[The comparison of the process of five different vitrification devices]. / Principe de la vitrification: cinétiques comparatives.

Over the last decade, several methods have been designed to improve the survival rate of vitrificated embryos. Although some teams have succeeded, the main remaining drawback of these methods is that they do not provide a leak proof environment for cryopreserved biological samples. To respond to that demand in respect with the European reglementation, the Cryo Bio System company (CBS) designed the HSV High Security Vitrification Kit (HSV). This system is composed of three distinct parts, a High Security thermal-autogenic sealed clear straw, a capillary with its extremity in form of a gutter, and an introducer that can be mounted on the manipulation rod before introduction into the straw. In this study, we confirmed that the CBS vitrification kit is a suitable method for vitrification in association with a small amount of cryoprotector enriched viscous media such as 25 microM Ficool 400, 750 mM Sucrose, 1% Bovine albumin, 20% Dimethyl Sulfoxide and 20% Ethylene glycol in a Phosphate buffered saline solution. We also evaluated the speed of the temperature decrease during vitrification in comparison with four other commercially available non-aseptic methods and showed the protective role of the CBS system during transfer. These physical data have now been confirmed biologically by P. Vanderzwalmen who obtained easily reproductible good results with human embryo using our method. Today, the HSV represents the unique aseptic alternative device (EC and FDA approved) for embryos, oocytes, and biological samples vitrification.

Plasma carotenoids as biomarkers of intake of fruits and vegetables: ecological-level correlations in the European Prospective Investigation into Cancer and Nutrition (EPIC).

OBJECTIVE: The aim of this study was to assess the ability of a single 24-h dietary recall (24HDR) and food questionnaires (FQ) to predict plasma carotenoid levels at the ecological level by assessing the relationship between mean plasma carotenoid levels and mean intake of fruit and vegetables measured by 24HDR and FQ across 16 European regions. DESIGN: A random subsample of 3089 subjects was included, stratified by age and gender. They provided blood samples and dietary information between 1992 and 2000 as part of the European Prospective Investigation into Cancer and Nutrition. RESULTS: Using Spearman's correlation coefficients, the correlations between mean regional 24HDR fruit and vegetable variables and corresponding mean plasma carotenoid levels were generally higher than the correlations using FQ means. The highest correlation was between the 24HDR citrus fruit variable and beta-cryptoxanthin (r = 0.90). For 24HDR, total fruits and vegetables were highly correlated with lutein, zeaxanthin, and beta-cryptoxanthin (r = 0.83-0.87), while vegetables were more closely related with lutein (r = 0.69) and zeaxanthin (r = 0.68), and fruits correlated with zeaxanthin (r = 0.87) and beta-cryptoxanthin (r = 0.84). Root vegetables (r = 0.81) and total carrots (r = 0.71) were well correlated with alpha-carotene. In the multivariate models adjusting for age, body mass index, and season, and using observations of means stratified by sex and region, the association was generally higher for 24HDR compared to FQ. CONCLUSION: Mean regional intakes of fruits and vegetables in several European countries were closely correlated with corresponding mean plasma levels of individual carotenoids. Fruits and vegetables measured by 24HDR were generally better able to predict plasma carotenoids at the ecological level.

Plasma carotenoids as biomarkers of intake of fruits and vegetables: individual-level correlations in the European Prospective Investigation into Cancer and Nutrition (EPIC).

OBJECTIVE: The aim in this study was to assess the association between individual plasma carotenoid levels (alpha-carotene, beta-carotene, lycopene, beta-cryptoxanthin, lutein, zeaxanthin) and fruit and vegetable intakes recorded by a calibrated food questionnaire (FQ) and 24-h dietary recall records (24HDR) in nine different European countries with diverse populations and widely varying intakes of plant foods. DESIGN: A stratified random subsample of 3089 men and women from nine countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC), who had provided blood samples and dietary and other lifestyle information between 1992 and 2000, were included. RESULTS: beta-Cryptoxanthin was most strongly correlated with total fruits (FQ r = 0.52, 24HDR r = 0.39), lycopene with tomato and tomato products (FQ r = 0.38, 24HDR r = 0.25), and alpha-carotene with intake of root vegetables (r = 0.39) and of total carrots (r = 0.38) for FQ only. Based on diet measured by FQ and adjusting for possible confounding by body mass index (BMI), age, gender, smoking status, alcohol intake, and energy intake, the strongest predictors of individual plasma carotenoid levels were fruits (R(partial)(2) = 17.2%) for beta-cryptoxanthin, total carrots ((partial)(2) = 13.4%) and root vegetables (R(partial)(2) = 13.3%) for alpha-carotene, and tomato products (R(partial)(2) = 13.8%) for lycopene. For 24HDR, the highest R(partial)(2) was for fruits in relation to beta-cryptoxanthin (7.9%). CONCLUSIONS: Intakes of specific fruits and vegetables as measured by food questionnaires are good predictors of certain individual plasma carotenoid levels in our multicentre European study. At individual subject levels, FQ measurements of fruits, root vegetables and carrots, and tomato products are, respectively, good predictors of beta-cryptoxanthin, alpha-carotene, and lycopene in plasma.

Plasma carotenoid levels in Dutch men and women, and the relation with vegetable and fruit consumption.

OBJECTIVE: Fruit and vegetable intake is inversely associated with cancer risk in many epidemiological studies. Accurate assessment of consumption of these foods is difficult, and biomarkers of intake would overcome several drawbacks of currently used dietary assessment methods. Therefore, we investigated the relation between plasma carotenoids and usual vegetable and fruit intake. DESIGN: Plasma carotenoid concentrations were measured and vegetable, fruit and juice consumption was assessed by a food frequency questionnaire (FFQ) in a random sample of 591 Dutch men and women aged 20-59 y from the MORGEN-project, one of the contributions to the European Prospective Investigation into Cancer and Nutrition (EPIC)-study. RESULTS: In this sample of the general Dutch population, in both genders, relative to the other carotenoids, plasma beta-cryptoxanthin was the best indicator for fruit intake, and for the sum of vegetable, fruit and juice intake, while lutein concentrations best reflected intake of vegetables, although quartiles of intake were not consistently separated. Since levels of lycopene were not associated with any of the main food groups examined, associations with total carotenoids improved when excluding lycopene, and monotonously increasing plasma levels were seen for intakes of vegetables, of fruits, and of the sum of vegetables, fruits and juices. Several vegetable types and orange/grapefruit juice were associated with plasma levels of one of the carotenoids. CONCLUSION: Plasma carotenoids were only crude indicators of vegetable and fruit intake as assessed by a FFQ; beta-cryptoxanthin for fruit intake and lutein for vegetable intake. None of the plasma carotenoids could distinguish all four quartiles of vegetables, fruit and/or juice intake.

Comparison of fatty acid profile in plasma phospholipids in women from Granada (southern Spain) and Malmö (southern Sweden).

We conducted a first pilot study on healthy women living in two countries with different dietary habits, Granada in the south of Spain and Malmö in the south of Sweden, in order to compare their levels of plasma phospholipid fatty acids, and to examine the relationship between the differences in food consumption. This study is part of a pilot study which is nested in the European Prospective Investigation into Cancer and Nutrition, a multi-centre prospective cohort study on diet, plasma concentrations of antioxidants and fatty acids, and markers of oxidative stress. Thirty-nine women in Granada and thirty-eight women in Malmö, aged 45-50 years (all pre-menopausal) were selected among the female participants in the cohorts from these two countries. Individual measurements of the women's habitual diet were obtained by a food frequency questionnaire. 24-hour diet recalls were used for the standardised measurement of diet at group level. Plasma phospholipid fatty acid composition was determined by capillary gas chromatography. We found a different fatty acid profile in plasma between the two populations, with higher mean levels of palmitic acid (16:0), palmitoleic acid (16:1) (n-7), oleic acid (18:1), alpha-linolenic acid (18:3) (n-3) and eicosapentaenoic acid (20:5) (n-3), and lower mean levels of stearic acid (18:0) in Malmö compared to Granada. Women in Malmö consumed more meat, alcoholic beverages and sugar, and less fish and shellfish than women in Granada. We conclude that the fatty acid composition in plasma phospholipids is different between women from the two European centres. For polyunsaturated fatty acids, differences were observed for (n-3) fatty acids. In relation to these differences, we observed that specific food intakes, particularly meat and fish, varied between the two centres.

Carotenoids, alpha-tocopherols, and retinol in plasma and breast cancer risk in northern Sweden.

OBJECTIVE: Using a nested case-referent design we evaluated the relationship between plasma levels of six carotenoids, alpha-tocopherol, and retinol, sampled before diagnosis, and later breast cancer risk. METHODS: In total, 201 cases and 290 referents were selected from three population-based cohorts in northern Sweden, where all subjects donated blood samples at enrolment. All blood samples were stored at -80 degrees C. Cases and referents were matched for age, age of blood sample, and sampling centre. Breast cancer cases were identified through the regional and national cancer registries. RESULTS: Plasma concentrations of carotenoids were positively intercorrelated. In analysis of three cohorts as a group none of the carotenoids was found to be significantly related to the risk of developing breast cancer. Similarly, no significant associations between breast cancer risk and plasma levels of alpha-tocopherol or retinol were found. However, in postmenopausal women from a mammography cohort with a high number of prevalent cases, lycopene was significantly associated with a decreased risk of breast cancer. A significant trend of an inverse association between lutein and breast cancer risk was seen in premenopausal women from two combined population-based cohorts with only incident cases. A non-significant reduced risk with higher plasma alpha-carotene was apparent throughout all the sub-analyses. CONCLUSION: In conclusion, no significant associations were found between plasma levels of carotenoids, alpha-tocopherol or retinol and breast cancer risk in analysis of three combined cohorts. However, results from stratified analysis by cohort membership and menopausal status suggest that lycopene and other plasma-carotenoids may reduce the risk of developing breast cancer and that menopausal status has an impact on the mechanisms involved.

Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum.

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37 degrees C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 +/- 13 nM) for plasma and 6.6% (658 +/- 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 +/- 7.5 nM) for total MDA, 3.2% (16 +/- 0.5 nM) for free MDA in plasma, and 1.6% (630 +/- 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93-108% (mean = 100%). Plasma levels in healthy women (n = 79, 45-51 years) are 162 +/- 51 and 24 +/- 15 nM for total and free MDA, respectively. In younger men (n = 19, 21-37 years) total and free MDA are, respectively, 138 +/- 28 and 19 +/- 9 nM.

Serum carotenoids as biomarkers of fruit and vegetable consumption in the New York Women's Health Study.

OBJECTIVE: To investigate the usefulness of serum carotenoids as biomarkers of fruit and vegetable consumption. DESIGN: : Reproducibility study on three repeat measurements of serum carotenoids. Correlation analysis of carotenoids and dietary food intake, and regression analysis of potential predictive parameters for serum carotenoid levels. SETTING: New York, USA. SUBJECT: : Women participating in the New York Women's Health Study, a prospective study of sex hormones, diet and breast cancer. Forty-eight women with three repeat blood samples and 302 women having a blood sample and a dietary history questionnaire (including 47 subjects from the reproducibility study). RESULTS: Serum carotenoid concentrations were highly reproducible between one- and two-year repeat samples. Estimated fruit and vegetable consumption was positively correlated with serum carotenoid concentrations but correlation coefficients were low. Consumption of fruit was predictive for serum levels of beta-carotene, alpha-carotene and beta-cryptoxanthin, while vegetable consumption was predictive for serum beta-carotene, lutein, zeaxanthin and lycopene. Serum concentrations of cholesterol and triglycerides were predictive for serum carotenoids but adjustment for their levels had little or no influence on the correlation between fruit and vegetable consumption and serum carotenoid concentrations. CONCLUSIONS: One single serum measurement of alpha-carotene, beta-carotene and lutein can accurately rank subjects according to their usual serum level. Serum concentrations, however, correlate only moderately with estimated dietary intake of fruits or vegetables and should therefore be used with caution as biomarkers of fruit and vegetable intake.

Serum carotenoids and breast cancer.

The consumption of vegetables and fruit may protect against many types of cancer, but research evidence is not compelling for breast cancer. Carotenoids are pigments that are present in most plants and have known antioxidant properties. Blood concentrations of carotenoids have been proposed as integrated biochemical markers of vegetable, fruit, and synthetic supplements consumed. In a case-control study (270 cases, 270 controls) nested within a cohort in New York during 1985-1994, the carotenoids lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, and beta-carotene were measured in archived serum samples using liquid chromatography. There was an evident increase in the risk of breast cancer for decreasing beta-carotene, lutein, alpha-carotene, and beta-cryptoxanthin. The risk of breast cancer approximately doubled among subjects with blood levels of beta-carotene at the lowest quartile, as compared with those at the highest quartile (odds ratio = 2.21; 95% confidence interval (CI): 1.29, 3.79). The risk associated with the other carotenoids was similar, varying between 2.08 (95% CI: 1.11, 3.90) for lutein and 1.68 (95% CI: 0.99, 2.86) for beta-cryptoxanthin. The odds ratio for the lower quartile of total carotenoids was 2.31 (95% CI: 1.35, 3.96). These observations offer evidence that a low intake of carotenoids, through poor diet and/or lack of vitamin supplementation, may be associated with increased risk of breast cancer and may have public health relevance for people with markedly low intakes.

Plasma carotenoids in relation to food consumption in Granada (southern Spain) and Malmö (southern Sweden).

We conducted a cross-sectional pilot study on healthy pre-menopausal women (aged 45-50 years) living in Granada, in the south of Spain (n = 39) and Malmö, in the south of Sweden (n = 38) in order to compare their plasma carotenoid levels and to investigate the relationship between the differences in food consumption. Plasma concentrations of six carotenoids were measured using high performance liquid chromatography, habitual diet (at individual level) was estimated by food frequency questionnaires and 24-hour diet recalls were used for standardised measurement of diet at group-level. We found that women in Granada consumed more fruit and vegetables than women in Malmö. Plasma concentrations of beta-cryptoxanthin, lycopene, zeaxanthin, total carotenoids and alpha-tocopherol were higher in Granada than in Malmö, although plasma concentrations of alpha-carotene and retinol were higher in Malmö. Both within and between study centres, consumption of fruit and vegetables correlated positively with plasma concentrations of different carotenoids. The study showed that differences in consumption of fruit and vegetables between the two European centres were reflected in plasma carotenoid concentrations.

Inverse correlation between alcohol consumption and lymphocyte levels of 8-hydroxydeoxyguanosine in humans.

In a cross-sectional study of 115 premenopausal non-smoking women, we examined the relationship between lymphocyte levels of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo) and habitual alcohol consumption. The study was conducted in four different regions of Europe, including Potsdam (Germany), Turin (Italy), Malmö (Sweden) and Granada (Spain). Mean 8-oxodGuo levels differed significantly across study centres (P = 0.001), with the highest levels in Granada [2.17 8-oxodGuox10(-6) 2'-deoxyguanosine (95% confidence interval 1.27-4.40)] and lowest levels in Turin [1.19 (0.36-4.29)]. Mean levels of total alcohol intake and of types of alcoholic beverages consumed (wine, fortified wines, beer and cider) also differed across the study centres (P < 0.05), with the highest total alcohol consumption in Turin, and the lowest intake in GRANADA: When combining all the data, but adjusting for study centre, individual 8-oxodGuo level correlated inversely with alcohol intake. This inverse association remained unaltered after further adjustment for Quetelet Index, fruit and vegetable consumption, and plasma carotenoid levels. Furthermore, the inverse association was also observed for each of the study centres separately, and for different beverage types, with the exception of Granada, where the majority of women were non-drinkers and where alcohol intakes were also very low for the consumers. Finally, on a group level, mean levels of 8-oxodGuo and alcohol intake were also inversely associated between the four study centres. The finding of a relationship between alcohol consumption and 8-oxodGuo in lymphocytes was unexpected and not based on a prior hypothesis. This finding consequently requires confirmation from a randomized intervention study.

Reliability of fatty acid composition in human serum phospholipids.

OBJECTIVE: We examined the reliability of the fatty acid composition of serum phospholipids in the New York University Women's Health Study, a prospective study of sex hormones, diet and breast cancer. DESIGN: Non-fasting serum samples collected at three yearly visits, in 46 healthy women, and stored at -80 degrees C for 7-12 y, were included in the study. Serum phospholipid fatty acid composition was measured by capillary gas chromatography. RESULTS: For the 20 individual fatty acids measured, the reliability coefficients were less than 0.50 for four, between 0.50 and 0.70 for nine, and greater than 0.70 for seven. Among the major fatty acids, arachidonic and alpha-linolenic acids had high reliability coefficients (0.71 and 0. 72, respectively), palmitic, oleic, linoleic, eicosapentaenoic and docosahexaenoic acids had intermediate coefficients (0.57, 0.69, 0. 62, 0.64 and 0.66, respectively), whereas stearic acid had the lowest coefficient (0.15). The reliability coefficients for total monounsaturated fats, omega-6 and omega-3 fatty acids were moderately high (0.66, 0.53 and 0.66, respectively), whereas the coefficients for total saturated fats and total polyunsaturated fats were low (0.31 and 0.43, respectively). CONCLUSIONS: These results indicate that the fatty acid composition of serum phospholipids can be a useful tool in epidemiologic studies, although for most fatty acids a single determination is associated with some error in measurement that should be taken into account at the design and analysis stages. Storage for up to 12 y at -80 degrees C preserved polyunsaturated fatty acids from oxidation very well.

Oxidative DNA damage in human lymphocytes: correlations with plasma levels of alpha-tocopherol and carotenoids.

In order to investigate whether oxidative damage is associated with differences in antioxidant intake, we measured the levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo) in lymphocytes and alpha-tocopherol and several carotenoids in plasma of women with different dietary habits. We found that women from Granada (Spain), a region with a typically Mediterranean diet, had significantly higher levels of 8-oxodGuo compared with Malmö (Sweden), a region with a Northern European dietary intake pattern (2.30 +/- 0.78 versus 1.59 +/- 1.01 8-oxodGuo/10(-6) deoxyguanosine). Levels of plasma alpha-tocopherol and carotenoids were higher in Granada and these values were significantly positively correlated with levels of 8-oxodGuo. Our results do not support the hypothesis that a Mediterranean diet rich in alpha-tocopherol and carotenoids protects cells against oxidative DNA damage. It is possible, however, that consumption of foods other than fruits and vegetables, including fats, are responsible for the higher levels of 8-oxodGuo in Granada. Further studies are warranted to better elucidate the role of antioxidants in the modulation of oxidative stress in vivo.

Fatty-acid composition in serum phospholipids and risk of breast cancer: an incident case-control study in Sweden.

The study of the relationship between dietary intake of fatty acids and the risk of breast cancer has not yielded definite conclusions with respect to causality, possibly because of methodological issues inherent to nutritional epidemiology. To evaluate the hypothesis of possible protection of n-3 polyunsaturated fatty acids (PUFA) against breast cancer in women, we examined the fatty-acid composition of phospholipids in pre-diagnostic sera of 196 women who developed breast cancer, and of 388 controls matched for age at recruitment and duration of follow-up, in a prospective cohort study in Umeâ, northern Sweden. Individual fatty acids were measured as a percentage of total fatty acids, using capillary gas chromatography. Conditional logistic-regression models showed no significant association between n-3 PUFA and breast-cancer risk. In contrast, women in the highest quartile of stearic acid had a relative risk of 0.49 (95% confidence interval, 0.22-1.08) compared with women in the lowest quartile (trend p = 0.047), suggesting a protective role of stearic acid in breast-cancer risk. Besides stearic acid, women in the highest quartile of the 18:0/18:1 n-9c ratio had a relative risk of 0.50 (95% confidence interval, 0.23-1.10) compared with women in the lowest quartile (trend p = 0.064), suggesting a decrease in breast-cancer risk in women with low activity of the enzyme delta 9-desaturase (stearoyl CoA desaturase), which may reflect an underlying metabolic profile characterized by insulin resistance and chronic hyper-insulinemia.

Structure of the standardized computerized 24-h diet recall interview used as reference method in the 22 centers participating in the EPIC project. European Prospective Investigation into Cancer and Nutrition.

A computerized 24-h diet recall interview program (EPIC-SOFT) was developed for use in a large European multi-center study, namely the European Prospective Investigation into Cancer and Nutrition (EPIC). This program, which was adapted for each participating country and translated into nine languages, was developed to standardize interviews between the 22 EPIC centers. Common rules were pre-entered into the system to describe, quantify and probe approximately 1500-2200 foods and 150-350 recipes. Common methods used to classify and export the EPIC-SOFT dietary data facilitate their exchange, comparison and analysis. So far, EPIC-SOFT is the only available computerized 24-h diet recall system developed to provide comparable food consumption data between several European countries.

Simultaneous measurement of seven carotenoids, retinol and alpha-tocopherol in serum by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative measurement in serum of seven carotenoids (lutein, zeaxanthin, canthaxanthin, beta-cryptoxanthin, lycopenes, alpha-carotene and beta-carotene), retinol, alpha-tocopherol and two internal standards (tocol and echinenone) has been developed. The geometric isomers, lutein and zeaxanthin, were completely separated as well as at least nine unidentified carotenoids. All compounds were resolved on an Adsorbosphere HS C18 (3 microm) column (250x4.6 mm I.D.) with a step gradient, 7.1 min after injection, from acetonitrile-methanol (60:40, v/v) containing 0.05% acetic acid to acetonitrile-methanol-dichloromethane (45.6:30.4:24, v/v) containing 0.04% acetic acid, in a total run time of 23 min. Chromatograms at four different wavelengths (292, 325, 450 and 473 nm) and spectra were monitored with a diode array detector. Because of its specificity and sensitivity for a large number of carotenoids, this procedure may be of interest for nutritional and epidemiological studies. Its speed and robustness make it suitable for analyses on large numbers of subjects.

Estimating the accuracy of dietary questionnaire assessments: validation in terms of structural equation models.

The validity and precision of questionnaire assessments of the habitual intake of individuals are usually evaluated by comparison with reference measurements that are supposed to provide a best possible substitute for the individuals' true intake values. In the present paper, a measurement error model is presented, defining different types of error--random or systematic, and within or between individuals--that may occur in dietary intake measurements. It is then discussed how simple latent variable models (structural equation models) can be used to estimate the average magnitude of these various types of error. So far, approaches described for the analysis of dietary validity studies have all been based on the assumption that the random errors of repeat reference measurements, taken by the same method on different occasions, are uncorrelated, so that the average of a sufficiently large number of repeat reference measurements will provide an accurate ranking of individuals by true intake level. In the present paper it is described how, by additional comparison with a third type of measurement such as a biochemical marker, the validity of dietary questionnaire measurements can be evaluated even in situations where the random errors of repeat reference measurements can no longer be assumed to be independent.