluble vitamins after obesity surgery.

BACKGROUND: The available evidence indicates a progressive increase in the incidence and severity of the deficiency of certain vitamins and related clinical conditions (i.e., metabolic bone disease). Because of the potential role of fat-soluble vitamins and carotenoids in bone metabolism, our aim was to assess the time-course changes of fat-soluble vitamins and serum markers of bone metabolism in candidates for obesity surgery and following two bariatric procedures. METHODS: Sixty-five candidates for bariatric surgery and 150 serum samples after obesity surgery (i.e., Roux-en-Y gastric bypass, n = 85; biliopancreatic diversion, n = 65) were consecutively analyzed over a period of more than 2 years. Retinol, α- and γ-tocopherol, 25-OH-vitamin D3, ß-cryptoxanthin, and ß-carotene were analyzed by high-performance liquid chromatography. Calcium, phosphorus, alkaline phosphatase, intact parathyroid hormone (iPTH), osteocalcin, beta-crosslaps, and N-terminal peptide of procollagen I (P1NP) were determined using commercial kits. RESULTS: Retinol, ß-cryptoxanthin, ß-carotene, and α- and γ-tocopherol levels were significantly lower in post-surgery samples while osteocalcin, b-crosslaps, and P1NP were significantly increased. Along the time and regardless of the surgical procedure, P1NP, b-crosslaps, and osteocalcin increased during the first 12-24 months but declined afterward. 25-OH-vitamin D increased during the first 12 months and tended to decrease afterward while iPTH remained constant or decreased but increased after 1 year in both groups. Vitamin A remained constant but α- and γ-tocopherol, ß-cryptoxanthin, and ß-carotene decreased in both groups. CONCLUSIONS: In addition to the nutritional assessment, regular monitoring of bone markers seems necessary in these patients and the early introduction of preventive strategies (i.e., the use of antiresorptive agents) should be evaluated.

Lutein bioavailability from lutein ester-fortified fermented milk: in vivo and in vitro study.

We assessed the bioavailability of lutein from lutein-fortified fermented milk using in vivo and in vitro approaches. Twenty-four volunteers were randomized to take lutein-fortified fermented milk at two levels of fortification. Single-dose bioavailability study (2x100 ml, ca. 8 or 16 mg of lutein) was performed using a three-point approach (baseline, 3.5 and 6.5 h). Multiple-dose study consisted of consuming one serving/day (ca. 4 or 8 mg/100 ml) for 14 days. Blood samples for biochemical, hematological and lutein analysis were drawn at baseline, Day 7 and Day 14. In vitro bioaccessibility was assessed by a static gastrointestinal digestion model. Lutein content, in vitro ester hydrolysis and micellarization, and lutein concentrations achieved in serum were analyzed by HPLC. In vivo, post-prandial response was higher using the high content fermented milk, but the percentage of absorption was not different according to the dose consumed. Net increments at Day 7 and Day 14 were significantly higher on consuming the high-dose milk as well. In vitro, lutein ester hydrolysis was incomplete regardless of the amount initially present. Free lutein released was higher using the high-dose fermented milk, but the percentage of hydrolysis was similar at both levels of fortification. In the micellar phase, the percentage of free and total lutein was not different according to the dose. Our results support the suitability of the fermented milk as a carrier of lutein esters and an in vivo dose-dependent effect upon regular consumption and suggest the usefulness of in vitro models to provide relevant information to predict in vivo responses.

Carotenoids: actual knowledge on food sources, intakes, stability and bioavailability and their protective role in humans.

Carotenoids are one of the major food micronutrients in human diets and the overall objective of this review is to re-examine the role of carotenoids in human nutrition. We have emphasized the attention on the following carotenoids present in food and human tissues: beta-carotene, beta-cryptoxanthin, alpha-carotene, lycopene, lutein and zeaxanthin; we have reported the major food sources and dietary intake of these compounds. We have tried to summarize positive and negative effects of food processing, storage, cooking on carotenoid content and carotenoid bioavailability. In particular, we have evidenced the possibility to improve carotenoids bioavailability in accordance with changes and variations of technology procedures.

Simultaneous measurement of retinol, alpha-tocopherol and six carotenoids in human plasma by using an isocratic reversed-phase HPLC method.

A simple and sensitive isocratic reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous determination of retinol, alpha-tocopherol and six carotenoids in human plasma was described. Sample preparation of the earlier published method was further developed by addition of ultrapure water, which enabled aqueous layer to freeze facilitating phase separation without pipetting thus also improving precision of the method. Developed method appeared to be less laborious and time consuming compared to the traditional extraction methods, which require removal of organic layer by pipetting. The recoveries (absolute and relative) were between 80% and 103%. The intra-assay CVs were 1.1-4.0% (normal level) and 3.3-9.0% (low level). Inter-assay CVs were 5.3-8.8%. Reference method for all these analytes was not available, but a comparison with another published method was carried out. The results of the comparison matched satisfactorily. The method is used routinely in our laboratory in a large population-based study.

Suitability of 3-point versus 7-point postprandial retinyl palmitate AUC in human bioavailability studies.

BACKGROUND: Bioavailability is a critical feature in the assessment of the role of micronutrients in human health. Although postprandial behaviour does not predict long-term responses and acute responses, it is accepted that the study of triglyceride-rich lipoprotein fractions reflects newly absorbed lipids from recent meals. AIM: To assess the predictive value of a 3-point versus 7-point post-prandial response (area under the curve) in nutrient bioavailability studies in humans. METHODS: We used results from a human bioavailability study (n = 19) that consisted of a single-dose pharmacokinetic assay involving three types of commercially available vitamin A and E fortified milk. RESULTS: Correlation coefficients between 3-point AUC (AUCp, predictive) versus 7-point AUC (AUCc, conventional) ranged between r = 0.81 (P < 0.001) for vitamin A-fortified skim milk and r = 0.95 (P < 0.001) for whole milk. Bland-Altman representations showed a good agreement between the two methods with 95% of the differences within the concordance limits. More than 90% of the subjects were correctly classified in the same or adjacent quartiles and he calculated relative absorption of vitamin A from the foods was, on average, <5% lower using the AUCp compared to that estimated using AUCc. CONCLUSION: The use of the 3-point approach may be a reliable alternative to assess the relative postprandial lipid response in human bioavailability studies. Nevertheless, since this approach has been studied considering one nutrient (i.e. preformed vitamin A) and one type of food (i.e. milk), its applicability to other nutrients and foods should be tested.

Comparative in vitro bioaccessibility of carotenoids from relevant contributors to carotenoid intake.

To compare the in vitro bioaccessibility of lutein, zeaxanthin, beta-cryptoxanthin, lycopene, and alpha-and beta-carotenes from relevant dietary contributors, a gastrointestinal model was used to assess the stability, isomerization, carotenol ester hydrolysis, and micellarization. Salivar, gastric, duodenal, and micellar phases were extracted, with and without saponification, and analyzed by using a quality-controlled HPLC method. The stability of carotenoids under digestion conditions was >75%, regardless of the food analyzed, whereas micellarization ranged from 5 to 100%, depending on the carotenoid and the food. cis-Isomers were maintained in processed foods, but increased in fresh foods. Xanthophyll ester hydrolysis was incomplete (<40%), and both free and ester forms were incorporated into supernatants, regardless of the xanthophyll involved and the food assessed. In vitro bioaccesibility varies widely both for different carotenoids in a given food and for a given carotenoid in different foods. Although in vitro bioaccesibility may not be enough to predict the in vivo bioavailability, it may be relevant for the food industry and for food-based dietary guidelines.