Feasibility of self-sampled dried blood spot and saliva samples sent by mail in a population-based study.

BACKGROUND: In large epidemiological studies it is often challenging to obtain biological samples. Self-sampling by study participants using dried blood spots (DBS) technique has been suggested to overcome this challenge. DBS is a type of biosampling where blood samples are obtained by a finger-prick lancet, blotted and dried on filter paper. However, the feasibility and efficacy of collecting DBS samples from study participants in large-scale epidemiological studies is not known. The aim of the present study was to test the feasibility and response rate of collecting self-sampled DBS and saliva samples in a population-based study of women above 50 years of age. METHODS: We determined response proportions, number of phone calls to the study center with questions about sampling, and quality of the DBS. We recruited women through a study conducted within the Norwegian Breast Cancer Screening Program. Invitations, instructions and materials were sent to 4,597 women. The data collection took place over a 3 month period in the spring of 2009. RESULTS: Response proportions for the collection of DBS and saliva samples were 71.0% (3,263) and 70.9% (3,258), respectively. We received 312 phone calls (7% of the 4,597 women) with questions regarding sampling. Of the 3,263 individuals that returned DBS cards, 3,038 (93.1%) had been packaged and shipped according to instructions. A total of 3,032 DBS samples were sufficient for at least one biomarker analysis (i.e. 92.9% of DBS samples received by the laboratory). 2,418 (74.1%) of the DBS cards received by the laboratory were filled with blood according to the instructions (i.e. 10 completely filled spots with up to 7 punches per spot for up to 70 separate analyses). To assess the quality of the samples, we selected and measured two biomarkers (carotenoids and vitamin D). The biomarker levels were consistent with previous reports. CONCLUSION: Collecting self-sampled DBS and saliva samples through the postal services provides a low cost, effective and feasible alternative in epidemiological studies.

Sense and sensitivity: responsiveness to offspring signals varies with the parents' potential to breed again.

How sensitive should parents be to the demands of their young? Offspring are under selection to seek more investment than is optimal for parents to supply, which makes parents vulnerable to losing future fitness by responding to manipulative displays. Yet, parents cannot afford to ignore begging and risk allocating resources inefficiently. Here, we show that parents may solve this problem by adjusting their sensitivity to begging behaviour in relation to their own likelihood of breeding again, a factor largely neglected in previous analyses of parent-offspring interactions. In two carotenoid-supplementation experiments on a New Zealand passerine, the hihi Notiomystis cincta, we supplemented adults to enhance their propensity to breed again, and supplemented entire broods to increase their mouth colour, thus enhancing their solicitation display. We found that adults that attempted two breeding attempts a season were largely insensitive to the experimentally carotenoid-rich gapes of their brood, whereas those that bred just once responded by increasing their rate of provisioning at the nest. Our results show that parents can strategically vary their sensitivity to begging in relation to their future reproductive potential. By restricting opportunities for offspring to influence provisioning decisions, parents greatly limit the potential for offspring to win parent-offspring conflict.

Nanoparticle-based polyacrylonitrile monolithic column for highly efficient micro solid-phase extraction of carotenoids and vitamins in human serum.

In this work, a biocompatible monolithic column based micro-solid-phase extraction (µ-SPE) method was developed for biological fluid analysis. A novel nanoparticle-based polyacrylonitrile monolithic column (C30 NP-PMC) was fabricated by incorporating triacontyl (C30) modified silica nanoparticles (NPs) into the polyacrylonitrile monolithic matrix through thermally induced phase separation. With efficient mass transfer and sorption capacity, C30 NP-PMC exhibited outstanding performance for the extraction of carotenoids and fat-soluble vitamins (FSVs) from human serum samples, superior to commercial C18 cartridges as well as liquid-liquid extraction (LLE) method. Under optimal conditions, the proposed µ-SPE method coupled with high-performance liquid chromatography-diode array detection (HPLC-DAD) achieved satisfactory limits of detection (LODs) (1.5-75.0 ng/mL) and good recoveries (85.0-106.5 %) with relative standard deviations (RSDs) of less than 12.1% by consuming lower sorbent (35.0 mg) and organic solvent (0.8 mL). Successful application of the developed method demonstrated the great potential of such monolithic sorbents for efficient isolation and preconcentration of trace analytes from blood samples.

Antioxidants and periodontitis in 60-70-year-old men.

OBJECTIVE: The aim was to investigate the association between periodontal health and the serum levels of various antioxidants including carotenoids, retinol and vitamin E in a homogenous group of Western European men. MATERIALS AND METHODS: A representative sample of 1258 men aged 60-70 years, drawn from the population of Northern Ireland, was examined between 2001 and 2003. Each participant had six or more teeth, completed a questionnaire and underwent a clinical periodontal examination. Serum lipid-soluble antioxidant levels were measured by high-performance liquid chromatography with diode array detection. Multivariable analysis was carried out using logistic regression with adjustment for possible confounders. Models were constructed using two measures of periodontal status (low- and high-threshold periodontitis) as dependent variables and the fifths of each antioxidant as a predictor variable. RESULTS: The levels of alpha- and beta-carotene, beta-cryptoxanthin and zeaxanthin were highly significantly lower in the men with low-threshold periodontitis (p<0.001). These carotenoids were also significantly lower in high-threshold periodontitis. There were no significant differences in the levels of lutein, lycopene, alpha- and gamma-tocopherol or retinol in relation to periodontitis. In fully adjusted models, there was an inverse relationship between a number of carotenoids (alpha- and beta-carotene and beta-cryptoxanthin) and low-threshold periodontitis. beta-Carotene and beta-cryptoxanthin were the only antioxidants that were associated with an increased risk of high-threshold severe periodontitis. The adjusted odds ratio for high-threshold periodontitis in the lowest fifth relative to the highest fifth of beta-cryptoxanthin was 4.02 (p=0.003). CONCLUSION: It is concluded that low serum levels of a number of carotenoids, in particular beta-cryptoxanthin and beta-carotene, were associated with an increased prevalence of periodontitis in this homogenous group of 60-70-year-old Western European men.

Serum Nutrient Levels and Aging Effects on Periodontitis.

Periodontal disease damages tissues as a result of dysregulated host responses against the chronic bacterial biofilm insult and approximately 50% of US adults >30 years old exhibit periodontitis. The association of five blood nutrients and periodontitis were evaluated due to our previous findings regarding a potential protective effect for these nutrients in periodontal disease derived from the US population sampled as part of the National Health and Nutrition Examination Survey (1999⁻2004). Data from over 15,000 subjects was analyzed for blood levels of cis-ß-carotene, ß-cryptoxanthin, folate, vitamin D, and vitamin E, linked with analysis of the presence and severity of periodontitis. Moderate/severe disease patients had lower cis-ß-carotene levels across all racial/ethnic groups and these decreased levels in moderate/severe periodontitis were exacerbated with age. ß-cryptoxanthin demonstrated lower levels in severe disease patients across the entire age range in all racial/ethnic groups. Folate differences were evident across the various age groups with consistently lower levels in periodontitis patients >30 years and most pronounced in females. Lower levels of vitamin D were consistently noted across the entire age range of patients with a greater difference seen in females with periodontitis. Finally, an analytical approach to identify interactions among these nutrients related to age and periodontitis showed interactions of vitamin D in females, and folate with race in the population. These findings suggest that improving specific nutrient intake leading to elevated blood levels of a combination of these protective factors may provide a novel strategy to affect the significant increase in periodontitis that occurs with aging.

Antioxidant Micronutrients and Oxidative Stress Biomarkers.

Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of life-style factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.

Multiple thermotropic state transitions in erythrocyte membranes. A laser-Raman study of the CH-stretching and acoustical regions.

1. We have examined the Raman scattering from erythrocyte ghosts at 2700 cm-1 -3000 cm-1 (CH-stretching region), that at low frequencies due to acoustical vibrations, as well as that due to v (-C=C-) and v (=C-C=) of beta-carotene, in the temperature range of -30 degrees C to +30 degrees C. For comparison we have also evaluated the Rama spectra of liposomes composed of lecithins+/-cholesterol. 2. Plots vs. temperature of the intensities (I) of the approx. 2880 cm-1 and 2932 cm-1 bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the (I2880/I2850) and (I2932/I2850) ratios, revealed discontinuities in both erythrocyte membranes and liposomes. 3. In erythrocyte membranes, plots of (I approximately 2880/I2850) and (I approximately 2932/I 2850) reveal several discontinuities. Those reported by (I approximately 2880/I 2850) occur at +17 degrees C and approx. -4 degrees C. The ratio (I approximately 2932/I 2850) reveals a transition between -20 degrees C and +10 degrees C, 1/2 maximal at -8 degrees C. The ratio (I 1527/I 1158), representing the relative intensity of v (-C=C-) vs. v (=C-C=) of membrane-bound beta-carotene, changes discontinuously with the 17 degrees C transition, but is stable over the rest of the temperature change studied. 4. The data indicate that a major membrane-state transition takes place below 0 degrees C but that an additional transition occurs at approx. 17 degrees C.

Consumption of carotenoids in photosensitized oxidation of human plasma and plasma low-density lipoprotein.

Human plasma and plasma low-density lipoprotein (LDL) were exposed to photoirradiation in the presence of methylene blue (water-soluble photosensitizer) or 12-(1-pyrene)dodecanoic acid (P-12, lipid-soluble photosensitizer). In methylene-blue-sensitized photooxidation of human plasma and LDL, endogenous carotenoids and tocopherols were consumed with the accumulation of cholesteryl ester hydroperoxides (CE-OOH). Xanthophylls (zeaxanthin and lutein) decreased faster than lycopene and carotenes in the case of human plasma. In P-12-sensitized photooxidation of human plasma and LDL, the decrease rate of xanthophylls was slower than that of lycopene and carotenes. A lower level of beta-carotene exerted the effective inhibition of lipid peroxidation and retarded the oxidative loss of alpha-tocopherol, when the phosphatidylcholine liposomes containing these two lipid-soluble antioxidants were subjected to methylene blue- or P-12-sensitized photooxidation. These results suggest that antioxidant activity of carotenoids in photosensitized oxidation (Type II) of human plasma LDL depends on the site of singlet oxygen (1O2) to be generated and that carotenoids can protect tocopherols from the oxidative loss by 1O2 in the plasma.

Vegetables, fruits, and carotenoids and the risk of cancer.

Low intake of vegetables, fruits, and carotenoids is consistently associated with increased risk of lung cancer in both prospective and retrospective studies. In addition, low levels of beta-carotene in serum or plasma are consistently associated with the subsequent development of lung cancer. The simplest explanation is that beta-carotene is protective. Since retinol (preformed vitamin A) is not related in a similar manner to lung cancer risk, beta-carotene appears to function through a mechanism that does not require conversion into vitamin A. However, the importance of other carotenoids and other constituents of vegetables and fruit has not been adequately explored. Both prospective and retrospective studies suggest that vegetable and fruit intake may reduce the risk of cancers of the mouth, pharynx, larynx, esophagus, stomach, colon, rectum, bladder, and cervix. But because of fewer studies and less consistency among studies, the epidemiologic evidence is at present less persuasive than for lung cancer.

A pilot study on the use of plasma carotenoids and ascorbic acid as markers of compliance to a high fruit and vegetable dietary intervention.

The authors examined the feasibility of using plasma carotenoids and ascorbic acid as markers of compliance for dietary intervention trials aimed at increasing the quantity and variety of the fruit and vegetable intake of free-living individuals. Nineteen former cancer patients who had been successfully treated for a stage I or II squamous cell carcinoma of the mouth, pharynx, larynx, or lung were recruited. Subjects served as their own controls. However, in order to detect any seasonal trends, 4 individuals among the 19 were randomized to a nonintervention group. Subjects in the intervention group were counseled by dietitians with the goal of increasing their intake of fruits and vegetables to eight servings/day (1 serving each of dark green vegetables, yellow-orange vegetables, tomato products, and other vegetables; 3 servings of vitamin C-rich fruits; and 1 serving of other fruits). Subjects in the nonintervention group were advised to follow their usual diet. Three-day measured food records kept at base line and after 3 months of intervention, as well as unannounced 24-h dietary recalls, documented an increase in mean fruit and vegetable intake from 4.2 to 9.5 servings daily in the intervention group. A concomitant increase of 29% was observed in total plasma carotenoids (P = 0.02), with increases of 25% for plasma lycopene (P = 0.06), 31% for plasma lutein (P = 0.002), 39% for plasma beta-carotene (P = 0.01), and 57% for plasma alpha-carotene (P = 0.01). Mean plasma levels of ascorbic acid increased by 27% (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Can alpha-tocopherol and beta-carotene supplementation reduce adverse radiation effects on salivary glands?

In this study, we evaluated whether supplementation with antioxidant vitamins can reduce the adverse effects of irradiation on the salivary glands in the rat. Four groups of adult Sprague-Dawley rats were given a basic diet providing 0.6 mg alpha-tocopherol and no beta-carotene per day. In two groups the basic diet was supplemented with 3.4 mg alpha-tocopherol and 6 mg beta-carotene per day from 14 days before irradiation until 12 days after completed irradiation. One group of rats given basic diet and one group given supplemented diet were irradiated with 7 Gy daily for five consecutive days. Isoproterenol and pilocarpine-stimulated whole saliva was collected from all rats 2, 4 and 26 weeks after irradiation. Vitamin-supplemented irradiated rats had higher secretion rates on all three occasions compared with those of irradiated rats given basic diet. The changes in saliva composition seen in irradiated rats were less accentuated in vitamin-supplemented, irradiated rats. The proportions of acinar cells were significantly decreased both in parotid and submandibular glands 26 weeks after irradiation. Supplementation with alpha-tocopherol and beta-carotene did not alter the morphology of the glands.

Chemoprevention of oral leukoplakia and chronic esophagitis in an area of high incidence of oral and esophageal cancer.

This intervention trial carried out in Uzbekistan (former USSR) in an area with a high incidence of oral and esophageal cancer involved random allocation of 532 men, 50 to 69 years old, with oral leukoplakia and/or chronic esophagitis to one of four arms in a double-blind, two-by-two factorial design, with active arms defined by the administration of (a) riboflavin; (b) a combination of retinol, beta-carotene, and vitamin E; or (c) both. Weekly doses were 100,000 IU of retinol, 80 mg of vitamin E, and 80 mg of riboflavin. The dose of beta-carotene was 40 mg/d. Men in the trial were followed for 20 months after randomization. The aim of the trial was to determine whether treatment with these vitamins or their combination could affect the prevalence of oral leukoplakia and/or protect against progression of oral leukoplakia and esophagitis, conditions considered to be precursors of cancer of the mouth and esophagus. A significant decrease in the prevalence odds ratio (OR) of oral leukoplakia was observed after 6 months of treatment in men receiving retinol, beta-carotene, and vitamin E (OR = 0.62; 95% confidence interval (CI): 0.39 to 0.98). After 20 months of treatment, no effect of vitamin supplementation was seen when the changes in chronic esophagitis were compared in the four different treatment groups, although the risk of progression of chronic esophagitis was lower in the subjects allocated to receive retinol, beta-carotene and vitamin E (OR = 0.65; 95% CI: 0.29 to 1.48) A secondary analysis not based on the randomized design revealed a decrease in the prevalence of oral leukoplakia in men with medium (OR = 0.45; 95% CI: 0.21 to 0.96) and high (OR = 0.59; 95% CI: 0.29 to 1.20) blood concentrations of beta-carotene after 20 months of treatment. Risk of progression of chronic esophagitis was also lower in men with a high blood concentration of beta-carotene, odds ratios being 0.30 (95% CI: 0.10 to 0.89) and 0.49 (95% CI: 0.15 to 1.58) for medium and high levels, respectively. A decrease in risk, also statistically not significant, was observed for high vitamin E levels (OR = 0.39; 95% CI: 0.14 to 1.10). These results were based on levels of vitamins in blood drawn after 20 months of treatment.

Carotenoids and vitamin A: the effect of hypocholesterolemic agents on serum levels.

Serum total carotenoid (STC) and vitamin A levels were done as part of the biochemical screening in comparative studies of lipid lowering agents in type Ila hyperlipoproteinemic patients. STC levels were reduced following bile acid sequestering agent administration (colestipol 30 g/d) by 30% (P less than 0.01). Clofibrate and avicel placebo had inconsistent and nonsignificant effects on the STC levels. Serum vitamin A levels were not significantly altered by any of the test agents. The STC level changes were not correlated with concomitant changes in low-density lipoprotein-cholesterol (LDL-C) during any of the treatment regimens. It is suggested that STC level changes are related to alterations in the absorption of carotenoids during bile acid sequestrant administration.

Coffee drinking influences plasma antioxidant capacity in humans.

Coffee and tea are widely consumed beverages, but only tea has been studied for its antioxidant capacity (AC) in vivo. The aim of this study was to compare the capacities of coffee and tea to affect plasma redox homeostasis in humans. The AC of plasma before and after supplementation with 200 mL of beverages (0, 1, and 2 h) was measured by the TRAP and crocin tests. The crocin test detected an increase in plasma AC only in subjects supplemented with coffee (+7% at peak time), whereas the TRAP method showed an increase in plasma AC after consumption of both coffee and tea (+6 and +4%, respectively, at peak time). Both beverages induced a significant increase in plasma uric acid (+5 and +7%, respectively). Uric acid strongly affects the results obtained by the TRAP test and does not affect those obtained by the crocin test. We can thus argue that uric acid is the main component responsible for the plasma AC increase after tea drinking, whereas molecules other than uric acid (probably phenolic compounds) are likely to be responsible for the increase in plasma AC after coffee drinking.

Solid-phase extraction of carotenoids.

In this work, solid-phase extraction (SPE) trapping performance of lutein and beta-carotene, which were used as the model molecules of carotenoids, was investigated. The absorption, elution, and enrichment of carotenoids on SPE cartridges with four different sorbents, i.e. C(30), C(18), diol, and silica, were compared respectively with the help of frontal analysis technique. The high retentions of both lutein and beta-carotene were achieved on the C(18) and C(30) cartridges. The diol and silica cartridges only had good retention for lutein. The optimized SPE method for sample pretreatment for the carotenoids analysis was obtained after the investigation of trapping performance. The method was applied successfully to the analysis of biological sample, i.e. serum and human breast milk. The recovery, accuracy, and precision of SPE method comparing with those of traditional liquid-liquid extraction (LLE) method for the sample pretreatment for the analysis of carotenoids owned a number of advantages such as rapid, no chloroform used, and accurate versus LLE.

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids, retinol, and tocopherols in human serum.

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 microm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for beta-cryptoxanthin, cis-trans-lycopene, alpha-carotene, beta-carotene, cis-beta-carotene, retinol, delta-tocopherol, gamma-tocopherol and alpha-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2-7.3% and 3.6-12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.

Condition dependence of nestling mouth colour and the effect of supplementing carotenoids on parental behaviour in the hihi (Notiomystis cincta).

Carotenoids are integument pigments that often reflect foraging efficiency, disease resistance and body condition. In contrast to the widespread attention this relationship has received in adult birds, the condition dependence of nestling colouration remains an understudied component of animal communication. Here we assess the condition dependence of carotenoid pigmentation in nestling hihi (Notiomystis cincta, an endangered New Zealand bird) and examine the influence of carotenoid supplementation on nestling quality and parental visitation rates. Our results show that carotenoids provided to breeding adult hihi were transferred to their offspring and resulted in an intensified orange-yellow flange colour. After accounting for carotenoid supplementation the parameter that most consistently explained variation in nestling flange colour was nestling tarsus length at 23 days, indicating condition dependence of this trait. We did not, however, detect direct effects of carotenoid supplementation on nestling mass or immune response (or any other fitness parameter measured). Carotenoid supplementation did, however, result in an increased paternal provisioning rate.