Biomarkers of carotenoid bioavailability.

The use of biomarkers constitutes an essential tool to assess the bioavailability of carotenoids in humans. The present article aims to review several methodological, host-related and modulating factors relevant on assessing and interpreting carotenoid bioavailability. Markers for carotenoid bioavailability can be broadly divided into direct, biochemical or "analytical" markers and indirect, physiological or "functional" indicators. Analytical markers usually refer to biochemical indicators of intake and/or status (short and long term exposure) while functional measures may be interpreted in terms of cumulative exposure, biological effect (bioactivity) or modification of risk factors. Both types of markers display advantages and limitations but, in general, a relationship exists among the type of marker, the biological specimen needed and the time required for a change. Humans may absorb a wide range of carotenes and xanthophylls and many of them may be found in serum and tissues. However, under physiological conditions, the several classes of dietary carotenoids may behave unequally leading to a different systemic profile and, moreover, they can be selectively accumulated at target tissues. In addition, some carotenoids may be chemically and enzymatically modified generating different oxidative metabolites and apocarotenoids. Quantitatively, the biological response upon carotenoid intervention (assessed by analytical and functional markers) is highly variable but the use of large doses and long-term protocols may lead to saturation effects and the loss of linearity in the response. Also, despite carotenoid exposition is considered to be safe, markers of overexposure include clinical signs (i.e. carotenodermia, corneal rings and retinopathy) and biochemical indicators (hypercarotenemia, xanthophyll esters). Overall, both host-related and methodological factors may influence analytical and functional markers to assess carotenoid bioavailability although the different subclasses of carotenoids may not be equally affected.

In vitro digestion-assisted development of a ß-cryptoxanthin-rich functional beverage; in vivo validation using systemic response and faecal content.

Bioavailability of carotenoids is low and significant amounts reach the colon where they may be biologically active. We aimed to optimize a previously developed beverage designed to improve cardiovascular and bone remodelling markers in post-menopausal women. By assessing different lipid emulsions (soy lecithin, milkfat globule membrane (MFGM) and olive oil) on the in vitro bioaccessibility of ß-Cryptoxanthin and phytosterols, a MFGM containing beverage was selected and resulted stable over time (recovery >95%) under in vitro digestion and simulated anaerobic conditions. This beverage was tested in a randomized human trial (n=38) by evaluating systemic response and the colonic availability of ß-Cryptoxanthin. Consumption for six weeks provoked an increment in serum ß-Cryptoxanthin of 38.9µg/dl (CI 95%; 31.0, 46.8; p<0.001). In faeces, free ß-Cryptoxanthin, tentatively identified ß-Cryptoxanthin esters and the ratio cis-/trans-ß-carotene approached the profile in the beverage and after in vitro digestion but it was different from serum. In conclusion, in vitro digestion-assisted approach appears adequate to develop functional foods although in vivo validation should consider both systemic response and the availability at the colon.

Prevalence of hypercalcemia related to hypervitaminosis D in clinical practice.

BACKGROUND & AIMS: Recent interest in vitamin D has led to a substantial increase in the use of vitamin D supplements. Vitamin D intoxication may be a concern as hypervitaminosis D can result in irreversible calcification of soft tissues so that it is important to detect early markers of vitamin D intoxication. Our aim was to assess the simultaneous presence of biochemical markers of vitamin D toxicity (i.e. hypervitaminosis D, hypercalcemia) and determine the concentrations of 25-OH-vitamin D at which the risk of hypercalcemia, and thus toxicity, might begin. METHODS: We evaluated retrospectively a 6-year period during which 25.567 samples were assessed for 25-OH-vitamin D status by UHPLC. Hypervitaminosis D was defined at serum 25-OH-vitamin D >160 nmol/L. Serum and urine calcium, phosphorus and iPTH were also recorded, if available. Medical history revision was performed in subjects displaying simultaneously hypervitaminosis D and hypercalcemia. RESULTS: Overall, hypervitaminosis D was found in 475 samples (1.86%) of which 51 displayed hypercalcemia (11.1%). A total of 382 samples were identified as the first record of hypervitaminosis D and 39 presented hypercalcemia (10.2%), most of them at 25-OH-vitamin D levels between 161 and 375 nmol/L. Only in 15 subjects, hypercalcemia could be directly attributed to vitamin D and serum 25-OH-vitamin D ranged between 164 and 1139 nmol/l. In no case, serum calcium achieved concentrations considered as critical values (>13 mg/dl). CONCLUSION: Hypercalcemia due to vitamin D represented <4% of the total hypervitaminosis D detected and <0.1% of the tests performed. However, a highly variable response was observed and most subjects presented hypercalcemia at serum concentrations of 25-OH-vitamin D < 375 nmol/L.

Analysis of microsamples of human faeces: a non-invasive approach to study the bioavailability of fat-soluble bioactive compounds.

INTRODUCTION: Bioavailability is a critical feature in the assessment of the role of micronutrients in human health. Poorly bioavailable micronutrients like carotenoids may reach significant concentrations in the gastrointestinal tract where they may exert biological actions. PURPOSE: We evaluated a simple collection protocol to determine vitamin A, E and carotenoids in microsamples of human faeces as a non-invasive approach for nutritional studies. METHODS: Microsamples of human faeces were collected using a commercially available device, extracted and analysed on two LC systems. Suitability of the protocol was assessed by evaluating several factors including the effect of simulated colonic conditions and two nutritional scenarios with different dietary components, chemical forms, nutritional goals and target groups. RESULTS: The protocol was reproducible and representative of a faeces sample. The major dietary and serum carotenoids, and several "unidentified" compounds (possibly metabolites) could be detected, and cis-/trans-ß-carotene profile reflected dietary intervention. In faeces of neonates, free retinol, retinyl and α-tocopheryl acetate (from infant formula), long-chain fatty acid retinyl esters (from human milk), free γ-tocopherol and α-tocopherol could be detected. CONCLUSION: Our results show that the analysis of vitamin A, E and carotenoids in microsamples of human faeces is a suitable, non-invasive approach that may provide relevant information regarding responsiveness, nutrient stability and metabolism and may help assess adequacy of chemical forms and delivery systems reaching the colon.

ß-Cryptoxanthin modulates the response to phytosterols in post-menopausal women carrying NPC1L1 L272L and ABCG8 A632 V polymorphisms: an exploratory study.

Phytosterol (PS) intake may be used for hypercholesterolaemia in some groups although the presence of non-responders is well known. Carotenoids and PS/cholesterol may compete for the same transporters during absorption. As part of a randomized, double-blind, crossover, multiple-dose supplementation study with ß-cryptoxanthin (ß-Cx) and PS, single and combined, polymorphisms of ABCG8 (A632V) and NCPL1 (L272L) were determined in 19 post-menopausal women. Subjects carrying CC polymorphism for NCP1L1 (L272L) showed a net increase in total cholesterol and LDL after PS intake but, interestingly, displayed a decrease in both lipid fractions after consuming PS plus ß-Cx. For the ABCG8 (A632V) gene, CT/TT carriers consuming PS also displayed an increase in total cholesterol and LDL, but this increment was much lower after the intake of PS plus ß-Cx. Additionally, in CC carriers for ABCG8 (A632V), a greater decrease in total cholesterol and LDL was found after the intake of PS plus ß-Cx compared to that observed after PS alone. Overall, our results suggest that ß-Cx improves the response to PS in individuals carrying specific genetic polymorphisms (i.e. non-responders), opening the possibility to modulate the response to PS by food technology. (ClinicalTrials.gov NCT01074723).

Effect of ß-cryptoxanthin plus phytosterols on cardiovascular risk and bone turnover markers in post-menopausal women: a randomized crossover trial.

BACKGROUND AND AIM: Post-menopausal women are at higher risk of cardiovascular disease and bone demineralization. Phytosterols (PS) may be used for hypercholesterolemia in some groups and ß-cryptoxanthin (ß-Cx) displays a unique anabolic effect on bone. Our aim was to assess the changes in cardiovascular and bone turnover markers from the oral intake of ß-Cx and PS in post-menopausal women. METHODS AND RESULTS: A randomized, double-blind, crossover study with ß-Cx (0.75 mg/day) and PS (1.5 g/day), single and combined, was performed in 38 postmenopausal women. Diet was supplemented with 1 × 250 mL milk-based fruit drink/day for 4 weeks with a wash-out period of 4-weeks in between. Serum ß-Cx and PS were determined by UPLC and CG-FID respectively. Outcome variables included markers of bone turnover and cardiovascular risk. Biological effect was assessed by paired t test and generalized estimating equations analysis that included the previous treatment, the order of intervention and the interactions. The intake of beverages containing ß-Cx and PS brought about a significant increase in serum levels of ß-Cx, ß-sitosterol and campesterol. Intervention caused changes in almost all the markers while the order, previous treatment and the interaction did not reach statistical significance. Only the intake of the beverage containing ß-Cx plus PS brought about significant decreases in total cholesterol, c-HDL, c-LDL and bone turnover markers. CONCLUSIONS: ß-Cx improves the cholesterol-lowering effect of PS when supplied simultaneously and this combination may also be beneficial in reducing risk of osteoporosis. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov number NCT01074723.

Assessment of 3-epi-25-OH-D3 in preterm and full term infant samples and its relationship to demographic, anthropometric and biochemical determinants.

OBJECTIVE: We aim to determine 3-epi-25-OH-D3 in 155 leftover infant samples (<12 months) and its relationship with demographic, anthropometric and biochemical markers. METHODS: Samples were randomly collected and assessed for 3-epi-25-OH-D3 by HPLC. Biochemical data were recorded, if available, including C-reactive protein, calcium, liver and renal markers. Season of birth, gestational age (pre-term vs full-term), weight and height were recorded for infants <1 month. RESULTS: 3-Epi-25-OH-D3 was quantified in 97% of the samples. Serum levels of the epimer increased at the end of the pregnancy and decreased over the first year of life. 3-Epi-25-OH-D3 was negatively associated with bilirubin and C-reactive protein but unrelated to gestational age, season, age after delivery, height and weight, and calcemia or renal function. CONCLUSIONS: Based on the present data, both liver function and acute phase reactants should be considered when measuring 3-epi-25-OH-D3 and assessing vitamin D status in infants.

Determinants of fat-soluble vitamin status in patients aged 65 years and over.

In the elderly, malnutrition is highly prevalent and a major contributor to increased morbidity and mortality. We aimed to evaluate the fat-soluble vitamin status and potential determinants in patients >65 years of age. Serum vitamins A, D and E were determined by liquid chromatography in 166 patients. Gender, age, season, hospitalization, nutritional markers (albumin and cholesterol), acute-phase reactants (ferritin and C-reactive protein) and renal function (creatinine and glomerular filtrate) were assessed as potential determinants. Prevalence of vitamin deficiency was highly variable, ranging from 0 (vitamin E/cholesterol ratio) to 94% (for vitamin D in hospitalized patients). Vitamin status did not differ according to gender, but age, season, hospitalization, a poor nutritional status and impaired renal function, and the presence of acute-phase response significantly affected serum levels of vitamin A, E and D. In conclusion, in subjects >65 years both demographic and clinical factors determined the fat-soluble vitamin status.

Independent sources of condition dependency and multiple pathways determine a composite trait: lessons from carotenoid-based plumage colouration.

Many colour ornaments are composite traits consisting of at least four components, which themselves may be more complex, determined by independent evolutionary pathways, and potentially being under different environmental control. To date, little evidence exists that several different components of colour elaboration are condition dependent and no direct evidence exists that different ornamental components are affected by different sources of variation. For example, in carotenoid-based plumage colouration, one of the best-known condition-dependent ornaments, colour elaboration stems from both condition-dependent pigment concentration and structural components. Some environmental flexibility of these components has been suggested, but specifically which and how they are affected remains unknown. Here, we tested whether multiple colour components may be condition dependent, by using a comprehensive 3 × 2 experimental design, in which we carotenoid supplemented and immune challenged great tit nestlings (Parus major) and quantified effects on different components of colouration. Plumage colouration was affected by an interaction between carotenoid availability and immune challenge. Path analyses showed that carotenoid supplementation increased plumage saturation via feather carotenoid concentration and via mechanisms unrelated to carotenoid deposition, while immune challenge affected feather length, but not carotenoid concentration. Thus, independent condition-dependent pathways, affected by different sources of variation, determine colour elaboration. This provides opportunities for the evolution of multiple signals within components of ornamental traits. This finding indicates that the selective forces shaping the evolution of different components of a composite trait and the trait's signal content may be more complex than believed so far, and that holistic approaches are required for drawing comprehensive evolutionary conclusions.

Serum levels of 3-epi-25-OH-D3 during hypervitaminosis D in clinical practice.

CONTEXT: Intoxication from vitamin D supplements has been rarely reported, but nowadays, it occurs more frequently. The presence of the C-3 epimer of 25-hydroxyvitamin D(3) (3-epi-25-OH-D(3)) is highly prevalent in adults, although there is little information regarding its in vivo relevance, if any, especially under pathological conditions. OBJECTIVE: Our aim was to assess the presence of the 3-epi-25-OH-D(3) in serum samples displaying 25-OH-D(3) concentrations indicative of hypervitaminosis D. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURE: A total of 58 samples displaying a wide range of concentrations of 25-OH-D(3) (>64-439 ng/ml) by ultrafast liquid chromatography were consecutively recruited and reassessed for the presence of 3-epi-25-OH-D(3) using a second chromatographic system. Data from additional biochemical tests performed as part of the patient evaluation were also recorded. RESULTS: Mean relative contribution of 3-epi-25-OH-D(3) was less than 4%, and concentrations ranged from 2-28.6 ng/ml. Serum levels of the C3 epimer, but not the relative contribution, correlate with serum 25-OH-D(3). Overall, in subjects with 25-OH-D(3) concentrations indicative of hypervitaminosis D, the presence of the C-3 epimer and its levels were apparently unrelated to age, serum markers of renal and liver function, acute-phase reactants, and the presence of hypercalcemia. 3-Epi-25-OH-D(3) did not correlate with PTH, but subjects displaying PTH suppression (<14 pg/ml) showed higher concentrations of 3-epi-25-OH-D(3). CONCLUSION: The relative contribution of 3-epi-25-D(3) was not significantly altered during hypervitaminosis D, although the absolute levels reached in serum may be biologically relevant. From a clinical viewpoint, although the small size of the group may affect the lack of relationships, the presence of 3-epi-25-OH-D(3) was apparently unrelated to serum markers of renal and liver function, acute-phase reactants, PTH, and the presence of hypercalcemia.

Hypercalcemia, hypervitaminosis A and 3-epi-25-OH-D3 levels after consumption of an "over the counter" vitamin D remedy. a case report.

Intoxication from vitamin D supplements has been rarely reported but, nowadays, it occurs more frequently. 3-epi-25-OH-D(3) is highly prevalent in adults and it is considered of biological relevance. We report a case of vitamin D toxicity with hypercalcemia, acute renal failure and hypervitaminosis A after consuming an over-the-counter vitamin D supplement. Our data suggest that the contribution of 3-epi-25-OH-D(3) is not altered during vitamin D toxicity, although the serum levels of 25-OH-D(3) and 3-epi-25-OH-D(3) may display a different rate of clearance. The patient also displayed hypervitaminosis A unrelated to diet, possibly caused by renal failure related to the hypercalcemia induced by vitamin D toxicity. Because of the increasing use of over-the-counter vitamin D supplements and the potential iatrogenic hypercalcemia related to hypervitaminosis A, the present case highlights the importance of evaluating both the use of (non-) prescribed medication and vitamin A status during vitamin D toxicity.

Suitability of ultra-high performance liquid chromatography for the determination of fat-soluble nutritional status (vitamins A, E, D, and individual carotenoids).

Our aim was to assess the suitability of ultra-high performance liquid chromatography (UHPLC) for the simultaneous determination of biomarkers of vitamins A (retinol, retinyl esters), E (alpha- and gamma-tocopherol), D (25-OH-vitamin D), and the major carotenoids in human serum to be used in clinical practice. UHPLC analysis was performed on HSS T3 column (2.1 x 100 mm; 1.8 microm) using gradient elution and UV-VIS detection. The system allows the simultaneous determination of retinol, retinyl palmitate, 25-OH-vitamin D, alpha- and gamma-tocopherol, lutein plus zeaxanthin, alpha-carotene, beta-carotene, alpha- and beta-cryptoxanthin and lycopene. The method showed a good linearity over the physiological range with an adequate accuracy in samples from quality control programs. Suitability of the method in clinical practice was tested by analyzing samples (n = 286) from patients. In conclusion, UHPLC constitutes a reliable approach for nutrient/biomarker profiling allowing the rapid, simultaneous and low-cost determination of vitamins A, E, and D (including vitamers and ester forms) and the major carotenoids in clinical practice.

25-OH-vitamin D assay variation and subject management in clinical practice.

OBJECTIVE: To compare two methods for assessing vitamin D status in clinical settings. DESIGN AND METHODS: 25-OH-vitamin D status was measured in 120 patients by HPLC and a commercial immunoassay. RESULTS: Only 53% of the subjects considered as vitamin D sufficient by immunoassay fell within this category according to HPLC although subjects with concentrations above 75 nmol/L, regardless of the method used, presented normal PTH concentrations. CONCLUSIONS: Both methods are not exchangeable to classify subjects based on unique cut-offs but they are comparable when interpreted in relation with PTH concentrations.

Bioavailability of carotenoids and alpha-tocopherol from fruit juices in the presence of absorption modifiers: in vitro and in vivo assessment.

The food industry is playing an increasing role in the development and marketing of new products although little is known regarding the bioavailability of the phytochemicals they contain. Our aim was to assess the effect of the presence of absorption modifiers (milk and iron) on the in vitro bioaccessibility and the serum response in vivo of carotenoids and alpha-tocopherol from fruit juices. Thirty-two young women participated in a three-period (21 d each) supplementation study with a 2-week wash-out in between. Subjects consumed consecutively 2 x 250 ml/d vitamin C-fortified juices supplied as fruit juice, fruit juice containing milk and fruit juice containing milk and iron. Fasting blood samples were collected before and after each supplementation period. In vitro bioaccessibility of carotenoids and alpha-tocopherol was assessed by a static digestion model. Vitamin E and carotenoids from both studies were determined by HPLC. In vitro, xanthophyll ester hydrolysis and transference of free xanthophylls and alpha-tocopherol into the micellar phase were higher in the presence of absorption modifiers. In vivo, consumption of the fruit juices provoked significant increments (within-subject) of alpha-tocopherol and some carotenoids in serum. Dose-adjusted increments in serum of some carotenoids were higher when subjects consumed juices with milk and milk plus iron, although differences did not reach statistical significance. In conclusion, the presence of milk and milk plus iron do not influence the bioavailability of carotenoids and alpha-tocopherol from fruit juices in vivo. Our results support the use of in vitro models to assess food-related factors affecting bioavailability of carotenoids and tocopherols from foods.

Hypocarotenemia after bariatric surgery: a preliminary study.

BACKGROUND: Dietary carotenoids have attracted a great deal of attention due to their potential clinical relevance in conditions such as age-related maculopathy, osteoporosis, cardiovascular disease, and cancer. Surgical procedures have become the primary treatment of severe obesity, although nutrient deficiencies are common and long-term metabolic sequelae remain unknown. Thus, our aim was to assess the carotenoid status in serum of subjects after obesity surgery. METHODS: We evaluated the status of lutein, zeaxanthin, alpha- and beta-cryptoxanthin, lycopene, alpha- and beta-carotene, and fat-soluble vitamins by a quality-controlled high-performance liquid chromatography method in serum of 53 patients. Subjects were consecutively included as they were monitored for nutritional status after Roux-en-Y gastric bypass (RYGBP) or biliopancreatic diversion (BPD). Average follow-up time was 18 and 14 months for each protocol, respectively. RESULTS: After obesity surgery, a consistent and continuous decline in all carotenoids to almost undetectable levels occurs, especially in those who underwent BPD diversion who, on average, displayed serum levels about one half to one third of those found in RYGBP patients. CONCLUSION: The hypocarotenemia observed after bariatric surgery may compromise the availability of carotenoids to tissues and the vitamin A status, reducing the fat-soluble antioxidant capacity and constituting an additional risk factor for several clinical conditions. Given the emerging role of carotenoids in disease prevention, dietary advice on carotenoid-rich and fortified foods or the use of supplements in these patients should be considered.

Consumption of restructured meat products with added walnuts has a cholesterol-lowering effect in subjects at high cardiovascular risk: a randomised, crossover, placebo-controlled study.

BACKGROUND: Diet and lifestyle are modifiable factors involved in the development and prevention of non-communicable diseases, including cardiovascular disease. Nut consumption, particularly walnut intake, has been inversely related to incident coronary heart disease (CHD) in observational studies and to improved lipid profiles in short-term feeding trials. OBJECTIVE: To assess the potential functional effect associated with the regular consumption of walnut-enriched restructured meat products in subjects at risk for cardiovascular disease (CVD). DESIGN: A crossover single-dose bioavailability study (n = 3) using gamma-tocopherol as exposure marker and a crossover unblinded dietary intervention study (5 weeks) in subjects at risk (n = 25). Dietary intervention consisted of regular consumption of the meat product, with or without walnuts, five times per week for five weeks with a 1-month washout in between. Overnight fasting blood samples were collected on days 0, 12, 21, 28 and 35, coinciding with blood pressure and body weight recordings. Participants were asked to complete a diet record throughout the study. The functional effects were assessed using clinically relevant and related biomarkers of CHD: serum total, HDL and LDL cholesterol, triacylglycerols, homocysteine, vitamins B(6) and B(12), folic acid, alpha-tocopherol and platelet function test (obturation time). RESULTS: The regular consumption of walnut-enriched meat products compared with that of the restructured meat products without added walnuts provokes a decrease in total cholesterol of 6.8 mg/dl (CI(95%): -12.8, -0.85). Compared to baseline (mixed diet), meat products with walnuts decreased total cholesterol (-10.7 mg/dl, CI(95%): -17.1, -4.2), LDL cholesterol (-7.6 mg/dl, CI(95%): -2.2, -13.0) and body weight (-0.5 kg, CI(95%): -0.1, -0.9) and increased gamma-tocopherol (8.9 mg/dl, CI(95%): 1.0, 16.8). CONCLUSIONS: The restructured meat products with added walnuts supplied in this study can be considered functional foods for subjects at high risk for CVD, as their regular consumption provokes a reduction in total cholesterol of 4.5% with respect to baseline values (mixed diet) and 3% with respect to the restructured meat without walnuts.

Seasonal variation of serum alpha- and beta-cryptoxanthin and 25-OH-vitamin D(3) in women with osteoporosis.

UNLABELLED: beta-Cryptoxanthin displays a unique anabolic effect on bone calcification. In women with osteoporosis, serum beta-cryptoxanthin and 25-OH-vitamin D(3) showed a weak but significant correlation and exhibited a complementary seasonal distribution. The potential role of beta-cryptoxanthin as a nutritional approach to improving bone health deserves further evaluation. INTRODUCTION: Dietary intake and serum levels of beta-cryptoxanthin have been inversely related to different bone and joint disorders and in vitro and animal studies have shown that beta-cryptoxanthin displays a unique anabolic effect on bone calcification. Due to the emerging role of beta-cryptoxanthin in bone biology, we aimed to assess the serum distribution and variability of beta-cryptoxanthin and their potential relation to 25-OH-vitamin D(3) in women with osteoporosis. METHODS: Serum concentrations of alpha- and beta-cryptoxanthin and 25-OH- D(3) in women with osteoporosis (N = 644) were analyzed using a quality-controlled high-performance liquid chromatography (HPLC) method. RESULTS: Overall, significant seasonal variations were found for the three analytes and inter-individual variation was also high (60-73%). beta-cryptoxanthin and 25-OH-vitamin D(3) exhibited a marked complementary seasonal distribution in serum, with vitamin D displaying the highest values in summer and beta-cryptoxanthin in winter. CONCLUSIONS: Given the anabolic effect of beta-cryptoxanthin on bone calcification and its complementary seasonal distribution with respect to 25-OH-vitamin D(3), the potential role of beta-cryptoxanthin as a sustainable nutritional approach to improving bone health deserves to be further evaluated.

Nutritional approach for designing meat-based functional food products with nuts.

Meat and meat products are essential components of diets in developed countries and despite the convincing evidence that relate them to an increased risk for CVD, a growing consumption of meat products is foreseen. Epidemiological studies show that regular consumption of nuts, in general, and walnuts in particular, correlates inversely with myocardial infarction and ischaemic vascular disease. We assess the nutritional basis for and technological approach to the development of functional meat-based products potentially relevant in cardiovascular disease (CVD) risk reduction. Using the available strategies in the meat industry (reformulation processes) and a food-based approach, we address the design and development of restructured beef steak with added walnuts, potentially functional for CVD risk reduction. Its adequacy as a vehicle for active nutrients is confirmed by a pharmacokinetic pilot study in humans using gamma-tocopherol as an exposure biomarker in chylomicrons during the post-prandial state. Effect and potential "functionality" is being assessed by a dietary intervention study in subjects at risk and markers and indicators related to CVD are being evaluated. Within the conceptual framework of evidence-based medicine, development of meat-based functional products may become a useful approach for specific applications, with a potential market and health benefits of great importance at a population level.

Assessment of carotenoid status and the relation to glycaemic control in type I diabetics: a follow-up study.

OBJECTIVE: To assess the carotenoid status in young type I diabetic patients and its relationship to the glycaemic control of the disease. DESIGN: A follow-up study. SETTING: Hospital Universitario Puerta de Hierro, Health Area VI of Madrid (Spain). SUBJECTS: Forty-seven type I diabetic patients, followed for 2.5 years. INTERVENTIONS: Coinciding with physical examination and laboratory tests, serum levels of carotenoids were analysed by HPLC, and dietary intake of carotenoids was evaluated by a semiquantitative food frequency questionnaire and 3-day prospective dietary records. RESULTS: In type I diabetic patients, average intake, serum levels and correlations between diet and serum levels of carotenoids were comparable to those in reference non-diabetic groups. Between-subjects seasonal variations were observed for beta-cryptoxanthin intake and serum levels (higher in winter) and serum lycopene (higher in summer). Significant within-subjects seasonal changes were shown for dietary and serum beta-cryptoxanthin and serum beta-carotene. Serum carotenoids were unrelated to glycaemic control markers. Subjects with clinically acceptable glycaemic control showed lower lycopene intake than those with unacceptable control. Intake of carotenoids did not explain variance in insulin dose, fasting glycaemia, fructosamine or HbA1c. With the exception of lycopene, serum carotenoids were predicted by dietary intake, but in no case by fasting glycaemia, HbA1c or fructosamine. CONCLUSION: In type I diabetic patients, serum carotenoid concentrations and their variance are determined by dietary intake patterns, and are unrelated to the glycaemic control of the disease, as assessed by biochemical markers.

Simultaneous determination of vitamins A, E and 25-OH-vitamin D: application in clinical assessments.

OBJECTIVE: To optimize an HPLC method for the simultaneous determination of vitamins A, E and 25-OH-vitamin D. METHODS: Modifications include sample volume, internal standard, temperature, detection and injection volume. RESULTS AND DISCUSSION: Validity was supported by performance in interlaboratory exercises. Utility and versatility in clinical practice were assessed in outpatients and hospitalized subjects. The method was shown to be accurate and reliable for the simultaneous determination of vitamins A, E and 25-OH-vitamin D in serum throughout the entire range of clinically relevant concentrations.