The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Evening preference correlates with regional brain volumes in the anterior occipital lobe.

Chronotype or diurnal preference is a questionnaire-based measure influenced both by circadian period and by the sleep homeostat. In order to further characterize the biological determinants of these measures, we used a hypothesis-free approach to investigate the association between the score of the morningness-eveningness questionnaire (MEQ) and the Munich chronotype questionnaire (MCTQ), as continuous variables, and volumetric measures of brain regions acquired by magnetic resonance imaging (MRI). Data were collected from the Baependi Heart Study cohort, based in a rural town in South-Eastern Brazil. MEQ and anatomical 1.5-T MRI scan data were available from 410 individuals, and MCTQ scores were available from a subset of 198 of them. The average MEQ (62.2 ± 10.6) and MCTQ (average MSFsc 201 ± 85 min) scores were suggestive of a previously reported strong general tendency toward morningness in this community. Setting the significance threshold at P > .002 to account for multiple comparisons, we observed a significant association between lower MEQ score (eveningness) and greater volume of the left anterior occipital sulcus (ß = -0.163, p = .001) of the occipital lobe. No significant associations were observed for MCTQ. This may reflect the smaller dataset for MCTQ, and/or the fact that MEQ, which asks questions about preferred timings, is more trait-like than the MCTQ, which asks questions about actual timings. The association between MEQ and a brain region dedicated to visual information processing is suggestive of the increasingly recognized fluidity in the interaction between visual and nonvisual photoreception and the circadian system, and the possibility that chronotype includes an element of masking.

Evaluation of the state-of-the-art measurement capabilities for selected PBDEs and decaBB in plastic by the international intercomparison CCQM-P114.

An international intercomparison involving eight national metrology institutes (NMIs) was conducted to establish their current measurement capabilities for determining five selected congeners from the brominated flame retardant classes polybrominated diphenyl ethers and polybrominated biphenyls. A candidate reference material consisting of polypropylene fortified with technical mixtures of penta-, octa- and decabromo diphenyl ether and decabromo biphenyl, which was thoroughly assessed for material homogeneity and stability, was used as study material. The analytical procedures applied by the participants differed with regard to sample pre-treatment, extraction, clean-up, employed calibrants and type of calibration procedure as well as regarding analytical methods used for separation, identification and quantification of the flame retardant congeners (gas chromatography coupled to an electron capture detector (GC-ECD), gas chromatography-mass spectrometry in the electron ionisation mode (GC-EI-MS), gas chromatography-mass spectrometry in the electron capture negative ionisation mode (GC-ECNI-MS), and liquid chromatography-inductive coupled plasma-mass spectrometry (LC-ICP-MS)). The laboratory means agreed well with relative standard deviations of the mean of means of 1.9%, 4.8%, 5.5% and 5.4% for brominated diphenyl ether (BDE) 47, 183 and 209 and for the brominated biphenyl (BB) congener 209, respectively. For BDE 206, a relative standard deviation of 28.5% was obtained. For all five congeners, within-laboratory relative standard deviations of six measurements obtained under intermediate precision conditions were between 1% and 10%, and reported expanded measurements uncertainties typically ranged from 4% to 10% (8% to 14% for BDE 206). Furthermore, the results are in good agreement with those obtained in the characterization exercise for determining certified values for the flame retardant congeners in the same material. The results demonstrate the state-of-the-art measurement capabilities of NMIs for quantifying representative BDE congeners and BB 209 in a polymer. The outcome of this intercomparison (pilot study) in conjunction with possible improvements for employing exclusively calibrants with thoroughly assessed purity suggests that a key comparison aiming at underpinning calibration and measurement capability (CMC) claims of NMIs can be conducted.

Regulation of mammalian circadian behavior by non-rod, non-cone, ocular photoreceptors.

Circadian rhythms of mammals are entrained by light to follow the daily solar cycle (photoentrainment). To determine whether retinal rods and cones are required for this response, the effects of light on the regulation of circadian wheel-running behavior were examined in mice lacking these photoreceptors. Mice without cones (cl) or without both rods and cones (rdta/cl) showed unattenuated phase-shifting responses to light. Removal of the eyes abolishes this behavior. Thus, neither rods nor cones are required for photoentrainment, and the murine eye contains additional photoreceptors that regulate the circadian clock.

Heritability of semantic verbal fluency task using time-interval analysis.

Individual variability in word generation is a product of genetic and environmental influences. The genetic effects on semantic verbal fluency were estimated in 1,735 participants from the Brazilian Baependi Heart Study. The numbers of exemplars produced in 60 s were broken down into time quartiles because of the involvement of different cognitive processes-predominantly automatic at the beginning, controlled/executive at the end. Heritability in the unadjusted model for the 60-s measure was 0.32. The best-fit model contained age, sex, years of schooling, and time of day as covariates, giving a heritability of 0.21. Schooling had the highest moderating effect. The highest heritability (0.17) was observed in the first quartile, decreasing to 0.09, 0.12, and 0.0003 in the following ones. Heritability for average production starting point (intercept) was 0.18, indicating genetic influences for automatic cognitive processes. Production decay (slope), indicative of controlled processes, was not significant. The genetic influence on different quartiles of the semantic verbal fluency test could potentially be exploited in clinical practice and genome-wide association studies.

Metabolic syndrome alters relationships between cardiometabolic variables, cognition and white matter hyperintensity load.

Cardiometabolic risk factors influence white matter hyperintensity (WMH) development: in metabolic syndrome (MetS), higher WMH load is often reported but the relationships between specific cardiometabolic variables, WMH load and cognitive performance are uncertain. We investigated these in a Brazilian sample (aged 50-85) with (N = 61) and without (N = 103) MetS. Stepwise regression models identified effects of cardiometabolic and demographic variables on WMH load (from FLAIR MRI) and verbal recall performance. WMH volume was greater in MetS, but verbal recall performance was not impaired. Age showed the strongest relationship with WMH load. Across all participants, systolic blood pressure (SBP) and fasting blood glucose were also contributors, and WMH volume was negatively associated with verbal recall performance. In non-MetS, higher HbA1c, SBP, and number of MetS components were linked to poorer recall performance while higher triglyceride levels appeared to be protective. In MetS only, these relationships were absent but education exerted a strongly protective effect on recall performance. Thus, results support MetS as a construct: the clustering of cardiometabolic variables in MetS alters their individual relationships with cognition; instead, MetS is characterised by a greater reliance on cognitive reserve mechanisms. In non-MetS, strategies to control HbA1c and SBP should be prioritised as these have the largest impact on cognition.

Certification of standard reference materials containing bitter orange.

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

Determination of HBCD, PBDEs and MeO-BDEs in California sea lions (Zalophus californianus) stranded between 1993 and 2003.

Blubber samples from male California sea lions (Zalphophus californianus) stranded between 1993 and 2003 were analyzed for 27 polybrominated diphenyl ether (PBDE) congeners, three isomers of hexabromocyclododecane (HBCD) and 14 methoxylated polybrominated diphenyl ether (MeO-BDE) congeners. Total PBDEs ranged from 450 ng/g to 4740 ng/g wet mass and total HBCD ranged from < 0.3 ng/g to 12 ng/g wet mass. The concentration of HBCD increased from 0.7 ng/g to12.0 ng/g wet mass in sea lion blubber between 1993 and 2003. However, no significant temporal trend was observed for any of the other brominated compounds over this 10 year period. Only one of the 14 MeO-BDE congeners was detected in the blubber samples, 6-methoxy-2,2',4,4'-tetrabromodiphenyl ether (6-MeO-BDE 47), and concentrations ranged from < 0.2 ng/g to 12 ng/g wet mass. A bromo-, chloro-heterocyclic compound, 1,1'-dimethyl-tetrabromo-dichloro-2,2'-bipyrrole (DBP-Br4Cl2), previously reported in marine species along the Pacific coast, was also identified in the sea lion blubber. DBP-Br4Cl2 ranged from 44 ng/g wet mass to 660 ng/g wet mass and was present at concentrations rivaling the dominant PBDE congener, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether). Concentrations of DBP-Br4Cl2 were positively correlated with 6-MeO-BDE 47 (r = 0.7; p < 0.05). Both of these compounds have been identified in marine algae and sponges, and studies suggest they are both produced from natural sources. This study demonstrates that brominated compounds from both anthropogenic and biogenic sources can accumulate to similar levels in marine mammals. In addition, HBCD concentrations appear to be increasing in California sea lion populations, whereas PBDE concentrations, between 1993 and 2003, were highly variable.

Timing and quality of sleep in a rural Brazilian family-based cohort, the Baependi Heart Study.

Sleep is modulated by several factors, including sex, age, and chronotype. It has been hypothesised that contemporary urban populations are under pressure towards shorter sleep duration and poorer sleep quality. Baependi is a small town in Brazil that provides a window of opportunity to study the influence of sleep patterns in a highly admixed rural population with a conservative lifestyle. We evaluated sleep characteristics, excessive daytime sleepiness, and chronotype using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale and Morningness-Eveningness Questionnaire questionnaires, respectively. The sample consisted of 1,334 subjects from the Baependi Heart study (41.5% male; age: 46.5 ± 16.2 y, range: 18-89 years). Average self-reported sleep duration was 07:07 ± 01:31 (bedtime 22:32 ± 01:27, wake up time: 06:17 ± 01:25 hh:min), sleep quality score was 4.9 + 3.2, chronotype was 63.6 ± 10.8 and daytime sleepiness was 7.4 ± 4.8. Despite a shift towards morningness in the population, chronotype remained associated with reported actual sleep timing. Age and sex modulated the ontogeny of sleep and chronotype, increasing age was associated with earlier sleep time and shorter sleep duration. Women slept longer and later, and reported poorer sleep quality than men (p < 0.0001). This study provides indirect evidence in support of the hypothesis that sleep timing was earlier prior to full urbanisation.

Fruit-developmental regulation of bell pepper knolle gene (cakn) expression(1).

A cDNA, cakn, orthologous to the Arabidopsis KN gene, which is involved in vesicle fusion during cell plate formation, was isolated from bell pepper fruit. cakn seems to be monogenic and its expression mainly transcriptionally regulated. During fruit development, transcript and protein levels increase significantly in the early stages in which numerous cell divisions occur, but in the stages corresponding to fruit growth by cell enlargement, whereas the messengers are undetectable, proteins are still faintly present, suggesting a different stability rate for the two types of macromolecules.

Fractionation of an anthocyanin-rich bilberry extract and in vitro antioxidative activity testing.

The incidence of chronic diseases increases with advancing age of the population. A commonly discussed cause of chronic diseases is oxidative stress, which occurs in the body when there is an imbalance between the formation and inactivation of so-called reactive oxygen species (ROS). Epidemiological data suggest that a 'healthy diet', with a high content of flavonoids indicates preventive properties and correlates with an inverse effect with respect to the risk of chronic diseases. Berries (especially bilberries, Vaccinium myrtillus L.) are an important source of these flavonoids. In this study, we investigated, in vitro, the antioxidative properties of fractions obtained from a commercially available anthocyanin-rich bilberry extract (BE). As markers for antioxidative activity, the intracellularly generated ROS levels, oxidative DNA damage, and total glutathione (tGSH) levels were determined in the human colon cell lines Caco-2 and HT-29. In Caco-2 cells, the ROS levels and, in both cell lines, the oxidative DNA damage, were significantly reduced in the presence of the original BE and phenolcarbonic acid-rich fraction. Total GSH levels were slightly increased after pretreatment with BE, phenolcarbonic acid and the polymeric fractions, but not with the anthocyanin fraction. In summary, the BE and the therefrom-isolated phenolcarbonic acid-rich fraction, showed the most potent antioxidative activity whereas the polymeric and anthocyanin-rich fraction, in total, were less active.

Extraocular light exposure does not suppress plasma melatonin in humans.

Light affects the circadian axis in at least two ways. It can cause the acute suppression of pineal melatonin synthesis, and/or a phase-shift of the circadian oscillator. As recent evidence has suggested that extraocular light exposure may cause phase-shifts of the circadian clock, we have investigated whether suppression of melatonin can be induced by the same type of light exposure. In the first study subjects' eyes were exposed to white light (2250 lux for 30 mins) via a fibre optic cable. As expected, suppression of nighttime plasma melatonin levels (61 +/- 6%) was observed. In the second study, light of the same quality but higher intensity (14,000 or 67,500 lux for 180 mins) was delivered in the same manner to the popliteal region behind the subjects' knees, whilst shielding their eyes. No suppression of plasma melatonin levels (4 +/- 7%) was detected in any of the subjects. Thus, extraocular photoreception, if it exists in mammals, does not affect the suprachiasmatic nucleipineal pathway.

Changes in ascorbate peroxidase activities during fruit ripening in Capsicum annum.

The ascorbate peroxidase (APX) system was studied in Capsicum annum during fruit ripening. A large increase in organelle APX activity was found during chloroplast-chromoplast transition whereas only a slight difference was detected in total fruit extracts. On native gels, four different isoforms were found in total fruit extracts but the patterns for red and green fruit were quite different. In isolated organelles, six isozymes were found and a comparison of the patterns showed significant differences. A cDNA encoding a cytosolic APX was cloned and sequenced. The corresponding transcript was shown to increase 3-4-fold during fruit ripening.

Characterization and gene expression of an annexin during fruit development in Capsicum annuum.

Several lines of evidence indicate that annexins, as calcium-dependent phospholipid-binding proteins, are involved in a variety of plant cellular processes. We were interested in determining if annexins are implicated in the highly regulated fruit development of bell pepper. By differential screening of several cDNA libraries, we isolated a full-length cDNA of 1180 bp encoding an annexin. Northern blot analyses show a differential expression pattern of the transcripts during the early stages of development and during ripening. Immunoblots using antiserum raised against p33/p35 from maize reveal that cross-reactive polypeptides of about 30 kDa are present at each stage of fruit development in bell pepper. We partially purified the annexins from seedlings and green fruits. At least one annexin of 32 kDa is present in these plant tissues.

Distribution of Purkinje cell-specific Zebrin-II/aldolase C immunoreactivity in the mouse, rat, rabbit, and human retina.

The developmental, genetic, and biochemical similarities that have been observed between the cerebellum and retina form the basis for ongoing investigations into retinal expression of cerebellar-specific proteins. We have examined the mouse, rat, rabbit, and human retina for expression of a protein that is present in parasagittal Purkinje cell strips and that is recognized by the antibody Zebrin-II. This protein has recently been identified as a member of the aldolase C isoenzymes. Western blotting and immunocytochemistry have been used. The monoclonal antibody Zebrin-II recognized a prominent 36 kDa protein band on immunoblots of both the cerebellum and the retina of the examined species. Immunocytochemistry showed that, in the three nonhuman species, cells were stained in the ganglion cell layer (GCL). In addition, in the mouse and rabbit, cells in the inner nuclear layer (INL) were also labeled. Except for the visual streak, there were more immunopositive cells in the rabbit GCL and INL than in corresponding areas of the mouse retina. In the human, in contrast to the other species, the photoreceptor cell layer was strongly aldolase C immunoreactive. In all species except for the rat, the photoreceptor inner segments also displayed a weak labeling. The results show that this aldolase C isoenzyme is another protein that is selectively expressed by the cerebellum and retina. Furthermore, the retinal expression is species specific, and this pattern seems to show a good correlation with the oxygenation level of the individual compartments. The indication that this aldolase C isoenzyme has specific developmental functions in the retina provides additional clues for our understanding of cerebellar organization.

Expression of phototransduction cascade genes in the ground squirrel retina.

PURPOSE: This study describes the expression and distribution of phototransduction cascade gene products in the cone-dominant retina of the ground squirrel Spermophilus tridecemlineatus. METHODS: Messenger RNA expression was studied by blot hybridization, and the distribution of the gene products was investigated by immunocytochemistry. RESULTS: RNA blot hybridization showed messages for the alpha 2, beta 1, and beta 3 subunits of transducin but was negative for rhodopsin, alpha 1-transducin, and the alpha, beta, and gamma subunits of cyclic guanosine monophosphate (cGMP) phosphodiesterase. Immunocytochemical labeling indicated that the approximate ratio of the photoreceptor types in ground squirrel retina is 90.6% for green cones, 6.3% for rod-like cells, and 3.1% for blue cones. Rod-like cells were immunopositive for rhodopsin and blue opsin. All photoreceptor elements were labeled by antibodies against alpha 1-transducin (which recognizes both the alpha 1 and alpha 2 isoforms), beta 3-transducin, and the rod gamma subunit of phosphodiesterase, whereas no cells were labeled by antibodies against the rod alpha and beta subunits of phosphodiesterase or against the rod cGMP-gated cation channel. Rod-like cells and blue cones were stained by antibodies against beta 1-transducin. CONCLUSIONS: The authors demonstrate new cone-like traits in the biochemical make-up of rod-like cells, and a distribution of the transducin beta subunit in the ground squirrel is different from that found in other mammals.

Expression of soluble phototransduction-associated proteins in ground squirrel retina.

PURPOSE: This study describes the expression and distribution of arrestin, phosducin, and recoverin in the cone-dominant retina of the ground squirrel Spermophilus tridecemlineatus. METHODS: mRNA expression was studied by blot hybridization of ground squirrel retinal RNA, with human and murine RNA as controls. The distribution of the gene products in the ground squirrel retina was investigated by immunocytochemistry using radial and consecutive tangential sections. RESULTS: Northern blot hybridization showed messages for arrestin (1.9 kb), phosducin (1.4 kb), and recoverin (1.2 kb) in ground squirrel retinal RNA. Both controls showed transcripts of the same or similar sizes. Rod-like cells and blue cones were stained by antibodies against arrestin and phosducin. The arrestin antiserum stained the whole cell bodies, most intensely in the myoid region, whereas phosducin immunoreactivity was confined to the outer and inner segments, which were stained with approximately equal intensity. The strongest immunoreaction was found in the photoreceptor plasma membrane. Recoverin antibodies recognized the entire soma of all photoreceptor cells. The myoid region and the synaptic pedicles were most heavily stained. No light-dependent migration was observed with either antiserum in any photoreceptor type. CONCLUSION: The presence of arrestin immunoreactivity in rod-like cells and blue cones is consistent with previous reports on other mammals. However, it has not been reported previously that phosducin immunoreactivity is distributed in the same way. The colocalization of arrestin and phosducin in rod-like cells and blue cones is yet another trait distinguishing blue cones from red and green cones.

Difference in PNA label intensity between short- and middle-wavelength sensitive cones in the ground squirrel retina.

PURPOSE: Peanut agglutinin lectin (PNA) is known for its selective binding to cone cells and to the cone domains of the interphotoreceptor matrix. In the current study, the authors investigated whether there is any difference in PNA binding between color-specific cones of the cone-dominant ground squirrel. METHODS: Consecutive serial sections of the retina of Spermophilus tridecemlineatus were reacted alternately with PNA and antivisual pigment antibodies. The PNA labels associated with short- and middle-wavelength-sensitive cones (S-cones and M-cones, respectively) were compared with fluorescent lectin cytochemistry. RESULTS: Although all rod-like cells were left unstained, the cones exhibited a specific lectin label. There was, however, a significant difference between the two cone types; the intensity of the ring-like PNA label in the matrix sheath around S-cones significantly exceeded that of the M-cones. CONCLUSIONS: The difference in PNA label intensity indicates a difference in the composition of the matrix sheaths surrounding the two respective cone types. To the authors' knowledge, this is the first report on lectin-cytochemical discrimination of cone matrix sheaths and the first lectin study in the ground squirrel retina leading to the observation that PNA can distinguish the three characteristic photoreceptor types in this animal. In this respect, the rod-like cells of the ground squirrel retina were shown to be no different from rod cells of other species.

Cloning of a cyclic GMP phosphodiesterase gamma subunit from the ground squirrel retina.

Recent evidence suggests that the two main classes of cones are not only equipped with different photopigments, but also exhibit differences in their downstream phototransduction cascade. An antibody against the gamma subunit of retinal cGMP phosphodiesterase (PDE) had previously been found to label all photoreceptors in the ground squirrel (Spermophilus tridecemlineatus). This property was utilized for the cloning of a cDNA fragment encoding the corresponding polypeptide. A lambda gt11 cDNA library was constructed and screened with the antibody. Positive clones were isolated, subcloned, and sequenced. Clones were used as probes in RNA blot hybridization, and the obtained sequence information was compared to other available gamma-PDE sequences. Four virtually identical cDNA clones were isolated. Both nucleic acid and amino acid sequence alignment placed this gene in the same group as bovine and human cone gamma-phosphodiesterase subunits. The predicted length of the translated protein was 84 amino acids, and its molecular weight 11 kD. The cDNA hybridized with an 0.6-kb transcript in retinal RNA from ground squirrel, Syrian golden hamster, and mouse, and also with a 1.4-kb transcript in the ground squirrel. We conclude that the isolated cDNA fragment encodes a gamma subunit of cone cGMP-PDE. This subunit is expressed in middle-wavelength sensitive cones (the predominant photoreceptor type in this species). It remains to be determined if this subunit is expressed in short-wavelength sensitive cones as well, or if these photoreceptors are equipped with another, unique gamma-PDE subunit.

Circadian oscillation of photopigment transcript levels in the mouse retina.

The mammalian retina contains an autonomous circadian oscillator that can sustain rhythms in outer segment disc shedding and melatonin synthesis even in the absence of cues from the central oscillator in the suprachiasmatic nucleus (SCN). The present investigation aimed to ascertain whether the steady-state levels of rod and cone opsin mRNA exhibited a circadian oscillation in the mouse, and if so, to characterise this oscillation with regard to phase. Retinas were collected from mice free-running in constant darkness at circadian times (CT) 0, 4, 12, 16, and 20. RNA was extracted for the production of Northern blots, which were sequentially hybridised with probes for alpha-tubulin (control), ultraviolet opsin, and rhodopsin. Whereas no significant oscillation was detected in the levels of alpha-tubulin, the levels of both ultraviolet opsin and rhodopsin transcripts oscillated with an amplitude of at least 3-fold. The highest levels were found at around CT12 (which corresponds to the subjective evening, the time of activity onset in the mouse). These results show that the levels of rod and cone photopigment mRNA are under circadian control. The significance of the rhythms and their phasing is discussed. These findings have potential clinical implications both with regard to nocturnal light treatment of phase asynchrony, and in the timing of chemo- and radiotherapy.