Fernando Granado-Lorencio

Simultaneous measurement of retinol, α-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.

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.

Time-Course Changes in Bone Turnover Markers and Fat-Soluble Vitamins After Obesity Surgery

BackgroundThe 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.MethodsSixty-five candidates for bariatric surgery and 150 serum samples after obesity surgery (i.e., Roux-en-Y gastric bypass, nu2009=u200985; biliopancreatic diversion, nu2009=u200965) were consecutively analyzed over a period of more than 2xa0years. Retinol, α- and γ-tocopherol, 25-OH-vitamin D3, β-cryptoxanthin, and β-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.ResultsRetinol, β-cryptoxanthin, β-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 12–24xa0months but declined afterward. 25-OH-vitamin D increased during the first 12xa0months and tended to decrease afterward while iPTH remained constant or decreased but increased after 1xa0year in both groups. Vitamin A remained constant but α- and γ-tocopherol, β-cryptoxanthin, and β-carotene decreased in both groups.ConclusionsIn 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.

Depletion of Serum Carotenoid and Other Fat-Soluble Vitamin Concentrations following Obesity Surgery

BackgroundObesity 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.MethodsSeventy-five candidates for bariatric surgery (17 men, 58 women; age 43u2009±u200910xa0years) and a total of 362 serum samples after obesity surgery (i.e., Roux-en-Y gastric bypass (nu2009=u2009187) and biliopancreatic diversion (nu2009=u2009175)) were consecutively collected and assessed. Retinol, α- and γ-tocopherol, 25-OH-vitamin D3, lutein, zeaxanthin, α- and β-cryptoxanthin, lycopene (trans and cis), α- and β-carotene (trans and cis) were analyzed by high-performance liquid chromatography.ResultsMean 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 β-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.ConclusionsThe 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.

Bioavailability of β-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 β-cryptoxanthin from β-cryptoxanthin-rich drinks with and without added phytosterols developed for this purpose. The developed drinks showed no difference in the content of β-cryptoxanthin, and they were stable over 6 months. In vitro, hydrolysis of β-cryptoxanthin esters and the amount of free β-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 β-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 β-cryptoxanthin is not significantly affected by the presence of phytosterols when they are simultaneously supplied in a drink.

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

BACKGROUND & AIMnVitamin 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.nnnMETHODSn25-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.nnnRESULTSnAll 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.nnnCONCLUSIONnThe prevalence of vitamin D deficiency in clinical practice is assay-dependent and physicians should be aware of the uncertainty associated with vitamin D assessment.

Hypocarotenemia After Bariatric Surgery: A Preliminary Study

BackgroundDietary 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.MethodsWe evaluated the status of lutein, zeaxanthin, α- and β-cryptoxanthin, lycopene, α- and β-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 14xa0months for each protocol, respectively.ResultsAfter 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.ConclusionThe 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.

β-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 β-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 β-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 β-cryptoxanthin effect, and further signaling experiments were conducted. Comet Assay was completed to evaluate the genotoxicity of the treatments. Results: β-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 β-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 β-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. Clin Cancer Res; 21(19); 4398–409. ©2015 AACR.

Seasonal variation of serum α- and β-cryptoxanthin and 25-OH-vitamin D3 in women with osteoporosis

Summaryβ-Cryptoxanthin displays a unique anabolic effect on bone calcification. In women with osteoporosis, serum β-cryptoxanthin and 25-OH-vitamin D3 showed a weak but significant correlation and exhibited a complementary seasonal distribution. The potential role of β-cryptoxanthin as a nutritional approach to improving bone health deserves further evaluation.IntroductionDietary intake and serum levels of β-cryptoxanthin have been inversely related to different bone and joint disorders and in vitro and animal studies have shown that β-cryptoxanthin displays a unique anabolic effect on bone calcification. Due to the emerging role of β-cryptoxanthin in bone biology, we aimed to assess the serum distribution and variability of β-cryptoxanthin and their potential relation to 25-OH-vitamin D3 in women with osteoporosis.MethodsSerum concentrations of α- and β-cryptoxanthin and 25-OH- D3 in women with osteoporosis (Nu2009=u2009644) were analyzed using a quality-controlled high-performance liquid chromatography (HPLC) method.ResultsOverall, significant seasonal variations were found for the three analytes and inter-individual variation was also high (60–73%). β-cryptoxanthin and 25-OH-vitamin D3 exhibited a marked complementary seasonal distribution in serum, with vitamin D displaying the highest values in summer and β-cryptoxanthin in winter.ConclusionsGiven the anabolic effect of β-cryptoxanthin on bone calcification and its complementary seasonal distribution with respect to 25-OH-vitamin D3, the potential role of β-cryptoxanthin as a sustainable nutritional approach to improving bone health deserves to be further evaluated.

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.