Susan Southon

The potential for the improvement of carotenoid levels in foods and the likely systemic effects

Carotenoids form one of the most important classes of plant pigments and play a crucial role in defining the quality parameters of fruit and vegetables. Their role in the plant is to act as accessory pigments for light harvesting and in the prevention of photo-oxidative damage, as well as acting as attractants for pollinators. Their function as antioxidants in the plant shows interesting parallels with their potential role as antioxidants in foods and humans. Carotenoids are products of the isoprenoid biosynthetic pathway. The enzymes leading to carotenoid biosynthesis have all been characterised, and more recently the genes encoding these enzymes have been cloned from bacteria, fungi and plants. New information on enzyme activities and the factors leading to the regulation of the pathway is reviewed. Vitamin A deficiency is a widespread problem in the developing world, causing blindness, particularly in the young. This has driven research into finding ways of introducing provitamin A carotenoids into staple crops, and this has recently been achieved in rice and canola through genetic manipulation. The fact that carotenoids show protective activity in vitro and in vivo against a variety of degenerative disease end points has also give impetus to studying whether increasing intakes of the commonly consumed carotenoids would have public health benefits in the developed world. Human intervention studies have been undertaken using supplements of β-carotene rather than utilising foods with enhanced carotenoid levels, but no potential benefit has been shown. Indeed, there is evidence of an increased health risk from the consumption of β-carotene supplements. These observations suggest that the threshold between the beneficial and adverse effects of some carotenoids is low and provides a strong stimulus to further understanding the functional effects of specific carotenoids. Specific needs for future research are identified in the review. © 2000 Society of Chemical Industry

A European carotenoid database to assess carotenoid intakes and its use in a five-country comparative study

A food frequency questionnaire (FFQ) and carotenoid database with information on alpha- and beta-carotene, lutein, lycopene and beta-cryptoxanthin was prepared and used to compare the carotenoid intakes in five European countries: UK, Republic of Ireland, Spain, France and The Netherlands. Eighty, age- (25-45 years) and sex-matched volunteers were recruited in each of the five countries. A FFQ and carotenoid database was prepared of the most commonly consumed carotenoid rich foods in the participating countries and the information was used to calculate frequency and intake of carotenoid-rich foods. The median total carotenoid intake based on the sum of the five carotenoids, was significantly higher (P < 0.05) in France (16.1 mg/day) and lower in Spain (9.5 mg/day,) than the other countries, where the average intake was approximately 14 mg/day. Comparison of dietary source of carotenoids showed that carrots were the major source of beta-carotene in all countries except Spain where spinach was most important. Likewise, carrots were also the main source of alpha-carotene. Tomato or tomato products, were the major source of lycopene. Lutein was mainly obtained from peas in Republic of Ireland and the UK, however, spinach was found to be the major source in other countries. In all countries, beta-cryptoxanthin was primarily obtained from citrus fruit. Comparing the data with that from specific European country studies suggests that the FFQ and carotenoid database described in the present paper can be used for comparative dietary intake studies within Europe. The results show that within Europe there are differences in the specific intake of some carotenoids which are related to different foods consumed by people in different countries.

Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries, and correlations with heart disease mortality rates

The ‘antioxidant hypothesis’ proposes that vitamin C, vitamin E, carotenoids, and other antioxidants occurring in fruit and vegetables afford protection against heart disease and cancer by preventing oxidative damage to lipids and to DNA, respectively. To test elements of this hypothesis, we have measured blood levels of dietary antioxidants, and 8‐oxodeoxyguanosine (8‐oxo‐dG) concentrations in lymphocyte DNA, in healthy men and women from five European countries: France, Ireland, The Netherlands, Spain, and the U.K. Volunteers, aged 25–45, all nonsmokers, gave blood samples before and after a 12‐wk carotenoid supplementation regime. Vitamin C was measured in plasma and vitamin E and carotenoids were measured in serum by high‐performance liquid chromatography (HPLC). 8‐oxo‐dG was assayed by HPLC (with coulometric detection) in DNA isolated from lymphocytes from the same blood samples. Mean values were calculated for groups of volunteers at each sampling time according to country, sex, and supplementation (between 9 and 24 individual samples contributing to each mean). We found that 8‐oxo‐dG levels in lymphocyte DNA vary significantly according to sex and country. A low mean 8‐oxo‐dG concentration is seen in DNA of women from all five countries, and of men from France and Spain. 8‐oxo‐dG is significantly higher (up to about threefold) in lymphocyte DNA from men in Ireland and the U.K. Oxidative DNA damage is not significantly affected by carotenoid supplementation; nor is there any association with mean baseline levels of antioxidants, which are generally similar in the five countries. The five countries sampled lie on an axis from northern to southern Europe with a steep gradient in terms of premature heart disease. There is a strong association between premature coronary heart disease mortality in men and the mean levels of 8‐oxo‐dG for the five countries (r = 0.95, P < 0.01). Women have low coronary heart disease mortality rates, which do not correlate with 8‐oxo‐dG. In terms of cancer deaths, only colorectal cancer in men shows a significant positive correlation (r = 0.91, P < 0.05), and stomach cancer in women is negatively correlated with DNA oxidation (r = −0.92, P = 0.01).—Collins, A. R., Gedik, C. M., Olmedilla, B., Southon, S., Bellizzi, M. Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries, and correlations with heart disease mortality rates. FASEB J. 12, 1397–1400 (1998)

Increased fruit and vegetable consumption within the EU: potential health benefits

Abstract In this EU (DG XII) funded project, a Core human study was conducted in Ireland, Northern Ireland, Spain, France and The Netherlands. Oxidative and antioxidant status, vegetable and fruit consumption, and carotenoid intake of volunteers from different countries was compared. Response to increased carotenoid intake was determined using a range of measurements. Attention was paid to whether the antioxidant capability of β-carotene, lutein and lycopene demonstrated in vitro, was apparent in relation to increased oxidation resistance of low-density-lipoprotein (LDL). Other (Complementary) studies were undertaken and included determination of: protective effects of carotenoid-rich foods against LDL and DNA oxidative damage; carotenoid absorbability; barriers to increased vegetable consumption; and carotenoid content of fruits and vegetables frequently consumed in Europe. Our results demonstrated that carotenoid supplementation did not increase LDL oxidation resistance. However, increased consumption of carotenoid-rich fruits and vegetables did increase LDL oxidation resistance, and higher plasma concentration of total and specific carotenoids (pre-supplementation) was associated with lower DNA damage and higher repair activity.

No Significant Effects of Lutein, Lycopene or β-Carotene Supplementation on Biological Markers of Oxidative Stress and LDL Oxidizability in Healthy Adult Subjects

Objective: The objective of this study was to determine the effect of individual carotenoid supplementation on biochemical indices of oxidative status in apparently healthy adult males. Methods:The study was a placebo controlled single blind study. Healthy male volunteers (n=175) were assigned to four groups. They received daily supplements of β-carotene (15 mg), lutein (15 mg), lycopene (15 mg) and placebo for three months. The effects of the supplementation on antioxidant status were monitored by plasma carotenoid, vitamin C and A levels, glutathione (GSH and GSSG) concentrations, protein SH groups, erythrocyte antioxidant enzyme activities (Cu-Zn SOD, Se-GSH-Px) and susceptibility of LDL to copper-induced oxidation. Results:β-carotene, lycopene and lutein supplementation led to significant plasma and LDL increases in each of these carotenoids, without modifications of other carotenoid levels in plasma or in LDL. The supplementation failed to enhance the resistance of LDL to oxidation or to modify the LDL polyunsaturated/saturated fatty acid ratio. Vitamin C, GSH, protein SH groups and antioxidant metalloenzyme activities were also unchanged. Conclusion: We did not observe beneficial or adverse effects of lutein, lycopene or β-carotene supplementation on biomarkers of oxidative stress. In apparently healthy subjects, carotenoid supplementation does not lead to significantly measurable improvement in antioxidant defenses.

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.

The effect of β-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers

Although there is strong epidemiologic evidence that diets rich in carotenoids such as beta-carotene are associated with a reduced incidence of cancer, the cellular mechanisms underlying this phenomenon remain unknown. This article describes the effect of dietary beta-carotene supplementation on both the expression of functionally associated surface molecules on human monocytes and on the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha) by monocytes, all of which are involved in the initiation and regulation of immune responses involved in tumor surveillance. A double-blind, placebo-controlled, crossover study was undertaken in which 25 healthy, adult male nonsmokers were randomly assigned to receive beta-carotene (15 mg daily) or placebo for 26 days, followed by the alternative treatment for a further 26 days. The expression of functionally related monocyte surface molecules was quantified by flow cytometry, and ex vivo secretion of TNF-alpha was quantified by an enzyme-linked immunosorbent assay, before and after each treatment period. After dietary supplementation there were significant increases in plasma levels of beta-carotene and in the percentages of monocytes expressing the major histocompatibility complex class II molecule HLA-DR and the adhesion molecules intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. In addition, the ex vivo TNF-alpha secretion by blood monocytes was significantly increased after supplementation. These findings suggest that moderate increases in the dietary intake of beta-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anticarcinogenic properties attributed to beta-carotene.

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.

Differences in intestinal protein synthesis and cellular proliferation in well-nourished rats consuming conventional laboratory diets

Male Wistar rats (100 g) were given a commercial pellet feed or a semi-synthetic diet ad lib. Although the pellet-fed group grew slightly faster than the other group during the early part of the feeding period, there was no significantly difference between the final weights of the groups. The fractional rates of protein synthesis in jejunum, proximal ileum and liver were measured by a technique based on the determination of L-[4-3H]phenylalanine incorporation over a short time period. Protein synthesis was higher in both jejunum and ileum of the pellet-fed rats compared with those eating the semi-synthetic diet, but there was no difference between the rates of protein synthesis measured in the livers of the groups. The rate of mucosal cell division was significantly faster in the ileal mucosa of the pellet-fed group compared with the other group, and there were significant differences in some aspects of mucosal morphology. The maintenance of higher rates of cell turnover and protein synthesis in animals given a commercial pellet feed is unexplained, but it may be related to the presence of non-absorbable polysaccharides or other complex plant materials in the pellet feed.

Dietary intake and micronutrient status of adolescents: effect of vitamin and trace element supplementation on indices of status and performance in tests of verbal and non-verbal intelligence

Relationships between micronutrient intake and status, and micronutrient status and performance in tests of intelligence were investigated in a group of adolescents (13-14 years old). Dietary intakes were assessed using a 7 d weighed dietary record method, coupled with the collection of duplicate diets. Vitamin and trace mineral intakes calculated using food composition tables were compared with those obtained by direct analysis of duplicate diets. Micronutrient status was judged via a range of biochemical indices measured in blood samples taken after a 12-15 h fast. Blood samples were taken both before and after a 16-week period of vitamin and trace mineral supplementation. Individual tests of verbal and nonverbal intelligence were also performed pre- and post-supplementation. The results of this study indicate that the use of food table data may lead to substantial over- or underestimation of the intake of several micronutrients. In general, the total calculated or analysed amount of a specific micronutrient consumed did not adequately predict status, as judged by a range of biochemical indices. There were significant changes in status measurements over the 16-week study period, irrespective of supplementation, and these changes were markedly influenced by the initial status of the subject. There was no effect of supplementation on performance in tests of intelligence. However, there was a significant association between plasma ascorbic acid and initial non-verbal intelligence quotient (IQ) in the boys, and between whole blood glutathione peroxidase (EC 1.11.1.9) activity and non-verbal and verbal IQ in both sexes. These findings are discussed in relation to other recent studies of the influence of micronutrient supplementation on the psychological performance of children.