Distribution and determinants of retinol in Norwegian adolescents, and its relation to bone mineral density: the Tromsø Study: Fit Futures.

BACKGROUND/OBJECTIVES: Sufficient vitamin A levels are important for many functions-and both too little and too much may have detrimental health effects. The aim of the study was to describe the distribution of retinol levels in Norwegian adolescents, the relation between lifestyle factors and retinol levels, and the relation between retinol levels and bone mineral density (BMD). SUBJECTS/METHODS: Serum retinol was measured in 414 girls and 474 boys aged 15-19 years, participating in the Tromsø Study: Fit Futures. Questionnaires regarding health and lifestyle factors were filled in, and physical examinations, body composition, and bone mineral density measurements (DEXA) performed. Multiple regression analyses were used to discover associations between retinol and exposure variables. RESULTS: Retinol levels ranged from 0.26 to 6.46 µmol/L with a median (2.5-97.5 percentile) of 2.35 (1.01-4.67) µmol/L. There was no gender difference. In the multivariate models, fat mass, albumin level, physical activity, and lunch habits were positively associated with retinol levels in boys. In girls, fat mass and height were negatively associated with retinol levels, and lean mass, vitamin D, calcium, total cholesterol, and the use of contraceptives were positively associated with retinol levels (p < 0.05). The models explained 18.3% and 14.6% of the variation (R2) in girls and boys, respectively. Retinol levels were not independently associated with BMD. CONCLUSION: Retinol levels in Norwegian adolescents are higher than reported elsewhere, and are to a low degree explained by lifestyle and physical measurements. No independent association with BMD was found.

Biomarkers for nutrient intake with focus on alternative sampling techniques.

Biomarkers of nutrient intake or nutrient status are important objective measures of foods/nutrients as one of the most important environmental factors people are exposed to. It is very difficult to obtain accurate data on individual food intake, and there is a large variation of nutrient composition of foods consumed in a population. Thus, it is difficult to obtain precise measures of exposure to different nutrients and thereby be able to understand the relationship between diet, health, and disease. This is the background for investing considerable resources in studying biomarkers of nutrients believed to be important in our foods. Modern technology with high sensitivity and specificity concerning many nutrient biomarkers has allowed an interesting development with analyses of very small amounts of blood or tissue material. In combination with non-professional collection of blood by finger-pricking and collection on filters or sticks, this may make collection of samples and analyses of biomarkers much more available for scientists as well as health professionals and even lay people in particular in relation to the marked trend of self-monitoring of body functions linked to mobile phone technology. Assuming standard operating procedures are used for collection, drying, transport, extraction, and analysis of samples, it turns out that many analytes of nutritional interest can be measured like metabolites, drugs, lipids, vitamins, minerals, and many types of peptides and proteins. The advantage of this alternative sampling technology is that non-professionals can collect, dry, and mail the samples; the samples can often be stored under room temperature in a dry atmosphere, requiring small amounts of blood. Another promising area is the potential relation between the microbiome and biomarkers that may be measured in feces as well as in blood.

Intrathecal levels of vitamin D and IgG in multiple sclerosis.

BACKGROUND: Intrathecal synthesis of IgG is a hallmark of multiple sclerosis (MS). Vitamin D may modulate B-cell function and dampen the synthesis of IgG. OBJECTIVE: To investigate the relation between vitamin D levels in cerebrospinal fluid and serum and intrathecal synthesis of IgG. METHODS: 25-hydroxyvitamin D (25(OH)D) and IgG were assessed in cerebrospinal fluid and serum in 40 patients with MS. RESULTS: There was no significant correlation between the IgG index and 25(OH)D levels in cerebrospinal fluid or serum. The levels of 25(OH)D in cerebrospinal fluid and serum did not differ between patients with and without intrathecal synthesis of IgG. There was a non-significant trend towards a positive correlation between the concentrations of 25(OH)D and IgG in the cerebrospinal fluid, but not in serum. CONCLUSION: Physiological variation in vitamin D does not exert a major impact on intrathecal synthesis of IgG in MS.

Bile retinoids imprint intestinal CD103+ dendritic cells with the ability to generate gut-tropic T cells.

Small intestinal lamina propria (SI-LP) CD103(+) dendritic cells (DCs) are imprinted with an ability to metabolize vitamin A (retinol), a property underlying their enhanced capacity to induce the gut-homing receptors CC chemokine receptor-9 and α4ß7 on responding T cells. In this study, we demonstrate that imprinting of CD103(+) DCs is itself critically dependent on vitamin A and occurs locally within the small intestine (SI). The major vitamin A metabolite retinoic acid (RA) induced retinol-metabolizing activity in DCs both in vitro and in vivo, suggesting a direct role for RA in this process. Consistent with this, SI-LP CD103(+) DCs constitutively received RA signals in vivo at significantly higher levels than did colonic CD103(+) DCs. Remarkably, SI CD103(+) DCs remained imprinted in mice depleted of dietary but not of systemic retinol. We found that bile contained high levels of retinol, induced RA receptor-dependent retinol-metabolizing activity in bone marrow-derived DCs, and imprinted these cells with the ability to generate gut-tropic T cells. Taken together, these results suggest a novel and unexpected role for bile in SI-LP CD103(+) DC imprinting.

Stability of whole blood and plasma ascorbic acid.

The objective of this study was to evaluate the impact of pre-analytical factors on the short and long term stability of ascorbic acid (AA), the main form of vitamin C in whole blood and plasma. The effects of various anticoagulants, acidification, storage temperature and time were tested. A recently developed fast and sensitive HPLC method was used to measure AA levels. AA baseline values observed in heparin plasma were significantly higher than values observed in EDTA, citrate and Stabilyte plasma, as well as in serum. pH and temperature were identified as additional critical pre-analytical factors during the short, medium and long term handling and storage. Thus, assessment of reliable and accurate AA status in biological samples demonstrates to be highly dependent on whether the initial conditions during sample handling are controlled. In conclusion, heparin tubes should be used for blood sample collection. As AA is rapidly degraded, sample collection should be followed by immediate centrifugation and plasma acidification. To avoid further degradation during sample handling, samples should be stored at -70 degrees C without delay and analyzed within 80 days.

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9-cis retinoic acid only detected after all-trans-retinoic acid levels are super-elevated experimentally.

Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [1998] Nature 395:398-402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [1998] J. Chromatogr. A 828:451-460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all-trans retinoic acid (at-RA), the ligand for RARs, and relate this to the expression of several retinoid-synthesizing enzymes. However, we do not detect 9-cis-retinoic acid (9-cis-RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at-RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9-cis-RA potentially is present and demonstrates that at-RA predominates over 9-cis-RA by at least 30- to 180-fold in different spinal cord regions.

Quantitative assessment of retinoid signaling pathways in the developing eye and retina of the chicken embryo.

Retinoid signaling has been implicated as an important regulator of retinal development and differentiation. We have used state of the art high-pressure liquid chromatography to identify and quantitate biologically active retinoids, immunohistochemistry to localize the retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 (RALDH2), and nucleic acid assays to quantitate and localize retinoid receptor gene transcripts in the developing eye and retina of the chicken. Our results demonstrate spatial distinctions in retinoid synthesis and signaling that may be related to laminar differentiation in the developing retina. Retinoic acids (RAs) and their precursor retinols (ROHs) are the predominant retinoids in the developing eye. All-trans-RA and all-trans-3,4-didehydro-RA are present in the neuroepithelium in approximately equal amounts from early stages of neurogenesis until shortly before hatching. The retinoid X receptor (RXR) ligand 9-cis-RA is undetectable at all stages; if present, it cannot exceed a small percentage of the total RA content. RAs are not detected in the pigment epithelium. All-trans-ROH is present in the neuroepithelium and pigment epithelium, whereas all-trans-3,4-didehydro-ROH is detected only in the pigment epithelium and/or the choroid and sclera. RALDH2 immunoreactivity is intense in the choroid, low or absent in the pigment epithelium, and moderate in the neuroepithelium, where it is highest in the outer layers. Transcripts of all five chicken retinoid receptor genes are present in the neural retina and eye throughout development. During the period of neurogenesis, at least three of the receptors (RAR gamma, RXR gamma, RXRalpha), exhibit dynamic patterns of differential localization within the depths of the neural retina.

Qualitative and quantitative liquid chromatographic determination of natural retinoids in biological samples.

Liquid chromatography continues to be the preferred method for determining retinoids in biological samples. The highly unstable nature of retinoids and the real possibility of artifacts or erroneous results have led to the development of rapid and highly automated protocols for retinoid extraction, separation and detection. Due to strong light absorbance in the ultraviolet region, UV detectors still predominate although mass spectrometric detection is gaining increased popularity. This paper reviews recent advances and provides major guidelines for using liquid chromatography to identify and quantify retinoids in biological samples.

Identification of endogenous retinoids, enzymes, binding proteins, and receptors during early postimplantation development in mouse: important role of retinal dehydrogenase type 2 in synthesis of all-trans-retinoic acid.

Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos.

New HPLC evidence on endogenous tauret in retina and pigment epithelium.

This investigation was improve the separation for tauret (retinylidene taurine) and to compare its content in the retina under dark and light adaptation. To prevent tauret hydrolysis, retinal samples were quickly frozen and lyophilized. Methanol extracts of dried retina and pigment epithelium from both dark- or light-adapted frogs, Rana ridibunda, were injected onto HPLC. Synthetic standard tauret appeared at 4.7 min after the solvent front. At the same time, an endogenous substance was eluted from the mixed retinal and pigment epithelial samples. The UV spectra of this endogenous compound matched with the spectra of synthetic tauret obtained under identical conditions, with lambda(max) = 446 nm at peak. We conclude that the HPLC system used permitted full separation of tauret from the methanol extracts of the retina and pigment epithelium. TLC and further HPLC analysis have shown that tauret quantities were several times higher in the retina and pigment epithelium of the frogs adapted to dark compared with those light-adapted (about 4 h under 1000 1x illumination). Tauret based vitamin A transport is probably involved in other systems as well, where along with its other known beneficial effects taurine probably is necessary to facilitate vitamin A transport.

Evidence for an essential role of megalin in transepithelial transport of retinol.

Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.

On-line solid-phase extraction and isocratic separation of retinoic acid isomers in microbore column switching system.

On-line solid-phase extraction coupled with micro-HPLC by column switching is an ideal technique for the analysis of retinoic acid in serum or plasma. The advantages are mainly contributed to an automated sample workup and low detection limits. On-line processing of the sample ensures minimal losses and full light protection during the entire procedure. Critical steps such as evaporation, extraction, and multiple transfers are avoided. Furthermore, the precision of highly automated methods is generally better than manual methods. We have successfully coupled a 2.1-mm I.D. analytical column with a 2.1-mm extraction column. This setup allows for large amounts of supernatant to be injected onto precolumns for concentration and cleanup. By means of column switching, this concentrate is transferred to the microcolumn with a highly reduced volume. The reduced diameter of the analytical column and the on-line solid-phase extraction allow for the fully automated quantification of as little as 100 fmol all-trans-retinoic acid in human serum. The detection limits obtained with these column switching techniques can compete with LC-MS. This new micro-HPLC method will be useful for the quantitation of endogenous retinoic acid metabolites, which are present at very low concentrations in biological material. Furthermore, more sensitive methods might also lead to the discovery of hitherto unknown retinoic acid metabolites. The combination of on-line SPE and micro-HPLC has, to our knowledge, not been used previously for retinoic acid analysis. The development of isocratic separation methods for retinoic acid isomers made this possible.

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection.

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.

Secretion of N-(4-hydroxyphenyl) retinamide-retinol-binding protein from liver parenchymal cells: evidence for reduced affinity of the complex for transthyretin.

The synthetic retinoid 4-HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4-HPR on the secretion of retinol-RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A-deficient diet or a normal diet supplemented with 4-HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron-derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph-derived [3H]retinol peaked after about 2 hr, whereas 4-HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4-HPR inhibits secretion of retinol-RBP from the liver. Therefore, we decided to study the effect of 4-HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4-HPR were found to induce the secretion of RBP. The medium from cells treated with 4-HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4-HPR. When the medium from cells incubated with either 4-HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4-HPR had a considerably reduced affinity for TTR. We conclude that 4-HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4-HPR-RBP complex has a reduced affinity for TTR. This observation may explain the 4-HPR-induced reduction of plasma retinol and RBP observed in in vivo studies.

Quantitative high-performance liquid chromatographic determination of retinoids in human serum using on-line solid-phase extraction and column switching. Determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-all-trans-retinoicacid and 4-oxo-13-cis-retinoic acid.

A fully automated isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, has been developed using on-line solid-phase extraction and a column switching technique allowing clean-up and pre-concentration in a single step. A 500-microliter sample of serum was diluted with 750 microliters of a solution containing 20% acetonitrile and the internal standard 9,10-dimethylanthracene. About 1000 microliters of this mixture was injected on a 20 x 4.6 mm I.D. poly ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak C18, 37-53 microns, 300 A particles. Proteins and very polar compounds were washed out to waste, from the pre-column, with 0.05% trifluoroacetic acid (TFA)-acetonitrile (8.5:1.5, v/v). More than 200 aliquots of diluted serum could be injected on this pre-column before elevated back-pressure enforces replacement. Components retained on the pre-column were backflushed to the analytical column for separation and detection at 360 nm. Baseline separation was achieved using a single 250 x 4.6 mm I.D. Suplex pKb-100 column and a mobile phase containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium acetate-glacial acetic acid. A total time of analysis of less than 30 min, including sample preparation, was achieved. Recoveries were in the range of 79-86%. The limit of detection was 1-7 ng/ml serum and the precision, in the concentration range 20-1000 ng/ml, was between 1.3 and 4.5% for all five compounds. The method was applied for the analysis of human serum after oral administration of 60 mg Roaccutan. The method is well suited for pharmacological studies, while the endogenous levels of some retinoic acid isomers are below the limit of quantitation.