Challenges to understanding and measuring carotenoid bioavailability.

Carotenoids are an excellent example of where poor understanding of food structure, complexity of behaviour during digestion, and inter-individual differences in response, lead to misinterpretation of study results. Four challenges associated with understanding and measuring carotenoid bioavailability are discussed: release of carotenoids from food structure and processing into an absorbable form (bioaccessibility), passage of carotenoids from gut lumen into the body (absorption), interpreting plasma response and inter-individual variation. Bioaccessibility of carotenoids is governed by characteristics of the food matrix, which affect the efficiency of physical, enzymic and chemical digestion. Carotenoids used as colorants are likely to be better absorbed because of the form in which they are dispersed in food. Extent of absorption of carotenoid supplements will depend on the proximity of dosing to the consumption of a fat-containing meal. Release of carotenoids from food plants occurs only when the plant cell is fractured and this occurs only during food preparation, processing and/or mastication, not during digestion. Following release from the food matrix, the major limiting factor is solubility of carotenoids in digesta. Absorption studies are best carried out by measuring chylomicron carotenoid excursion, with modelling of chylomicron turnover rate. In this way, inter-individual differences in lipoprotein metabolism can, in part, be taken into account before formulating conclusions on the rate and extent of absorption.

Kinetics of gastro-intestinal transit and carotenoid absorption and disposal in ileostomy volunteers fed spinach meals.

BACKGROUND: Reports of low carotenoid absorption from food sources has undermined their postulated 'protective' role as one of the active agents in diets rich in vegetable matter. AIM OF THE STUDY: This study quantified beta-carotene and lutein absorption from a representative green vegetable with different degrees of processing, using both mass balance and metabolic modelling of triglyceride-rich lipoprotein plasma fraction (TRL) response. METHODS: Whole or chopped-leaf cooked spinach was fed to volunteers (n = 7, paired) with vegetable oil (40 g) in yoghurt. Blood and ileal effluent samples were collected for up to 24 h. Effluent and TRL samples were analysed for lutein and beta-carotene by HPLC. A digesta transit model was used to describe meal transit and a single compartment model used to predict percentage absorption from the plasma TRL response. RESULTS: Mass balance showed 25% of lutein and beta-carotene were absorbed from chopped spinach, compared with 25% beta-carotene and 40 % lutein from whole-leaf spinach. Increased lutein absorption correlated to slower gastrointestinal (GI) transit for the whole-leaf meal. An area under the curve (AUC) response for the TRL fraction, found in 50% of cases, was not confined to those with the greatest percentage absorption. Absorption by mass balance and TRL AUC indicate a half-life of newly absorbed carotenoid around 11 min CONCLUSION: GI residence time appears to have an effect on the absorption of lutein but not beta-carotene. Rapid clearance is probably the main reason for absence of measurable plasma concentration excursions. Lack of plasma response cannot be interpreted as lack of carotenoid absorption without knowledge of the absorption and disposal kinetics.

DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with beta-carotene, lutein or lycopene at a range of concentrations (0.00-8.00 micromol/l) using a liposome delivery method. Uptake was dose-dependent. beta-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2.00 micromol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 micromol H2O2/l, 5 min, 4 degrees C) following incubation with carotenoids between 0.50 and 1.00 micromol/l; at >1.00 micromol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, beta-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma beta-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes.

Epidemiological evidence has shown that the habitual consumption of diets high in fruits and vegetables is associated with reduced risk of cancers. The challenge is to identify causal mechanisms of effect. The aim of the current study was to determine whether an increase in rate of removal of DNA single-strand breaks (SSB) following oxidative challenge could be provoked ex vivo in peripheral blood lymphocytes (PBL). The PBL were isolated from apparently healthy volunteers following dietary intervention with: (1) a mixed carotene capsule; (2) a daily portion of cooked minced carrots; (3) a matched placebo; (4) a portion of mandarin oranges; (5) vitamin C tablets. Single-cell gel electrophoresis was employed to measure baseline levels of SSB and DNA susceptibility to oxidative damage, and to monitor the number of SSB over 4 h, in both unchallenged and H2O2-treated PBL. The enzymatic capacity for repair of different types of DNA oxidative lesions was also measured using two related cell-free assays. There was no evidence that any of the dietary supplementation regimens altered baseline levels of SSB, provided any direct antioxidant protection or altered DNA repair capacity, with two exceptions: the number of SSB following exposure to H2O2 decreased more rapidly in PBL from volunteers given the mixed carotene capsules and repair patch synthesis activity in PBL increased from volunteers given the cooked carrots. These results suggest that carotenoids and carotenoid-rich foods can influence DNA damage:repair by modulation of discrete stages in the DNA repair mechanisms.

Plasma and buccal mucosal cell response to short-term supplementation with all trans-beta-carotene and lycopene in human volunteers.

Despite interest in the health-beneficial role of carotenoids little is known about the specific storage metabolism and mechanisms involved in various target tissues. The aim of the study was to search for a relatively simple non-invasive method to detect and determine the cellular effects of supplemented dosage of beta-carotene and lycopene to peripheral tissues such as the buccal mucosa in relation to the plasma concentrations. Subjects (30) were allocated into five different subgroups of 6 volunteers. The change in concentration of all-trans-beta-carotene and lycopene in plasma and in buccal mucosal cells was measured in groups of volunteers supplemented with either 15 mg, 30 mg or placebo capsules in a randomised double blind study for a period of 7 days. With the exception of supervised high fat (40 g carotenoid free sunflower oil) breakfasts and capsule ingestion the volunteers ate their habitual diets. Plasma lycopene and beta-carotene concentrations were determined at baseline and following one week of capsule ingestion. In all the supplemented groups the plasma carotenoid levels were significantly higher than in the placebo group indicating absorption of the supplement. Carotenoid concentrations, expressed per unit protein, assayed in buccal mucosal cells before (at baseline) and at the end of the study were found to be significantly higher in the groups supplemented at 30 mg/d, of either carotenoid as compared to the 15 mg/d or placebo supplemented groups. We conclude that buccal mucosal cells respond readily to changes in plasma beta-carotene and lycopene concentration. These observations suggest that dietary carotenoids are quickly incorporated into rapidly turning over mucosal tissues. It is not clear if the change in carotenoid content of the plasma is reflected in existing cells or only in those concurrently produced during the elevated plasma concentration. If desquamated buccal mucosal cells reflect habitual plasma carotenoid concentration then it is not an appropriate tissue for the measurement of acute changes.

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry.

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.

Use of an oral/intravenous dual-label stable-isotope protocol to determine folic acid bioavailability from fortified cereal grain foods in women.

Folic acid fortification, mandatory in the United States, is currently being considered by the UK. The hypothesis that the matrix of some cereal-product vehicles may result in low fortificant bioavailability was tested using a dual oral/intravenous (i.v.) isotopic-label approach, which was evaluated concurrently. Fifteen women received 225 microg oral folate (capsules, fortified white bread and fortified branflakes), mainly as folic acid labeled with (13)C on 6 carbons of the benzoyl ring ((13)C(6)-PteGlu), followed by i.v. injection of 100 microg folic acid labeled with (2)H on 4 hydrogens of the glutamic acid group ((2)H(4)-PteGlu). The urinary excretion ratio (UER) in intact folate of the percentage of labeled oral dose excreted divided by the percentage of i.v. dose excreted was used as the primary index of absorption. The geometric mean (95% confidence interval) UER for folic acid capsules was 3.68 (1.90, 7.14) at 24 h and 2.18 (1.24, 3.83) at 48 h. Because these were significantly in excess of 1.0, indicative of 100% absorption of the oral dose, it was concluded that oral and i.v. labeled folic acid are handled differently by the body and that "absolute" absorption cannot be calculated. Compared with the 48-h UER for folic acid capsules, the "relative" 48-h UER for white bread and branflakes was 0.71 and 0.37, respectively, indicating that some cereal-based vehicles may inhibit absorption of fortificant. However, even the validity of this "relative" approach is questioned.

Comparison of LDL fatty acid and carotenoid concentrations and oxidative resistance of LDL in volunteers from countries with different rates of cardiovascular disease.

Within Europe there are differences in cardiovascular disease (CVD) risk between countries and this might be related to dietary habits. Oxidative modification of LDL is suggested to increase the risk of CVD and both the fatty acid and antioxidant content of LDL can affect its oxidation. In the present study, concentration of LDL fatty acid and antioxidant micronutrients (tocopherols and carotenoids) and ex vivo oxidative resistance of LDL (lag phase) was compared in volunteers from five countries with different fruit and vegetable intakes and reported rates of CVD. Eighty volunteers (forty males, forty females per centre), age range 25-45 years, were recruited from France, Northern Ireland, UK, Republic of Ireland, The Netherlands, and Spain, and their LDL composition and lag phase were measured. There were some differences in LDL carotenoid and alpha-tocopherol concentrations between countries. alpha-Tocopherol was low and beta- + gamma-tocopherol were high (P<0.001) in the Dutch subjects. Beta-Carotene concentrations were significantly different between the French and Spanish volunteers, with French showing the highest and Spanish the lowest concentration. LDL lycopene was not different between centres in contrast to lutein, which was highest in French (twofold that in the Dutch and Spanish and threefold that in Northern Ireland and the Republic of Ireland, P<0.001). However absolute LDL saturated, monounsaturated, polyunsaturated and total unsaturated fatty acid concentrations were different between countries (P<0.001, total unsaturated highest in Northern Ireland) there was little difference in unsaturated:saturated fatty acid concentration ratios and no difference in polyunsaturated:saturated fatty acid concentration ratios. LDL from the Republic of Ireland (a region with a high rate of CVD) had greater resistance to Cu-stimulated oxidation than samples obtained from volunteers in other countries. In conclusion, LDL composition did not predict resistance to Cu-stimulated oxidation, nor is there evidence that LDL from volunteers in countries with lower rates of CVD have greater resistance to oxidation.

A European multicentre, placebo-controlled supplementation study with alpha-tocopherol, carotene-rich palm oil, lutein or lycopene: analysis of serum responses.

Increased levels of oxidative stress have been implicated in tissue damage and the development of chronic diseases, and dietary antioxidants may reduce the risk of oxidative tissue damage. As part of a European multicentre project, several studies were undertaken with the aim of testing whether the consumption of foods rich in carotenoids reduces oxidative damage to human tissue components. We describe here the serum response of carotenoids and tocopherols upon supplementation with carotenoids from natural extracts (alpha-carotene+beta-carotene, lutein or lycopene; 15 mg/day) and/or with alpha-tocopherol (100 mg/day) in a multicentre, placebo-controlled intervention study in 400 healthy male and female volunteers, aged 25-45 years, from five European regions (France, Northern Ireland, Republic of Ireland, The Netherlands and Spain). Supplementation with alpha-tocopherol increased serum alpha-tocopherol levels, while producing a marked decrease in serum gamma-tocopherol. Supplementation with alpha- + beta-carotene (carotene-rich palm oil) resulted in 14-fold and 5-fold increases respectively in serum levels of these carotenoids. Supplementation with lutein (from marigold extracts) elevated serum lutein (approx. 5-fold), zeaxanthin (approx. doubled) and ketocarotenoids (although these were not present in the supplement), whereas lycopene supplementation (from tomato paste) resulted in a 2-fold increase in serum lycopene. The isomer distributions of beta-carotene and lycopene in serum remained constant regardless of the isomer composition in the capsules. In Spanish volunteers, additional data showed that the serum response to carotenoid supplementation reached a plateau after 4 weeks, and no significant side effects (except carotenodermia) or changes in biochemical or haematological indices were observed throughout the study. This part of the study describes dose-time responses, isomer distribution, subject variability and side effects during supplementation with the major dietary carotenoids in healthy subjects.

Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge.

Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.