Abstract: Polymethoxylated flavones (PMFs) and flavanone glycosides (FGs) were analyzed in hand-pressed juice and the peeled fruit of 'Sainampueng' tangerines ( Citrus reticulata Blanco cv. Sainampueng) grown in northern Thailand. The tangerines were collected from a citrus cluster of small orchard farmers and were cultivated as either agrochemical-based (AB), agrochemical-safe (AS), or organic grown fruits. Juice samples were also measured on contents of carotenoids, ascorbic acid, and tocopherols. The peel-deriving PMFs tangeretin, nobiletin, and sinensetin were found with high concentrations in juice as a result of simple squeezing, whereas amounts of those PMFs were negligibly low in peeled tangerine fruit. In contrast, the mean concentrations of the FGs narirutin, hesperidin, and didymin were several fold higher in peeled fruit than in tangerine juice and significantly higher in organic than AS and AB tangerines. Narirutin and hesperidin in juice from organic produces as well as narirutin in juice from AS produces were significantly higher than respective mean concentrations in juice from AB produces. beta-Cryptroxanthin was the predominant carotenoid beside zeaxanthin, lutein, lycopene, and beta-carotene in tangerine juice. Ascorbic acid concentrations were not predicted by the type of cultivation, whereas alpha-tocopherol was significantly higher in juice from organic than AS produces. In summary, hand-pressed juice of C. reticulata Blanco cv. Sainampueng serves as a rich source of PMFs, FGs, carotenoids, and antioxidants: 4-5 tangerine fruits ( approximately 80 g of each fruit) giving one glass of 200 mL hand-pressed juice would provide more than 5 mg of nobiletin and tangeretin and 36 mg of hesperidin, narirutin, and didymin, as well as 30 mg of ascorbic acid, >1 mg of provitamin A active beta-cryptoxanthin, and 200 microg of alpha-tocopherol.
Abstract: Photodynamic therapy (PDT) is a promising antitumor treatment strategy. However, effectiveness of PDT is limited due to an initiation of rescue responses in tumor cells, including the induction of heme oxygenase-1 (HO-1). Furthermore, the main sources of free radical production in PDT-induced oxidative stress are not clear. Here, human melanoma cells were loaded with the photosensitizer 5-aminolevulinic acid and exposed to non-thermal light of 420-800 nm at different doses. It was shown that inhibition of HO-1 activity by zinc protoporphyrin IX increased PDT-induced cytotoxicity in a dose-dependent manner. Interestingly, the cytotoxic effects were not diminished by the simultaneous application of the iron chelator desferrioxamine. Importantly, PDT together with non-toxic doses of hemin increased the number of dead cells. From these results can be concluded that heme but not iron act as the main source of free radicals in PDT treatment. This is supported by the fact that during PDT ferritin is readily up-regulated, able to bind excess iron formed by the HO-1 action. The combined treatment of photosensitizers with HO-1 inhibitors might increase the effectiveness of PDT in tumor treatment.
Abstract: The aim of this article is to emphasize the importance of malnutrition and micronutrient deficiencies in Alzheimer's disease and discuss recent supplementation trials. Alzheimer's disease (AD) is a devastating neurodegenerative disease with increasing socio-economic impact. It leads to cognitive decline over the years, finally resulting in brain atrophy and gradually destroying a person's ability to learn, reason, make judgments, and communicate. Most of the cases are sporadic and risk factors evolve. There is evidence that malnutrition, oxidative stress, and homocysteine-related vitamins play a role in the pathogenesis of AD. A plethora of epidemiologic studies have explored the associations between nutrients and AD. In addition, more and more data from recent trials are evolving to analyze the impact of micronutrient supplementation in AD and incipient AD concerning B vitamin status and antioxidants. Available data do not support definitive conclusions regarding specific recommendations on micronutrient supplementation for the prevention or treatment of AD; however, more data from prospective trials are needed. Approaches with multiple nutritional components might be promising.
Abstract: In the present study, the effects of various conventional shelling methods (oil-bath roasting, direct steam roasting, drying, and open pan roasting) as well as a novel "Flores" hand-cracking method on the levels of bioactive compounds of cashew nut kernels were investigated. The raw cashew nut kernels were found to possess appreciable levels of certain bioactive compounds such as beta-carotene (9.57 microg/100 g of DM), lutein (30.29 microg/100 g of DM), zeaxanthin (0.56 microg/100 g of DM), alpha-tocopherol (0.29 mg/100 g of DM), gamma-tocopherol (1.10 mg/100 g of DM), thiamin (1.08 mg/100 g of DM), stearic acid (4.96 g/100 g of DM), oleic acid (21.87 g/100 g of DM), and linoleic acid (5.55 g/100 g of DM). All of the conventional shelling methods including oil-bath roasting, steam roasting, drying, and open pan roasting revealed a significant reduction, whereas the Flores hand-cracking method exhibited similar levels of carotenoids, thiamin, and unsaturated fatty acids in cashew nuts when compared to raw unprocessed samples.
Abstract: Many non-surgical tumor treatments induce reactive oxygen species (ROS) which result in cell damage. This study investigated the impact of ROS induction on the expression of adhesion molecules and whether alpha-tocopherol pre-treatment could have a protective effect. Experimental rat DS-sarcomas were treated with a combination of localized 44 degrees C-hyperthermia, inspiratory hyperoxia and xanthine oxidase which together lead to a pronounced ROS induction. Further animals were pre-treated with alpha-tocopherol. The in vivo expression of E- and N-cadherin, alpha-catenin, integrins alpha v, beta 3 and beta 5 as well as of the integrin dimer alpha v beta 3 was assessed by flow cytometry. The expression of alpha v-, beta 3-integrin, of the alpha v beta 3-integrin dimer and of E-cadherin was significantly reduced by the ROS-inducing treatment. This effect was partially reversible by alpha-tocopherol, indicating that ROS play a role in this process. N-cadherin, alpha-catenin and beta 5-integrin expression were unaffected by ROS. These results indicate that the expression of several adhesion molecules is markedly reduced by ROS and may result in a decrease in the structural stability of tumor tissue. Further studies are needed to clarify the impact of ROS induction on the metastatic behavior of tumors.
Abstract: The efficacy and safety of bioactive compounds depend on a few known and unknown parameters. What is a physiologic dose and how can that dose be defined in cases of bioactive compounds with a poor knowledge of supply and distribution? What safety sets are needed? How can individual aspects such as polymorphisms or differences in absorption be considered? A group of experts tried to answer these and related questions during the 23rd Hohenheim Consensus Meeting at the University of Hohenheim in Stuttgart.
Abstract: Patients with parenteral nutrition depend on an adequate supply of micronutrients, in particular, antioxidant vitamins and cofactors such as selenium. In cases of oxidative stress (eg, chronic inflammation, sepsis, lung distress syndrome, and organ failure), there is a higher need for antioxidants. One of the most important antioxidant vitamins is vitamin E. For very low birth weight infants the plasma level is an indicator for adequate supply and for safety. Safe and effective blood levels are between 23 and 46 micromol/L, maintained with a dose of 2.8 IU/kg body weight (1-2 mg/day). For safety reasons a plasma level of 80 micromol/L should not be exceeded. For adults, 10 IU/day (9.1 mg/day) are recommended. Whether this dose is sufficient to ensure body stores and sufficient antioxidant activity is controversial. If parenteral lipid emulsions are supplied there is an additional need for vitamin E to protect the lipids (polyunsaturated fatty acids) from lipid peroxidation and to deliver additional vitamin E. Dietary guidelines for healthy adults recommend an intake of polyunsaturated fatty acids equal to 10% of total energy and an intake of alpha-tocopherol greater than 0.4 mg/g of polyunsaturated fatty acids. Randomized clinical trials are performed using special formulations of vitamin E solutions because vitamin E is available only in lipid emulsions to protect lipids, but not in an isolated solution for parenteral supply.
Abstract: A close cooperation between medical teams is necessary when calculating the fluid intake of parenterally fed patients. Fluids supplied parenterally, orally and enterally, other infusions, and additional fluid losses (e.g. diarrhea) must be considered. Targeted diagnostic monitoring (volume status) is required in patients with disturbed water or electrolyte balance. Fluid requirements of adults with normal hydration status is approximately 30-40 ml/kg body weight/d, but fluid needs usually increase during fever. Serum electrolyte concentrations should be determined prior to PN, and patients with normal fluid and electrolyte balance should receive intakes follwing standard recommendations with PN. Additional requirements should usually be administered via separate infusion pumps. Concentrated potassium (1 mval/ml) or 20% NaCl solutions should be infused via a central venous catheter. Electrolyte intake should be adjusted according to the results of regular laboratory analyses. Individual determination of electrolyte intake is required when electrolyte balance is initially altered (e.g. due to chronic diarrhea, recurring vomiting, renal insufficiency etc.). Vitamins and trace elements should be generally substituted in PN, unless there are contraindications. The supplementation of vitamins and trace elements is obligatory after a PN of >1 week. A standard dosage of vitamins and trace elements based on current dietary reference intakes for oral feeding is generally recommended unless certain clinical situations require other intakes.
Abstract: Biomarkers and their role in evaluating efficacy and safety were the topic of the 23rd Hohenheim Consensus Meeting at the University of Hohenheim in Stuttgart. Scientists who had published and reviewed scientific and regulatory papers on the topic were invited, among them basic researchers, toxicologists, clinicians, and nutritionists. The participants were presented with 11 questions (in bold font), which were discussed and answered (in italic font) at the workshop, with the aim of summarizing the current state of knowledge on the subject. The explicatory text accompanying the short answers was produced and agreed on after the conference and was backed up by corresponding references.
Abstract: Lycopene is a promising nutritional component for chemoprevention of prostate cancer (PCa). A possibly beneficial role of lycopene in patients diagnosed with benign prostate hyperplasia (BPH), who are at increased risk of developing PCa, has been suggested, although clinical data are lacking. Therefore, this pilot study aimed to investigate the effects of lycopene supplementation in elderly men diagnosed with BPH. A total of 40 patients with histologically proven BPH free of PCa were randomized to receive either lycopene at a dose of 15 mg/d or placebo for 6 mo. The effects of the intervention on carotenoid status, clinical diagnostic markers of prostate proliferation, and symptoms of the disease were assessed. The primary endpoint of the study was the inhibition or reduction of increased serum prostate-specific antigen (PSA) levels. The 6-mo lycopene supplementation decreased PSA levels in men (P < 0.05), whereas there was no change in the placebo group. The plasma lycopene concentration increased in the group taking lycopene (P < 0.0001) but other plasma carotenoids were not affected. Whereas progression of prostate enlargement occurred in the placebo group as assessed by trans-rectal ultrasonography (P < 0.05) and digital rectal examination (P < 0.01), the prostate did not enlarge in the lycopene group. Symptoms of the disease, as assessed via the International Prostate Symptom Score questionnaire, were improved in both groups with a significantly greater effect in men taking lycopene supplements. In conclusion, lycopene inhibited progression of BPH.
Abstract: The term "nutritargeting" in analogy to the term "drug targeting" means targeting nutrients to specific "target" tissues. What is the rationale for this idea? Some tissues obviously are able to accumulate micronutrients selectively and to use them predominantly for specific functions. It has, for instance, been known for a long time that the accumulation of beta-carotene in the skin does not only provide a "golden-yellow" color but considerable antioxidative protection as well. Yet beta-carotene is only one of many antioxidants, which can be detected in the skin. Other carotenoids, for example, lutein and zeaxanthine, are preferentially found in the macula lutea, the so-called yellow spot in the eye. Here, carotenoids are subject to a metabolism typical for that tissue, which cannot be found in other tissues (e.g., formation of meso-zeaxanthine). In addition, they can specifically be absorbed into the macula. In the macula, they protect the retinal pigment epithelial cells against oxidative damage from UV light. Indeed, these two carotenoids can be protective against age-dependent macula degeneration. Another example is the tissues that are particularly rich in vitamin C, for example, the cortex of the suprarenal gland or the lens: here, vitamin C fulfills both antioxidative functions and metabolic ones as it helps in the formation of collagen structures. Approximately 40% of the body's ascorbate is stored in skeletal muscle because this tissue is relatively abundant and its cellular concentration is tenfold higher than the plasma level. Similarly, the intracellular ascorbate concentration in the brain (3 mM) greatly exceeds the level in the extracellular fluid (200-400 muM). The majority of ascorbate is stored in the astroglial cells that are capable of reducing great quantities of DHAA to ascorbate, which then becomes available for release back into the extracellular fluid. Thus, the accumulation of vitamins respectively micronutrients in single tissues is not limited to a pure storage process like the storage of vitamin A in the liver, but is often connected with important and tissue-specific metabolic functions. When single micronutrients are applied for prevention or even intervention in diseases of organs or tissues, they are usually administered in higher doses for a longer period of time. The hope is to accumulate it this way sufficiently in the tissue and to thus be able to ensure the therapeutic success. This procedure, however, leads to a "flooding" of the whole organism with micronutrients and their potential enrichment in tissues which would usually not accumulate the respective micronutrient. Thus, unexpected side effects may occur. An attractive solution to these problems in the future could be to wrap up or apply micronutrients in such a way that they can selectively reach the targeted tissue. For this approach, called "drug targeting" by pharmacologists, one could introduce the analogous expression "nutritargeting" with respect to micronutrients. For such a nutritargeting there are already a lot of examples and developments which show that it is possible to accumulate micronutrients in target tissues while simultaneously circumventing or protecting other tissues. A substantial requirement for the development of "carriers" for nutritargeting is the availability of procedures or specific carriers, which allow the selected nutrients to bypass the main barriers that are encountered when, for example, circumventing the enteral route in the targeting process. The entrance areas for such a targeting are the nasal mucosa, the oral mucosa, the cornea, the skin, or the lung. In the case of enteral application of proteins, the packaging has to resist gastric digestion and the body must be able to absorb the particles through the intestinal mucosa without hydrolyzing the proteins in order for them to reach the systemic circulation. Another field in which nutritargeting may play an important role is the diseases where either systemic absorption is not possible (e.g., malabsorption/maldigestion) or where local deficits occur, which may not or only inadequately be supplied by systemic application.
Abstract: PURPOSE OF REVIEW: Parenteral ascorbic acid has been frequently used to overcome problems of vitamin C deficiency in haemodialysis patients. The benefits of vitamin C supplementation in clinical studies have been controversial and did not consider toxicological aspects. The review summarizes recent findings of the effects of parenteral ascorbic acid and discusses toxicological effects. RECENT FINDINGS: Vitamin C deficiency in haemodialysis patients, which has been frequently described, cannot be improved with oral supplementation due to limited absorption of high dosages. To avoid consequences of vitamin C deficiency, parenteral vitamin C solutions should be administered because this intervention is the only way to guarantee a sufficient supply to the cells. A beneficial consequence of parenteral vitamin C on the recombinant human erythropoietin resistance is an additional therapeutic effect, which contributes to the prevention of iron deficiency anaemia in haemodialysis patients. Thus, large amount of supplemental vitamin C are required for extended periods of time (up to 500 mg 3 times a week). To avoid hyperoxaluria, plasma oxalate levels should be monitored on a regular basis, for example, once a week. SUMMARY: Parenteral administration of ascorbic acid may be an approach that can overcome problems of vitamin C deficiency in haemodialysis patients - in particular problems of iron overload, erythropoetin resistance, and chronic inflammation.
Abstract: Vitamin A and its active metabolite retinoic acid (RA)(1) play a major role in development, differentiation, and support of various tissues and organs of numerous species. To assure the supply of target tissues with vitamin A, long-lasting stores are built in the liver from which retinol can be transported by a specific protein to the peripheral tissues to be metabolized to either RA or reesterified to form intracellular stores. Vitamin A cannot be synthesized de novo by animals and thus has to be taken up from animal food sources or as provitamin A carotenoids, the latter being converted by central cleavage of the molecule to retinal in the intestine. The recent demonstration that the responsible beta-carotene cleaving enzyme beta,beta-carotene 15,15'-monooxygenase (Bcmo1) is also present in other tissues led to numerous investigations on the molecular structure and function of this enzyme in several species, including the fruit fly, chicken, mouse, and also human. Also a second enzyme, beta,beta-carotene-9',10'-monooxygenase (Bcmo2), which cleaves beta-carotene eccentrically to apo-carotenals has been described. Retinal pigment epithelial cells were shown to contain Bcmo1 and to be able to cleave beta-carotene into retinal in vitro, offering a new pathway for vitamin A production in another tissue than the intestine, possibly explaining the more mild vitamin A deficiency symptoms of two human siblings lacking the retinol-binding protein for the transport of hepatic vitamin A to the target tissues. In addition, alternative ways to combat vitamin A deficiency of specific targets by the supplementation with beta-carotene or even molecular therapies seem to be the future.
Abstract: Fortified food claiming to exert a special function are increasingly offered on the market. Antioxidants (vitamin C, E and beta-carotene) are the major compounds in fortified foods. Due to the fact that the fortified vitamins do not exceed the recommended daily allowances within a food there is no risk for the consumer. Even in speculative cases of accumulation of antioxidants from different fortfied food sources there is no real risk. However, fortified food should not be taken to compensate an unbalanced and unhealthy diet. This risk is far more real because fortified food contains only a few antioxidants whereas a balanced diet contains hundreds. Whether the consumer may have a benefit from fortified food has not yet been evaluated with respect to antioxidant vitamins.
Abstract: Carotenoids lutein and zeaxanthin are proposed to protect ocular tissues from free-radical damage that can cause cataract and age-related macular degeneration (AMD). They accumulate selectively in the lens and macular region of the retina. Changes in the retinal pigment epithelium are characteristic in AMD. Efficient uptake is essential to study the intracellular effects of carotenoids in cell cultures. For in vitro experiments carotenoids are often dissolved in organic solvents like tetrahydrofuran (THF), dimethylsulfoxide (DMSO) and n-hexane, but difficulties have been associated with these application methods. Recently, O'Sullivan et al. (SM O'Sullivan et al., Br J Nutr 91 (2004) 757) developed a method whereby carotenoids could be delivered to cultured cells without the cytotoxic side effects often observed when organic solvents are used. We modified this method and investigated the effects of different carotenoid-formulations (ethanol/Tween40, methanol/tween40 and acetone/Tween40) on the uptake of lutein and zeaxanthin by differentiated ARPE-19 cells, cell viability and the expression of the "stress" gene HO-1, which is easily induced by a range of stimuli including chemical and physical agents. Micelle formulations prepared with ethanol/Tween40 resulted in the lowest LDH release, the highest carotenoid uptake and the lowest stress response (changes in HO-1 mRNA expression).
Abstract: OBJECTIVE: Update of the Hohenheim consensus on monosodium glutamate from 1997: Summary and evaluation of recent knowledge with respect to physiology and safety of monosodium glutamate. DESIGN: Experts from a range of relevant disciplines received and considered a series of questions related to aspects of the topic. SETTING: University of Hohenheim, Stuttgart, Germany. METHOD: The experts met and discussed the questions and arrived at a consensus. CONCLUSION: Total intake of glutamate from food in European countries is generally stable and ranged from 5 to 12 g/day (free: ca. 1 g, protein-bound: ca. 10 g, added as flavor: ca. 0.4 g). L-Glutamate (GLU) from all sources is mainly used as energy fuel in enterocytes. A maximum intake of 6.000 [corrected] mg/kg body weight is regarded as safe. The general use of glutamate salts (monosodium-L-glutamate and others) as food additive can, thus, be regarded as harmless for the whole population. Even in unphysiologically high doses GLU will not trespass into fetal circulation. Further research work should, however, be done concerning the effects of high doses of a bolus supply at presence of an impaired blood brain barrier function. In situations with decreased appetite (e.g., elderly persons) palatability can be improved by low dose use of monosodium-L-glutamate.
Abstract: BACKGROUND: An adequate supply of vitamin A during pregnancy and breastfeeding plays an important role for development of foetus and neonate, especially in lung development and function. AIM OF THE STUDY: Aim of this pilot study was to analyze vitamin A and beta-carotene status and to investigate the contribution of nutrition to the vitamin A and beta-carotene supply in mother-infant pairs of gemini or births within short birth intervals. METHODS: Twenty-nine volunteers aged between 21 and 36 years were evaluated for 48 h after delivery. During this time frame a food frequency protocol considering 3 months retrospective was obtained from all participants. In order to establish overall supply retinol and beta-carotene levels were determined in maternal plasma, cord blood and colostrum via HPLC analysis. RESULTS: Regardless of the high to moderate socio-economic background, 27.6% of participants showed plasma retinol levels below 1.4 micromol/l which can be taken as borderline deficiency. In addition, 46.4% showed retinol intake <66% of RDA and 50.0% did not consume liver at all although liver contributes as a main source for preformed retinol. Despite high total carotenoid intake of 6.9 +/- 3.6 mg/d, 20.7% of mothers showed plasma levels <0.5 micromol/l beta-carotene. Retinol and beta-carotene levels were highly significantly correlated between maternal plasma versus cord blood and colostrum. In addition, significantly lower levels were found in cord blood (31.2 +/- 13.0% (retinol), 4.1 +/- 1.4% (beta-carotene) compared with maternal plasma. CONCLUSIONS: Despite the fact that vitamin A and beta-carotene rich food is generally available, risk groups for low vitamin A supply exist in the western world.
Abstract: Many epidemiological studies predict a role for homocysteine (HCys) in cardiovascular disease occurrence, progression, and risk factors. In vitro studies demonstrated that HCys is an atherogenic determinant that promotes oxidant stress, inflammation, endothelial dysfunction and cell proliferation. This study originally attempted to examine the mechanism by which exposure of endothelial cells to HCys (0-250 microM) initiates inflammatory reaction and oxidative stress, by (i) investigating whether physiological and pathophysiological concentrations of HCys exhibit a prooxidative activity in vitro, (ii) examining the interaction of monocyte adhesion (Mono Mac 6) to monolayers of human microvascular endothelial cells (HMEC-1) exposed to different HCys concentrations, and (iii) examining if adherent monocytes increase reactive oxygen species either in endothelial cells or in monocytes themselves. However, our results demonstrate that HCys had neither prooxidative nor cytotoxic effects on endothelial cells. Only a moderate time- and concentration-dependent increase in monocyte adhesion up to 28.3 +/- 5.5% was achieved relative to control after 4 h of HCys stimulation. This effect was accompanied by an increased VCAM and ICAM-1 mRNA expression. This "proinflammatory" effect appeared also when HMEC-1 cells were incubated with cysteine or glutathione at the concentration range 0-250 microM, demonstrating a non-specific rather than a specific HCys effect. In addition, adherent monocytes did not increase ROS formation neither in endothelial cells nor in monocytes themselves, indicating no direct or indirect cytotoxic or prooxidative effects of HCys.
Abstract: Vitamin A is essential for growth and differentiation of a number of cells and tissues. Notably during pregnancy and throughout the breastfeeding period, vitamin A has an important role in the healthy development of the fetus and the newborn, with lung development and maturation being particularly important. The German Nutrition Society (DGE) recommends a 40% increase in vitamin A intake for pregnant women and a 90% increase for breastfeeding women. However, pregnant women or those considering becoming pregnant are generally advised to avoid the intake of vitamin A rich liver and liver foods, based upon unsupported scientific findings. As a result, the provitamin A carotenoid beta-carotene remains their essential source of vitamin A. Basic sources of provitamin A are orange and dark green vegetables, followed by fortified beverages which represent between 20% and 40% of the daily supply. The average intake of beta-carotene in Germany is about 1.5-2 mg a day. Assuming a vitamin A conversion rate for beta-carotene for juices of 4:1, and fruit and vegetables between 12:1 and 26:1; the total vitamin A contribution from beta-carotene intake represents 10-15% of the RDA. The American Pediatrics Association cites vitamin A as one of the most critical vitamins during pregnancy and the breastfeeding period, especially in terms of lung function and maturation. If the vitamin A supply of the mother is inadequate, her supply to the fetus will also be inadequate, as will later be her milk. These inadequacies cannot be compensated by postnatal supplementation. A clinical study in pregnant women with short birth intervals or multiple births showed that almost 1/3 of the women had plasma retinol levels below 1.4 micromol/l corresponding to a borderline deficiency. Despite the fact that vitamin A and beta-carotene rich food is generally available, risk groups for low vitamin A supply exist in the western world. It is therefore highly critical to restrict the beta-carotene supply from diet, particularly from sources of beta-carotene with high consumer acceptance such as fortified juices (e.g. "ACE juices") or dietary supplements (e.g. multivitamins for pregnant women). For the part of the population unable to meet vitamin A requirements according to the DACH recommendations, sufficient intake of beta-carotene may be crucial to help improve and maintain adequate vitamin A status and prevention of developmental disorders. At this time it has to be urgently advised against restricting the beta-carotene supply or putting warning labels on beta-carotene fortified products. It is, however, highly recommended to improve the available data on nutrient intakes in Germany, especially for pregnant and breastfeeding women. For them, recommendations to be aware of potential nutrient intake inadequacies might prove useful.
Abstract: PURPOSE OF REVIEW: For more than 20 years polyphenols, food-derived bioactive compounds in fruits and vegetables, are claimed to help prevent cancer, degenerative diseases and chronic and acute inflammation. Modern methods in cell and molecular biology allow us to understand the interactions of different polyphenols with basic mechanisms of inflammatory response. This review summarizes recent papers dealing with the effect of polyphenols on modulators of the inflammatory cascade. RECENT FINDINGS: The majority of papers deal with the effects of different antioxidants on the redox sensitive transcription factor nuclear factor kappaB, inducible nitric oxide synthase expression and cyclooxygenase inhibition. Understanding the regulatory steps and the multiple potential actions of polyphenols within the inflammatory cascade may help to define special polyphenols to be used for prevention and intervention in clinical conditions of inflammation. The relation between formation of advanced glycation end products as a consequence of high glucose and the activation of defence mechanisms through polyphenols opens new fields in the prevention and treatment of oxidative stress and hyperglycemia. SUMMARY: Polyphenols are promising compounds that may help to control oxidative stress and consequently inflammatory response. To date, however, clinical studies are missing and should be carried out with specific polyphenols.
Abstract: Based on the observation that 5-aminolevulinic acid (ALA) induces the expression of heme oxygenase-1 (HO-1) in cultured melanoma cells, the role of HO-1 on the effectiveness of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) was examined. Transcriptional activation of the HO-1 gene is considered to be an adaptive response to oxidative and cellular stress and confers a protective capacity against cell and tissue injury, which could affect the responsiveness to ALA-PDT. A time-dependent accumulation (0-16 h) of protoporphyrin IX (PPIX) within melanoma cells was seen after incubation with ALA (0.5 mM ALA). Over the same time interval, a significant increase (up to 25-fold) in HO-1 protein expression was observed. Thus, the production and degradation of PPIX (via heme by HO-1) were simultaneously enhanced, leading to a reduced intracellular concentration of the photodynamically active substance PPIX. Diminishing HO-1 activity by the HO-1 inhibitor tin protoporphyrin IX (SnPPIX) significantly enhanced the formation of PPIX up to 1.8 fold. A further strong increase in HO-1 protein expression (up to 128-fold) was seen after ALA-PDT treatment. Induction of HO-1 is an essential step in the 'rescue response' of tumor cells. The pharmacological inhibition of HO-1 activity by SnPPIX leads to a considerable increase in the sensitivity of tumor cells to ALA-PDT treatment. At low radiation doses (0.42 J/cm(2)), the percentages of death cells increased significantly from 7.3+/-1.3% to 43.7+/-6.4%. This effect could be further intensified by cellular depletion of HO-1 mRNA by siRNA. The combination of pharmacological inactivation of HO-1 with gene silencing led to an increase in the death rate of up to 54.1+/-8.6%. The results presented indicate that HO-1 can play a protective role against ALA-PDT mediated cytotoxicity so that a specific inhibition of HO-1 activity and/or expression might be used to increase the efficacy of ALA-based photodynamic therapy.
Abstract: This review presents the rationale for the therapeutic use of antioxidants in treating critically ill patients; it is not a systematic review of the clinical evidence that has been assessed recently by others. Clinical and nonclinical evidence is presented to support the notion that natural antioxidants are of therapeutic value in treating cardiovascular shock. Oxidative stress is a major promoter and mediator of the systemic inflammatory response. The microcirculation is particularly susceptible to oxidative stress that causes hemodynamic instability, leading to multiple organ failure due to systemic inflammatory response syndrome. Vitamin C is the antioxidant used experimentally to demonstrate oxidative stress as a key pathophysiologic factor in septic shock. Pharmacologic studies reveal that vitamin C (as ascorbate), at supraphysiologic doses, significantly affects the bioavailability of nitric oxide during acute inflammation, including inhibiting nitric oxide synthetase induction. Parenteral high-dose vitamin C inhibits endotoxin-induced endothelial dysfunction and vasohyporeactivity in humans and reverses sepsis-induced suppression of microcirculatory control in rodents. In severe burn injury, in both animals and patients, parenteral high-dose vitamin C significantly reduces resuscitation fluid volumes. Therefore, a significant body of pharmacologic evidence and sound preliminary clinical evidence supports the biological feasibility of using the exemplary antioxidant, vitamin C, in the treatment of the critically ill.
Abstract: Many tumor treatment modalities such as ionizing radiation or some chemotherapy induce reactive oxygen species (ROS) resulting in therapeutic cell damage. The aim of this study was to analyze whether such ROS induction may affect the mechanical stability of solid tumor tissue by degradation of the extracellular matrix proteins or by a loss of cell adhesion molecules. Additionally, the protective impact of alpha-tocopherol treatment on these processes was studied. Experimental DS-sarcomas in rats were treated with a combination of localized 44 degrees C hyperthermia, inspiratory hyperoxia and xanthine oxidase in order to induce pronounced oxidative stress. A second group of animals were pretreated with alpha-tocopherol. The in vivo expression of E- and N-cadherin, alpha-catenin, integrins alphav, beta3 and beta5 as well as the expression of the integrin dimer alphavbeta3 were assessed by flow cytometry. The activity of the matrix metalloproteinases MMP-2 and -9 and the activity of the urokinase-type plasminogen activator (uPA) were determined by zymography. The expression of E-cadherin, the alphav-, beta3-integrin and the alphavbeta3-integrin dimer was significantly reduced by ROS induction, an effect which was at least partially reversible by alpha-tocopherol. N-cadherin, alpha-catenin and the beta5-integrin expression was not affected by ROS. In addition, MMP-2, MMP-9 and uPA activities were markedly reduced immediately after hyperthermia. Whereas 24 h later the effects on MMP-2 and -9 were no longer evident, for uPA the impact of oxidative stress became even more pronounced at this time. These results show that several processes responsible for the structural stability of the tumor tissue are affected by therapeutic ROS generation. Changes in some of the markers assessed suggested a decrease in tissue stability upon ROS induction, whereas others indicated changes which could lead to a more stable tumor cell cluster. Depending on the individual tumor entity ROS may therefore influence the mechanical stability of solid tumors and by this affect metastatic behavior.
Abstract: Epidemiological studies show that dietary products rich in carotenoids delay the progression of age-related macular degeneration. Experimental evidence from cellular studies on the antioxidant actions of carotenoids in the retinal pigment epithelium is still, however, fragmentary. The present study examined the uptake and protective potential of dietary carotenoids from tomato on the human retinal pigment epithelial cell line ARPE-19. ARPE-19 cells were incubated in medium supplemented with tomato extract containing high levels of beta-carotene, lycopene and traces of lutein. The cellular uptake of carotenoids was analysed by reverse-phase HPLC. Oxidative stress was induced by treatment with 1 mm-H2O2. Nitrotyrosine was detected by immunocytochemistry, and oxidised proteins (protein carbonyls) were measured by a quantitative ELISA method. Lipid peroxidation was assessed by quantifying thiobarbituric acid reactive substances. ARPE-19 cells preferentially accumulated lutein and beta-carotene rather than lycopene. Nitrotyrosine formation was considerably reduced in cells incubated with tomato extract compared with controls after H2O2 treatment. Protein carbonyls were reduced by 30 % (P = 0.015), and the formation of thiobarbituric acid-reactive substances was reduced by 140 % (P = 0.003) in cells incubated with tomato extract. The present study provides the experimental evidence for protective effects of dietary tomatoes rich in carotenoids on oxidative stress in the retinal pigment epithelium.
Abstract: Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation, and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells on 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-alpha (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis-inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1-fold increased cell survival rate on ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT and may therefore be contraindicated in patients undergoing tumor treatment.
Abstract: Populations living in endemic malaria areas maybe exposed simultaneously to DDT and malaria infection. DDT may impair status of vitamins, which are implicated in the immunity and pathophysiology of malaria. To explore possible interactions, DDT residues, retinol, alpha-tocopherol, beta-carotene and cholesterol were measured in plasma samples of malaria-infected pregnant women (cases, n=50) and age matched malaria-free controls (n=58). DDT residues were found in all samples: mean (sd) total DDT levels of 29.7 and 32.7 ng/ml in cases and controls, respectively. Mean (sd) p,p'-DDT was higher in the controls than the cases (13.5 vs. 9.5 ng/ml, p=0.006). Malaria infection was associated with lower mean (sd) plasma retinol (0.69 vs. 1.23 micromol/L) and cholesterol (2.62 vs. 3.48 mmol/L) compared to controls (p<0.001). Mean (sd) plasma alpha-tocopherol (7.65 vs. 15.58 micromol/L) and alpha-tocopherol/cholesterol ratio (2.3 vs. 6.7 micromol/L/mmol/L) were significantly lower among the controls (p<0.001). Mean (sd) plasma beta-carotene was low (<0.3 micromol/L) in both groups, but higher among malaria cases (0.19 vs. 0.15 micromol/L). Plasma retinol among the controls showed highly significant positive correlations with individual DDT compounds, particularly with p,p'-DDT (r=0.51, p<0.001). Plasma alpha-tocopherol and beta-carotene seemed not to be affected by DDT residues.
Abstract: BACKGROUND: Bioavailability of fat-soluble vitamins from conventional oral supplements is insufficient in some conditions in which fat digestion and absorption are chronically impaired (e. g. cystic fibrosis). AIM OF THE STUDY: We used a water-soluble form of fatsoluble vitamin E (AQUANOVA solubilisate) to create a nutritional supplement (NS) in the form of vitaminized gummi bears (with micellised water-soluble alpha-tocopheryl acetate (100 IU) and 400 mg crystalline vitamin C). We assessed the bioavailability of the NS in comparison to conventional preparations. METHODS: The trial consisted of three study days (d0: NS sucked; d10: NS swallowed; d20: reference products swallowed). A total of 14 subjects (6 male/8 female), aged 25.3 (22.7-35.3) years, BMI 24.3 (19.0-31.7) kg/m(2) participated in the study. They had blood samples drawn after fasting for >or=12 hours and then 1, 5, 15, 30, 60, 120, 180, 240, 300 and 320 minutes after ingesting the vitamins. HPLC and a colorimetric method were used to determine vitamin E and vitamin C, respectively. Areas under the curve (AUC(0-320min)) and maximum increases in plasma concentrations (Delta concentration) were calculated to assess bioavailability. RESULTS: The AUCs(0-320min) of alpha-tocopherol from d0 were significantly larger (p = 0.016) when compared to d20. Moreover, the maximum increase in alpha-tocopherol plasma concentrations was significantly higher for d0 (p = 0.023) and d10 (p = 0.002) when compared to d20. CONCLUSIONS: Short-term bioavailability of AQUANOVA micellised fat-soluble vitamin E from our NS was significantly higher than from regular supplements. The NS will now be tested for its clinical efficacy in a randomized double-blind controlled intervention trial with CF patients.
Abstract: The 22 supersetnd Hohenheim Consensus Workshop took place in at the University of Stuttgart-Hohenheim. The subject of this conference was vitamin C and its role in the treatment of endothelial dysfunction. Scientists, who had published and reviewed scientific and regulatory papers on that topic were invited, among them basic researchers, toxicologists, clinicians and nutritionists. The participants were presented with eleven questions, which were discussed and answered at the workshop, with the aim of summarising the current state of knowledge. The explicatory text accompanying the short answers was produced and agreed on after the conference and was backed up by corresponding references. The therapeutic relevance of administration of the physiological antioxidant vitamin C in high parenteral doses in Endothelial Dependent Pathophysiological Conditions (EDPC) was discussed. Endothelial dysfunction is defined as including disturbed endothelial dependant relaxation of resistance vessels, breakdown of the microvascular endothelial barrier and/or loss of anti-adhesive function. It occurs in severe burn injury, intoxications, acute hyperglycemia, sepsis, trauma, and ischemic-reperfusion tissue injury and is induced by oxidative stress. Reduced plasma ascorbate levels are a hallmark of oxidative stress and occur in severe burns, sepsis, severe trauma, intoxication, chemotherapy/radiotherapy and organ transplantation. Vitamin C directly enhances the activity of nitric oxide synthase, the acyl CoA oxidase system and inhibits the actions of proinflammatory lipids. There is experimental evidence that parenteral high-dose vitamin C restores endothelial function in sepsis. In vitro, supraphysiological concentrations (> 1mM) of ascorbate restore nitric oxide bioavailability and endothelial function. Only parenterally, can enough vitamin C be administered to combat oxidative stress. There is no evidence that parenteral vitamin C exerts prooxidant effects in humans. Theoretical concerns in relation to competitive interactions between vitamin C and glucose cellular uptake are probably only relevant for oxidised vitamin C (dehydroascorbate).
Abstract: The relative bioavailability of coenzyme Q10 (CoQ10) is markedly influenced by its delivery systems. The aim of this study was to compare four standard CoQ10 supplements available on the market with a novel solubilizate formulation of CoQ10 (Solu Q10). Pharmacokinetic parameters were assessed in 54 healthy volunteers after single and multiple intakes of 60 mg CoQ10 over a time period of 14 days. Solubilizates showed earlier flooding compared with oily dispersions and crystalline CoQ10, resulting in significantly elevated area under the curve between 0 and 4 h (P<0.01 solubilizates versus crystalline). The difference in the pharmacokinetic parameters of maximum plasma concentration, time to reach the peak plasma concentration and area under the curve between 0 and 12 h was not statistically significant between formulations. Long-term supplementation resulted in significantly higher plasma levels (P<0.01) for all formulations, with Solu Q10 performing best. Intracellular CoQ10 levels measured in buccal mucosa cells were increased (P<0.05) in response to supplementation when starting within the physiological range. In summary, solubilizates were clearly superior to oily dispersions and crystalline CoQ10 in their overall bioavailability, with the best absorption characteristics seen for the novel Solu Q10 solubilizate.
Abstract: PURPOSE OF REVIEW: The impact of vitamin C on oxidative stress-related diseases is moderate because of its limited oral bioavailability and rapid clearance. Parenteral administration can increase the benefit of vitamin C supplementation as is evident in critically ill patients. The aim here is to assess recent evidence of the clinical benefit and underlying effects of parenteral vitamin C in conditions of oxidative stress. RECENT FINDINGS: In critically ill patients and after severe burns, the rapid restoration of depleted ascorbate levels with high-dose parenteral vitamin C may reduce circulatory shock, fluid requirements and oedema. SUMMARY: Oxidative stress is associated with reduced ascorbate levels. Ascorbate is particularly effective in protecting the vascular endothelium, which is especially vulnerable to oxidative stress. The restoration of ascorbate levels may have therapeutic effects in diseases involving oxidative stress. The rapid replenishment of ascorbate is of special clinical significance in critically ill patients who experience drastic reductions in ascorbate levels, which may be a causal factor in the development of circulatory shock. Supraphysiological levels of ascorbate, which can only be achieved by the parenteral and not by the oral administration of vitamin C, may facilitate the restoration of vascular function in the critically ill patient.
Abstract: Although strong evidence is available suggesting that microenvironmental parameters play a role in lymphogenic or hematogenic metastasis, the underlying mechanisms are still unclear and further investigations of this topic are needed. For such a study however, an appropriate model of metastasis for in vivo analysis of this process would be required. An in vivo model of a solid tumor (rat DS sarcoma) has therefore been established to enable monitoring of the steps involved in tumor metastasis. Rat DS sarcoma cells were transfected with the pTracer-SV40 plasmid, containing the super-GFP and zeocin resistance genes. DS sarcoma cells showing high and stable expression of GFP (DSGFP cells) were selected by cell sorting and in vitro culturing with zeocin. To establish in vivo growth, DSGFP cells were subsequently injected intraperitoneally (i.p.) without additional selection by zeocin and GFP expression was monitored by flow cytometry. Using DSGFP ascites cells, solid tumors were implanted subcutaneously into the hind foot dorsum of rats. The expression of GFP was assayed by fluorescence microscopy. The detection of circulating DSGFP sarcoma cells in the blood was performed using the PCR technique. GFP expression in vitro was stable for more than 40 passages. Cell sorting, however, did not enable selection of a DSGFP cell population with a higher long-term stable GFP expression. After i.p. cell implantation, GFP expression in DSGFP ascites cells was maintained over at least 19 passages. Solid tumors implanted by injection of DSGFP ascites cells showed stable GFP expression. The growth rate of solid DSGFP sarcomas was slightly slower compared to that of non-transfected cell lines. The detection limit for circulating DS sarcoma cells in blood was 100 DSGFP cells/ml whole rat blood. Micrometastases in loco-regional lymph nodes, lung and liver were detectable by immunohistology and real-time PCR. This in vivo model showing stable expression of GFP could be useful for analyzing the mechanisms of metastasis, particularly where micrometastases or circulating tumor cells are to be identified.
Abstract: Dermatitis is a group of highly pruritic chronic inflammatory skin diseases which represents a major public-health problem worldwide. The prevalence of dermatitis has increased in recent years affecting up to 20% of the general population. Acute skin lesions are characterized by extensive degrees of intercellular edema of the epidermis (spongiosis) and a marked perivenular inflammatory cell infiltrate in the dermis. Keratinocytes within eczematous lesions exhibit a modified expression of proinflammatory cytokines, chemokines and cell-surface molecules. The pathophysiological puzzle of dermatitis is far from being elucidated completely, but skin infiltration of activated memory/effector T cells are thought to play the pivotal role in the pathogeneses. The aim of this study was the set-up of organotypic models mimicking the symptoms of eczematous dermatitis to provide a tool for therapeutic research in vitro. Therefore activated T cells (ATs) were integrated in organotypic skin and epidermis equivalents (SE, EE). These models enabled the reproduction of several clinical hallmarks of eczematous dermatitis: (1) T cells induce keratinocyte apoptosis, which leads to a reduced expression of the adhesion molecule E-cadherin (E-cad) and disruption of the epidermal barrier. (2) Expression of intercellular adhesion molecule-1 (ICAM-1) allows the attachment of leukocytes to epidermal cells. (3) Upregulation of neurotrophin-4 (NT-4) in the epidermis is thought to mediate pruritus in lesions by supporting nerve outgrowth. (4) Elevated levels of pro-inflammatory cytokines (IL-1alpha and IL-6) and chemokines (IL-8, IP-10, TARC, MCP-1, RANTES and eotaxin) amplify the inflammatory response and lead to an influx of secondary immunocells into the skin. The therapeutics dexamethasone and FK506 markedly reduce cytokines/chemokines production and epidermal damaging in these models. These data underline that activated memory/effector T cells induce eczematous changes in this HaCaT cell based organotypic skin equivalent. Furthermore it can be concluded that these models make it possible to investigate targets of therapeutics in skin.
Abstract: BACKGROUND:Iron is an essential micronutrient but also a major catalyst of oxidative and inflammatory reactions. OBJECTIVE:To evaluate the potential utility of selected biomarkers in blood or urine to indicate in vivo oxidative or inflammatory response to oral iron intake at pharmacological doses. METHODS:Three healthy volunteers provided morning, fasting samples of blood and urine on up to 13 study days--3 before, 7 during and 3 following a 7-consecutive-day period of receiving 120 mg of iron per day as ferrous sulfate in commercially available syrup. A series of 23 biomarkers were measured on each collection of biological fluids to monitor iron-responsive changes in biomarkers related to hematological or iron status, inflammation and in vivo oxidation. RESULTS:Among the inflammatory biomarkers measured, white blood cells, serum CRP and urinary neopterin showed no response to iron dosing. Only circulating interleukin-4 (IL-4) and TNF-alpha had abnormal responses with a time association to the oral iron intake. Among the oxidative biomarkers, expression of blood superoxide dismutase (SOD), hemoxygenase-1, catalase as well as circulating thiobarbituric acid reactive substances (TBARS), total oxidative capacity and carbonyl proteins were stable in response to iron exposure. Only urinary TBARS, 8-hydroxy-2-desoxyguanosine and isoprostanes evidenced consistent or suggestive responses to ingestion of the iron challenge. Serum hepcidin concentration increased dramatically in all three subjects after only the first 120 mg dose of iron, and remained elevated even 9 days after cessation of the iron intervention. CONCLUSIONS:Most of the candidate biomarkers show very limited promise as response-indicators to oral iron dosing at the 120 mg dosages or lower, but circulating IL-4, TNF-alpha as well as urinary TBARS, 8-hydroxy-2-desoxyguanosine and isoprostanes showed potential utility as reliable indicators of oxidative and inflammatory response to oral ferrous sulfate.
Abstract: PURPOSE: Vitamin A is essential for vision. The key step in the vitamin A biosynthetic pathway is the oxidative cleavage of beta-carotene into retinal by the enzyme beta,beta-carotene-15,15'-monooxygenase (BCO). The purpose of the study was to investigate beta-carotene metabolism and its effects on BCO expression in the human retinal pigment epithelial (RPE) cell line D407. METHODS: BCO mRNA and protein expression were analyzed by real-time quantitative PCR and Western blot analysis, respectively. BCO activity was assayed in protein extracts isolated from D407 cells. The conversion of beta-carotene to retinoids was determined by measuring retinol levels in D407 cells on beta-carotene supplementation. RESULTS: By RT-PCR, BCO mRNA was detected in D407 cells, bovine RPE, and retina. Western blot analyses revealed the presence of BCO at the protein level in D407 cells. Exogenous beta-carotene application to D407 cells resulted in a concentration (75% at 0.5 microM and 96% at 5 microM; P < 0.05)- and time (127% at 2 hours and 97% at 4 hours in 5 microM beta-carotene, P < 0.05)-dependent upregulation of BCO mRNA expression. Application of exogenous retinoic acid downregulated BCO mRNA levels at higher concentrations (1 microM; -96%, P < 0.0005) and upregulated it at a lower concentration (0.01 microM; 399%, P < 0.005). The RAR-a-specific antagonist upregulated BCO expression by sixfold (P < 0.005). Tests for enzymatic activity demonstrated that the mRNA upregulation resulted in enzymatically active BCO protein (7.3 ng all-trans-retinal/h per milligram of protein). Furthermore, D407 cells took up beta-carotene in a time-dependent manner and converted it to retinol. CONCLUSIONS: The results suggest that BCO is expressed in the RPE and that beta-carotene can be metabolized into retinol. beta-Carotene cleavage in the RPE may be an alternative pathway that would ensure the retinoid supply of photoreceptor cells.
Abstract: Several non-surgical tumor treatment modalities produce their cytotoxic activity by generating reactive oxygen species (ROS). Anti-oxidative enzymes such as superoxide dismutase (SOD) or exogenously supplied antioxidants may therefore reduce the efficacy of these treatments. The aim of the present study was to analyze the impact of (i) inhibiting SOD using 2-methoxyestradiol (2-ME), or (ii) application of alpha-tocopherol, on the cellular damage induced by hyperthermia (HT) in experimental tumors. DS-sarcoma cells grew either in culture or as solid tumors subcutaneously implanted in rats. In vitro, DS-cells were incubated with 2-ME, and cell proliferation, ROS formation, lipid peroxidation and apoptosis were measured. In vivo, DS-sarcomas were treated with a ROS-generating hyperthermia combined with 2-ME or alpha-tocopherol application. Inhibition of SOD by 2-ME in vitro induced pronounced oxidative injury resulting in reduced proliferation. In vivo, ROS-generating hyperthermia led to local tumor control in 23% of the animals. The additional inhibition of SOD by 2-ME increased the control rate by approximately 50%. Application of alpha-tocopherol was found to have no effect on local tumor control, either in combination with ROS-generating hyperthermia or when 2-ME was additionally applied. Inhibition of SOD during ROS-generating hyperthermia results in pronounced cell injury and an improved local tumor control whereas exogenously applied vitamin E seems not to have an impact on oxidative stress.
Abstract: The effect of non-selective (theophylline) inhibition of cyclic AMP breakdown on norepinephrine stimulated lipolysis rate was investigated in subcutaneous adipose tissue of obese subjects. In addition, changes in interstitial glucose and lactate concentration were assessed by means of the microdialysis technique. The interaction of endogenous released insulin and theophylline on adipocyte metabolism was determined. Theophylline and norepinephrine alone increased glycerol outflow significantly. When both agents were perfused in combination, interstitial glycerol concentration increased further. The enhanced glycerol level due to theophylline application was slightly decreased by insulin. In the presence of theophylline, extracellular glucose concentration increased, in contrast to the catecholamine. Norepinephrine decreased interstitial glucose level. When both drugs were added in combination, the level of interstitial glucose increased to about 1 mM, greater than with theophylline alone. With each intervention, lactate was synthesized. Local adipose tissue blood flow was increased by theophylline and theophylline plus norepinephrine. In conclusion, post-receptor mechanisms increased norepinephrine maximal stimulated lipolysis rate in subcutaneous adipose tissue. Glucose uptake was inhibited by the non-specific inhibitor of phosphodiesterase. The effect of insulin on inhibition of lipolysis was modest but sustained in the presence of high theophylline (10(-4) M) concentration. Phosphodiesterase activity may be relatively low in obese subjects in comparison with lean subjects. In lean subjects theophylline caused a transient reversal of the antilipolytic effect of insulin.
Abstract: OBJECTIVE: To investigate the involvement of alpha1-adrenoceptors in the sympathetic regulation of glucose uptake in human adipocytes. RESEARCH METHODS AND PROCEDURES: Twenty-four severely obese subjects participated in this study. The microdialysis technique was used to determine interstitial glucose concentration after stimulation of abdominal subcutaneous adipose tissue with the alpha1-agonist norfenefrine, the alpha1,2beta-agonist norepinephrine, and both agents in combination with the alpha1-antagonist urapidil. The effect of beta-adrenoceptor stimulation was assessed by orciprenaline. Changes in local blood flow were determined using the ethanol escape technique. RESULTS: Both norfenefrine and norepinephrine induced a concentration-dependent decrease of interstitial glucose concentration, with a greater decrease observed with norepinephrine. Preperfusion of adipose tissue with urapidil inhibited glucose decrease. The inhibition was overcome with high concentrations of norfenefrine and norepinephrine, respectively. Both adrenergic agents induced tachyphylaxia. Urapidil enhanced extracellular glucose level at high concentration. Blood flow decreased in the presence of norfenefrine and norepinephrine but increased in response to urapidil. The accelerated blood flow due to urapidil was counteracted by norepinephrine and norfenefrine. Orciprenaline decreased interstitial glucose concentration and increased nutritive blood flow. The observed changes in blood flow induced by adrenergic agents were not related to glucose uptake. DISCUSSION: The stimulatory effect of the sympathetic nerves on glucose uptake in subcutaneous adipose tissue appears to be mediated by the alpha1-adrenoceptor. Norepinephrine enhances glucose entry into adipocytes independently of insulin action. In obese subjects with insulin resistance, the alpha1-adrenergic receptor may provide an important alternative pathway for glucose uptake.
Abstract: OBJECTIVE: We studied alterations of circulating antioxidant nutrients in patients with classic dengue fever in the tropical lowlands of Guatemala. METHODS: In nine patients with dengue fever and 12 healthy Guatemalan control subjects, we assessed plasma concentrations of retinol, alpha-tocopherol, beta-carotene, glutathione, taurine, thiobarbituric acid-reactive species, and total antioxidant status. Control subjects were assessed twice within 48 h to account for day-to-day variations. Febrile patients with dengue fever were examined on the day of admission to the hospital, at discharge after defervescence (approximately 5 d after admission), and 7 d thereafter. RESULTS: In patients with dengue fever, increases in plasma concentrations of retinol and beta-carotene were seen, whereas decreases were observed for glutathione and total antioxidant status. As compared with the reference group, patients with dengue fever had lower retinol concentrations in the acute phase of the disease and lower glutathione concentrations 7 d after discharge. Further, thiobarbituric acid-reactive species levels were higher in the dengue fever patients as analyzed by unpaired t test. CONCLUSION: Using dengue fever as a model for the metabolic response to an acute, self-limited tropical viral infection, the present findings suggest slight turbulence of the antioxidant system that may be a response to or a consequence of the viral inflammatory process.
Abstract: OBJECTIVE: High-dose vitamin C therapy might mediate beneficial clinical effects by counteracting reactive oxygen species. However, concerns are raised whether this approach might provoke diametrical (ie pro-oxidative) effects. The objective was to determine ascorbyl free radical (AFR) concentrations and potential variables of pro-oxidative damage. DESIGN: Crossover study; six healthy males received daily infusions of 750 or 7500 mg vitamin C for six consecutive days. Fasting concentrations of vitamin C and AFR were determined daily. On day 1, concentrations of vitamin C and AFR were measured at 0.25, 0.5, 1, 2, 4 and 8 h post infusion. Plasma concentrations of thiobarbituric acid-reactive substances (TBARS), tocopherol and urine concentrations of 8-oxoguanosine were determined on days 1 and 6. RESULTS: Kinetic studies on day 1 showed that concentrations of vitamin C and AFR displayed parallel dose- and time-dependent kinetics and elimination was highly efficient. Vitamin C and AFR fasting concentrations on days 2-6 were slightly above the baseline, suggesting new, stable steady states. TBARS decreased in both groups, whereas tocopherol and 8-oxoguanosine concentrations remained unchanged. CONCLUSION: Kinetics of AFR largely depend on plasma vitamin C concentrations and AFR is eliminated efficiently. Our data do not support induction of pro-oxidative effects in healthy volunteers given intravenous high-dose vitamin C. SPONSORSHIP: Pascoe Pharmazeutische Präparate GmbH, Giessen, Germany.
Abstract: The anti-cancer potential of the natural estrogen metabolite 2-methoxyestradiol is associated with microtubuli interaction, anti-angiogenetic effects and inhibition of superoxide dismutase leading to apoptosis. The effectors of apoptotic signaling through 2-methoxyestradiol, however, are cell type-dependent. We investigated the effect of 2-methoxyestradiol on several events associated with apoptosis in rat DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to mitochondria was identified as an initial apoptotic event that was accompanied by a decrease in mitochondrial transmembrane potential and the formation of reactive oxygen species (ROS) followed by mitochondrial release of apoptosis inducing factor and endonuclease G. In addition, 2-methoxyestradiol treatment caused upregulation of death receptor ligands FasL and TNFalpha and induced caspase-8 activation. The pan caspase inhibitor Z-VAD-FMK did not suppress apoptotic cell death, however, indicating that the major pro-apoptotic effect of 2-methoxyestradiol is mediated by a caspase-independent mechanism. Furthermore, ROS do not seem to play a pivotal role in the toxic/apoptotic effect of 2-methoxyestradiol in DS-sarcoma cells because supplementation with various antioxidants provided only limit protection. Colony formation was not affected by antioxidants. Therefore, in DS-sarcoma cells, the breakdown of mitochondrial integrity with the subsequent release of mitochondrial nucleases is the main factor in 2-methoxyestradiol mediated cell death.
Abstract: The adherence of program participants to periodic vitamin A capsule (VAC) supplementation among children aged 1-5 years (n = 677) in Central Java, Indonesia was assessed. Fourteen villages from five sub-districts and one ward from one sub-district in Central Java were included in the study to represent rural and suburban areas. All questions about demographic factors, socioeconomic conditions, current dietary practice and healthcare-seeking attitudes for common childhood illnesses, previous breastfeeding experience, their knowledge about vitamin A and adherence to the VAC program after capsule distribution (two periods in 2000) were asked. Caretakers with limited knowledge about the health benefits of vitamin A, households with more than one preschool child, and households with older children (> 36 months) were associated with a decreased likelihood of regular participation in the program with odds ratios of 0.38, 0.55, and 0.26, respectively (p < 0.01). The percentage of caretakers who utilized community health centers, village health posts or midwives' practices in rural areas, was significantly higher (86.6 per cent, p < 0.001) than in suburban areas (62.8 per cent). Living in a rural location was associated with an increased adherence to participate in the program regularly with an odds ratio of 2.02 (p < 0.01). In conclusion, nutritional education and periodic social marketing should be re-emphasized and other potential delivery channels, such as private healthcare practices, could also contribute to an increase adherence of supplementation program.
Abstract: The so-called Mediterranean diet is claimed to be preventive with respect to diet-related degenerative diseases. It is suggested that the preventive effect especially with respect to diabetes might be the low glycemic index of this kind of diet. However, nutrients with a high GI are more frequently consumed in the Mediterranean diet than in other European countries. The major difference seems to be the higher amount of fiber and a higher intake of unsaturated fat together with a higher intake of fruits and vegetables. Based on recent studies from the nurses health and physicians health study, a diet which is similar to the Mediterranean diet, physical exercise and a BMI < 25 protects from the development of diabetes type 2.
Abstract: BACKGROUND AND AIMS: Liver surgery usually involves ischemia and reperfusion (I/R) which results in oxidative stress and cell damage. The administration of antioxidants should diminish or prevent this damage. The purpose of this study was to investigate the effect of the antioxidant vitamin E on I/R injury. METHODS: We carried out a placebo-controlled double-blind study on 68 patients undergoing elective, tumor-related, partial liver resection. 47 patients were qualified for the per protocol population based evaluation. The patients were randomly assigned to two groups. The day before surgery one group received three infusions containing vitamin E (600 IU=540 mg vitamin E emulsion). The other group received three infusions of placebo. RESULTS: Length of stay in the intensive care unit (ICU) was significantly shorter in the verum group than in the placebo group (P<0.05). There were signs of improvement for AUC AST (P<0.05), ALT and GLDH in the verum group after surgery. Serum vitamin E concentration increased after administration of vitamin E infusion and declined in both treatment groups after surgery (P<0.01). In the verum group vitamin E deficiency was prevented while vitamin E concentration remained low in the placebo group (P<0.01). CONCLUSIONS: The findings from this study indicate that preoperative administration of vitamin E is safe and that this treatment may have beneficial effects by reducing the impact of I/R injury in liver surgery.
Abstract: Vitamin A and its active metabolites are important for growth and differentiation of a variety of cells, mainly in mucosa-associated epithelia, where they exhibit a wide spectrum of activities. Vitamin A, stored as retinyl esters (REs), is delivered from liver stores into the bloodstream as retinol bound to retinol binding protein. This process is regulated homeostatically, ending up in a more or less constant plasma retinol level. In situations of a high vitamin A demand (e.g., inflammation, diseases, prenatal period), this supply can be insufficient because of delayed production of retinol binding protein, leading to local deficiencies and impairment of structure and function in the respective tissues. This delay may be overcome by cellular RE stores. Several cell types, including buccal mucosa cells, can take up RE. Retinyl palmitate is taken up when it is applied topically to either metaplastically mutated rat vaginal epithelium (as a gel) or to human meta- and dysplastic bronchial epithelia (via inhalation) that have a vitamin A deficiency. In rats and humans, the modified epithelia can be normalized, at least in part. In conclusion, topically applied retinyl esters may be a promising therapy for local retinol deficiencies and may reverse the morphological alterations of the epithelium in tissues that are vitamin A deficient.
Abstract: This study assesses the initiation and duration of exclusive/almost exclusive breastfeeding (Ex/AEx-BF) versus partial breastfeeding (P-BF) and its relationship to infant growth and maternal body mass index (BMI) in Ethiopian infants up to 12 months of age (Tigray n = 471; Gonder n =596). Initiation of breastfeeding within 1 hr after birth was 1.7 times more common in Tigray. In Gonder 19% of the mothers started breastfeeding on the third day of delivery and consequently, a significant higher proportion of newborns were offered prelacteal feeds (P<0.001). At six months, 52.2% and 61.5% of mothers in Tigray and Gonder respectively practised Ex/AEx-BF. Perceived lack of breastmilk was a major factor for offering complementary foods before six months. More than 25% of infants in both regions were Ex/AEx-BF up to the eighth month and 16.4% and 15.7% of infants in Tigray and Gonder were still Ex/AEx-BF at the end of the first year. Growth faltered before six months of age but less frequently in Ex/AEx-BF compared to P-BF infants. Malnutrition became obvious after eight months in both groups. Malnourished mothers (BMI < 18.5) offering mixed feeding to their malnourished children (<-2 z-score) below six months of age was higher in both regions compared to the Ex/AEx-BF group (in Gonder 25% vs 5.9%; in Tigray 42.9% vs 33.3%). Nutrition education to raise awareness of mothers regarding initiation of exclusive breastfeeding directly after birth, the value of colostrum and avoidance of pre-postlacteal feeds, needs to be implemented. A new health package to be implemented could be a proper vehicle to reach the rural population, which doesn't have access to health services. The relationship between infant growth, mode of feeding and mothers nutritional status should be further investigated, particularly in populations with a high prevalence of maternal and infant nutrition and where long term breastfeeding is practiced.
Abstract: The measurement of vitamin A (VA) and iron status is very important in the assessment of nutritional deficiencies. The objective of this research was to develop a sandwich ELISA technique for the simultaneous measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein (CRP) as indicators for VA and iron status. The inclusion of CRP as marker of infection allows for more accurate interpretation of VA and iron status. This is accomplished in a 30-microL serum or plasma sample using an ELISA with different capture and detection antibodies and different dilutions of the sample. Commercially available clinical serum controls were used for calibration purposes. The developed assays were compared to commercially available traditional tests. Regression coefficients comparing both assays were better than 0.84 (P < 0.001). Using a limited sample set, the sandwich ELISA assay produced very similar specificity and sensitivity compared to traditional methods when common cutoff values were applied. Intra- and interassay variability was between 5 and 14% for all tests. The cost of the materials for all 5 measurements decreases to less than $1/sample if a large number of samples is analyzed. Due to the low cost, high throughput, and comparability to traditional tests, this procedure has several advantages for assessing VA and iron status in population surveys.
Abstract: BACKGROUND: Patients on dietary, weight-reducing treatment commonly are advised against alcohol consumption. In light of the widespread use of alcoholic beverages and the well-established benefits of light to moderate alcohol consumption in risk reduction, a revision of dietary treatment recommendations may be warranted. OBJECTIVE: To investigate whether daily consumption of moderate amounts of alcohol influences the effectiveness of an energy-restricted diet in overweight and obese subjects. DESIGN: A prospective randomized clinical trial was conducted, with a 3-months intervention period and two isocaloric dietary regimens containing 6.3 MJ (1500 kcal) each, one with 10% of energy from white wine and one with 10% of energy from grape juice. The trial was performed in obese subjects being recruited from the Obesity Outpatient Clinic at the University Hospital, Ulm, who all habitually consumed moderate amounts of alcohol. Out of 87 patients, 49 were eligible to participate and 40 completed the study (age 48.1+/-11.4 y, BMI 34.2+/-6.4 kg/m(2)). Efficacy parameters were body weight and biomarkers of good health. RESULTS: All subjects achieved significant body weight reduction. Weight loss in the grape juice group and white wine group was 3.75+/-0.46 and 4.73+/-0.53 kg, respectively. Percent body fat, waist circumference, blood pressure, blood glucose, insulin, triglycerides, and cholesterol were reduced. The antioxidant status was unchanged, as were liver enzyme activities and other safety parameters. There were no significant differences between the groups. CONCLUSIONS: An energy-restricted diet is effective in overweight and obese subjects used to drinking moderate amounts of alcohol. A diet with 10% of energy derived from white wine is as effective as an isocaloric diet with 10% of energy derived from grape juice.
Abstract: BACKGROUND: Little is known about age- and disease-related changes in prooxidant and antioxidant systems in patients with cystic fibrosis (CF). OBJECTIVE: We investigated changes in antioxidant concentrations and oxidative stress in plasma, buccal mucosal cells (BMCs), and breath condensate in patients with CF in relation to age and disease progression. DESIGN: We recruited 22 patients with CF as well as 35 healthy control subjects and conducted a cross-sectional study by dividing the participants into 4 age groups (<6 y, 6-11 y, 12-17 y, > or =18 y). We collected fasting blood samples, BMCs, and breath condensate. Carotenoids, alpha-tocopherol, vitamin C, protein carbonyls, thiobarbituric acid-reactive substances, and F(2)alpha-isoprostane were assessed. RESULTS: In patients with CF, plasma vitamin C concentrations, plasma and BMC alpha-tocopherol concentrations, and forced expiratory volume in 1 s (percentage predicted) decreased significantly with age. Plasma beta-carotene, beta-cryptoxanthin, and total lycopene were significantly lower in patients than in control subjects in all age groups. Furthermore, alpha-tocopherol and vitamin C plasma concentrations as well as alpha-tocopherol concentrations in BMCs were significantly lower in CF patients > or =18 y old, whereas all indicators of oxidative stress assessed were significantly higher than those same indicators in control subjects. CONCLUSIONS: Adult patients with CF in particular showed distinct vitamin deficits and elevated indicators of oxidative stress in plasma, BMCs, and breath condensate along with a progression of clinical status. We suggest that early in life dietary habits should be improved and that innovative supplementation strategies should be applied to optimize the antioxidant status of patients with CF.
Abstract: Retinoids have been reported to produce regressions in metaplastic changes of the mucosal epithelium. In order to define the role of these micronutrients in the prevention of squamous metaplasia of the oral cavity, it is necessary to measure their uptake in target tissues such as the buccal mucosal epithelium. We demonstrated in a trial that retinyl palmitate applied topically via a toothpaste is taken up by buccal mucosal cells in young healthy volunteers. In the randomised, parallel-designed, placebo-controlled and double-blind trial, forty volunteers divided in two groups cleaned their teeth either with a placebo toothpaste or a retinyl palmitate-containing toothpaste (1 mg/g) for 56 d. Buccal mucosal cells samples were taken from the healthy volunteers during the retinyl palmitate application and the following wash-out phase to determine the concentration of retinyl palmitate and retinol by HPLC. Supplementary blood samples were taken from the volunteers on days 0 and 56 to investigate changes in plasma retinyl palmitate and retinol concentrations. Results from only thirty participants (sixteen placebo and fourteen treated subjects) were used in the statistical evaluation as the remaining sample results were spoiled by a technical defect during the HPLC analysis. A significant (P<0.05) uptake of retinyl palmitate in buccal mucosal cells after 7 d and a significant (P<0.05) increase of plasma retinol after 17 d was demonstrated in our present study. The uptake of retinyl palmitate and the following hydrolysis to retinol led to an enrichment of vitamin A in buccal mucosal cells.
Abstract: OBJECTIVE: The aim of this study was to evaluate the effectiveness of a widespread vitamin A supplementation programme and to describe indicators of compliance with the programme in Indonesia. DESIGN: Prospective cohort study. Children's anthropometric data were gathered at baseline (June 2000) and 4 months later (2 months after supplementation in August 2000). Serum retinol, haemoglobin, ferritin, alpha1-acid glycoprotein and C-reactive protein were measured at baseline and at follow-up. Caregivers of the children were interviewed using a questionnaire. SETTING: Semi-urban and rural areas of Semarang district, Central Java, Indonesia. SUBJECTS: Children aged 1-5 years. RESULTS: After the supplementation, the proportion of children with a low concentration of retinol decreased in recipients from 18.8 to 14.5%. However, in non-recipients, the prevalence of vitamin A deficiency increased from 31.9 to 37.5%, this prevalence being significantly higher than in recipients. A significant decrease occurred in the proportion of recipients with low ferritin (26.5 to 16.2%) and haemoglobin (25.7 to 15.3%), whereas the proportions did not show a significant change after supplementation for non-recipients. Modest linear growth was detected in recipients after supplementation but there was no effect on ponderal growth. The coverage rate of the supplementation in the study areas was 60%. There was an association between compliance of the caregivers and their knowledge about the potential benefit of vitamin A supplementation, the place where sick children were taken and age of the children. CONCLUSIONS: The vitamin A supplementation programme marginally decreased the proportion of vitamin A deficiency and had a marginal effect on the nutritional status of recipients. More than one micronutrient intervention is needed to increase the effectiveness of the supplementation programme. To increase compliance and coverage in the supplementation programme, nutrition communication and private healthcare practices need to be included in the programme.
Abstract: A lower intake of carotenoids is associated with an increased risk of colorectal cancer. In order to take advantage of the chemopreventive properties of carotenoids, it is necessary to determine carotenoid concentration at the target tissue. As early stages in the adenoma-carcinoma sequence of colorectal cancer might be susceptible to chemoprevention, we sought to determine carotenoid concentrations in biopsies from colorectal adenomas. METHODS: Biopsies from colorectal adenomas and non-involved mucosa were taken from seven patients. For controls, biopsies were obtained from the ascending and descending colon of patients without polyps (n = 5). Concentration of carotenoids (alpha-, beta-carotene, lutein, lycopene, zeaxanthin, beta-cryptoxanthin) were determined by optimizing gradient HPLC-analysis. Results are expressed as pmol/microg DNA. RESULTS: Except for alpha-carotene, all carotenoids could reliably be detected in all specimens. In control patients carotenoid concentrations were highest in the ascending colon, being followed by the descending colon and non-involved mucosa from polyp-carriers. In colorectal adenomas all carotenoids were significantly reduced as compared to-non-involved mucosa (beta-carotene: 0.37 vs 0.19, P<0.03; lycopene: 0.34 vs 0.21, P<0.06, beta-cryptoxanthin: 0.14 vs 0.09, P<0.03, zeaxanthin: 0.18 vs 0.09, P<0.02; lutein: 0.18 vs 0.13,P <0.02). CONCLUSION: All carotenoids investigated are reduced in colorectal adenomas, suggesting that mucosal carotenoids could serve as biomarkers for predisposition to colorectal cancer. Moreover, anti-tumor activity exerted by carotenoids is limited due to mucosal depletion. We speculate that supplementation of a larger array of carotenoids might be beneficial for patients with colorectal adenoma.
Abstract: Oxidative stress-related changes in tumors upon localized hyperthermia (HT), 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) and their combination (ALA+HT) were examined after the observation that the antitumor effects of ALA-PDT could be significantly enhanced upon simultaneous application of HT. Rats bearing s.c. DS-sarcomas (0.6-1.0 ml) on the hind foot dorsum were anesthetized and underwent one of the following treatments: (i) ALA-PDT (375 mg/kg 5-ALA i.v.); (ii) localized HT, 43 degrees C for 60 min; (iii) combined ALA-PDT and HT [=ALA+HT]. Appropriate control experiments were also performed. After treatment, tumors were excised and rapidly frozen for later analysis of nitrosative stress (protein nitration), apoptotic events (TUNEL, caspase activation, DNA and RNA fragmentation), expression of heat shock proteins (hsp70 and HO-1), glutathione (GSH) levels and glutathione peroxidase (GPx) activity. Protein nitration was found to increase upon treatment, being especially pronounced in the ALA+HT group, and could partially be related to areas surrounding microvessels. The extent of nitrosative stress also correlated well with the appearance of the markers of apoptosis and the inhibition of in vivo tumor growth as seen in a previous study. GSH levels decreased upon treatment, the reduction being most prominent in the ALA-PDT and ALA+HT groups. GPx activity, however, showed a significant decrease only in the ALA-PDT group. Whereas hsp70 expression increased upon HT, ALA-PDT caused a decrease, and these opposing effects were nullified with ALA+HT. The results obtained point to a number of cellular mechanisms-including effects on cellular defense mechanisms and an abrogation of the heat shock defense mechanism-that may interact to achieve the potentiated tumor response rate seen in vivo upon combined treatment.
Abstract: BACKGROUND: Extensive exercise may promote the formation of reactive oxygen species and subsequently contribute to tissue damage. A compound which can protect cells and tissues is vitamin E. The vitamin E radical, formed during the radical scavenging process, can be reduced by vitamin C resulting in a higher level of the vitamin C radical (semidehydroascorbate free radical). An increase of the vitamin C radical, however, is assumed to exert prooxidative effects. AIM OF THE STUDY: To elucidate whether supplementation of vitamin E and exhaustive exercise lead to an increase of the vitamin C radical in human plasma. METHODS: A placebo controlled, cross over study with 13 male volunteers was carried out. After an 8 day supplementation period with 500 I.U. D-alpha-tocopherol, the subjects performed two exhaustive treadmill runs. Blood samples were collected at rest, 0, 0.25, 1, 3, 24 and 48 h after exercise. Serum was separated and concentrations of D-alpha-tocopherol and ascorbic acid were determined by HPLC. Vitamin C radical levels in plasma were assessed by electron paramagnetic resonance (EPR). RESULTS: Vitamin E and C both showed a tendency to decrease between 3 h and 24 h after exercise. Vitamin C radical level remained stable during the whole period. Neither vitamin E supplementation nor exercise had any influence on the plasma concentration of the vitamin C radical. CONCLUSIONS: Vitamin E supplementation under conditions of mild oxidative stress does not result in an increased vitamin C radical concentration.
Abstract: To elucidate the scientific state of the art with respect to the role of nutrition in skin ageing, nine experts from different disciplines discussed the role of micronutrients on 'oxidative and premature skin ageing'. In this 25th Hohenheim Consensus Meeting, 13 questions were discussed and, based on published valid data, answered by mutual agreement. The consensus answers achieved during the meeting are justified by a scientific background text. The importance of in vitro and in vivo models regarding oxidative and premature skin ageing was critically evaluated. There was a special focus on prevention and intervention of skin ageing with nutrition. Finally, the paper summarizes the scientific background from different areas related to oxidative and premature skin ageing.
Abstract: OBJECTIVE: The aim of this study was to consider the risk of micronutrient deficiencies and approaches for intervention, and to summarize existing knowledge and identify areas of ignorance. DESIGN: Experts from a range of relevant disciplines received and considered a series of questions related to aspects of the topic. INTERVENTION: The experts met and discussed the questions and arrived at a consensus. CONCLUSION: Though healthy balanced diet is available for the general European population, a few defined groups are at risk of micronutrient deficiencies. In addition, the intake of specific micronutrients such as iron, folic acid, vitamin D and vitamin B12 are often marginal. To overcome these deficiencies, either selected micronutrients or a mixture of different micronutrients might be recommended. However, to define and detect micronutrient deficiencies, specific biomarkers are only available for a few micronutrients (e. g. vitamin D, folic acid, vitamin C, iron). The definition of a risk group, based on scientific data, might be an appropriate way to justify intervention with supplements.
Abstract: Vitamin-A is essential for growth and development of cells and tissues. In its active form, retinoic acid, it controls the regular differentiation as a ligand for retinoic acid receptors (RAR, RXR) and is involved in the integration (gap junction formation) of cell formations [Nature 37 (1994) 528; International Review of Cytology. San Diego Academic Press, 1-31]. Vitamin-A plays a substantial role, especially in the respiratory epithelium and the lung. During moderate vitamin-A-deficiency, the incidence for diseases of the respiratory tract is considerably increased and repeated respiratory infections can be influenced therapeutically by a moderate vitamin-A-supplementation [Aust. Paediatr. J. 22 (1986) 95; Lancet 338 (1991) 67]. In addition to the importance of the vitamin for the lung function, vitamin-A is also responsible for the development of many tissues and cells as well as for the embryonic lung development. Recent studies proved that the control occurs by different expressions of retinoid receptors as well as by time-dependent changes of the vitamin-A-metabolism respectively via cellular vitamin-A-binding proteins (CRBP: cytoplasmatic retinol binding protein; CRABP: cytoplasmatic retinoic acid binding protein).
Abstract: The uptake of alpha-tocopherol and beta-carotene and their antioxidative effect in plasma and buccal mucosal cells after oral application in twelve subjects is demonstrated in our study. The effect on the antioxidative status was evaluated using a modified thiobarbituric acid-reactive substance (TBARS) method. As expected, the supplement of 134.2 mg alpha-tocopherol/d and 25 mg beta-carotene/d for 7 d resulted in a significant increase of alpha-tocopherol and beta-carotene concentration in plasma (P<0.05). In buccal mucosal cells, the concentration of beta-carotene increased after supplementation (P<0.05), whereas the concentration of alpha-tocopherol remained constant. A decrease in TBARS (P<0.05) was found in buccal mucosal cells but not in plasma. In conclusion, an uptake of the supplemented antioxidants was detected in plasma and in buccal mucosal cells. There was significant change in beta-carotene concentration and oxidative stress as measured using a modified TBARS test in buccal mucosal cells, but not in the plasma.
Abstract: Copper (Cu) is an essential trace element with many physiological functions. Homeostatic mechanisms exist to allow Cu to act as a cofactor in enzymatic processes and to prevent accumulation of Cu to toxic levels. The aim of this commentary is to better understand the role of dietary Cu supply in deficiency and under physiological and pathological conditions. The essentiality of Cu can be attributed to its role as a cofactor in a number of enzymes that are involved in the defence against oxidative stress. Cu, however, has a second face, that of a toxic compound as it is observed with accumulating evidence in hepatic, neurodegenerative and cardiovascular diseases. The destructive potential of Cu can be attributed to inherent physico-chemical properties. The main property is its ability to take part in Fenton-like reactions in which the highly reactive and extremely deleterious hydroxyl radical is formed. Diseases caused by dietary Cu overload could be based on a genetic predisposition. Thus, an assessment of risk-groups, such as infants with impaired mechanisms of Cu homeostasis regarding detoxification, is of special interest, as their Cu intake with resuspended formula milk may be very high. This implies the need for reliable diagnostic markers to determine the Cu status. These topics were introduced at the workshop by the participants followed by extensive group discussion. The consensus statements were agreed on by all members. One of the conclusions is that a re-assessment of published data is necessary and future research is required.
Abstract: Based on epidemiological studies it is assumed that meat, especially red meat, enhances risk for cancer, particularly of the colon, breast and prostate. Meat and meat products are important sources of protein, some micronutrients and fat. High fat intake has been blamed for correlation with different diseases, including cancer. Meat protein is reported to contribute to cancer formation. However, meat, including liver, is not only composed of fat and protein, it contains essential nutrients which appear exclusively in meat (vitamin A, vitamin B12) and micronutrients for which meat is the major source because of either high concentrations or better bioavailability (folate, selenium, zinc). In particular, vitamin A, folate and selenium are reported to be cancer-preventive, with respect to colon, breast and prostate cancer. Taken together, meat consists of a few, not clearly defined cancer-promoting and a lot of cancer-protecting factors. The latter can be optimized by a diet containing fruit and vegetables, which contain hundreds of more or less proven bioactive constituents, many of them showing antioxidative and anticarcinogenic effects in vitro.
Abstract: The free radical theory of the aging process is based on the hypothesis that with increasing age, mutations of the mitochondrial DNA will accumulate and will at least lead to a loss of function with subsequent acceleration of cell death. Even if this theory is widely accepted, the reactive-oxygen-species-induced mutations of mitochondrial DNA, the accumulation of mitochondrial DNA and the role of antioxidants are not fully understood. Based on this theory, supplements with unproven mixtures of antioxidants or hormones, such as melatonin with antioxidant properties, are widely recommended and cover a big market. However, we are far away from understanding their specific role and we have to consider that, based on the free radical theory of aging, the balance of antioxidants and prooxidants in both directions is of importance in maintaining the physiological function of both reactive oxygen species and antioxidants.
Abstract: We report in our present pilot study on the uptake of retinyl palmitate and its formation to retinol in buccal mucosal cells. Retinoids are able to change metaplastic lesions in several tissues. Prior to any clinical evaluation it is necessary to measure its uptake in target tissues such as the buccal mucosal epithelium. In this pilot study 12 volunteers creamed the inside of their cheeks lightly with a 0.1% retinyl palmitate containing toothpaste for 10 days followed by a wash out phase. On day 0, 3, 7, 10, 17 and 21, buccal mucosal cell samples were taken from volunteers and the uptake of retinol and retinyl palmitate was determined by HPLC analysis. An uptake of vitamin A was demonstrated in all volunteers. Comparing day 0 to day 3 a significant uptake of retinyl palmitate (p<0.05) was detected and comparing day 3 to 17 and 21 a significant decrease (p<0.05) during growth and differentiation of the mucosal epithelium could be seen. The subsequently formed retinol showed an increase but no statistical significance was determined. - The uptake of retinyl palmitate and the formation to retinol led to an enrichment of vitamin A in buccal mucosal cells. In this way metaplastic alterations of the buccal mucosal epithelium might be prevented or reversed by the application of topical retinyl palmitate.
Abstract: We describe an improved method for the measurement of retinol in dried blood spots (DBS) on filter paper. Retinol in human DBS on filter paper was analyzed by normal phase HPLC after a simple extraction method. Retinol associated with its binding protein was eluted from the paper into aqueous solution facilitated by ultrasonic agitation. Retinol associated with retinol binding protein was denatured with acetonitrile, and then retinol was isolated in a single hexane extract and analyzed directly by HPLC. When analyzing DBS, the individual plasma volume of the spots was calculated by measuring the sodium content or by weighing the blood spots. The described method yielded low intra- and interassay variability (<6%), with sufficient sensitivity (detection limit, 0.1 micromol/L) and good recovery (97% spike). Compared with matching plasma samples, DBS retinol consistently decreased 18-23% during the 1st wk of storage. After 1 wk, retinol remained stable in the blood spots at 23 degrees C for >3 mo. In conclusion, the analysis of retinol in DBS by HPLC is comparable to retinol analysis in serum. The variability of the method was reduced by using sodium concentration to estimate sample volume. Collection of DBS for retinol analysis is appropriate under field conditions, where it is difficult to centrifuge or freeze blood samples.
Abstract: The objective of this preliminary uncontrolled study was twofold: First, to assess the feasibility of retinyl palmitate inhalation and second, to analyze the changes of metaplastic lesions of the respiratory epithelium (metaplasia or dysplasia) following retinyl palmitate inhalation. The response to a daily dose of 18.000 I.U. retinyl palmitate by inhalation over a period of 3 month was assessed in 11 subjects (9 smokers, 2 ex-smokers). Using white-light bronchoscopy combined with autofluorescence bronchoscopy, bronchial biopsies were taken before and after a 3 month-period. The biopsy samples were evaluated blind by a referee lung pathologist. The overall response rate (remission or partial remission) was 56% (95% CI 0.30 0.79; p<0.05). These data suggest that inhalation of retinyl esters could be a promising therapeutical approach for chemoprevention of lung cancer. Vitamin A; chemoprevention; lung cancer; squamous metaplasia; dysplasia; retinoids
Abstract: In an investigation of the antitumor effects of 2-methoxyestradiol (2-ME) in combination with other reactive oxygen generating treatments, 2-ME (0.5 microM) was found to completely inhibit cell proliferation of rat DS-sarcoma cells in vitro, with 71% of cells dying after exposure to 5 microM 2-ME. Concentration-dependent increases in ROS-formation, lipid peroxidation and mitochondrial changes were also observed, and an elevation in caspase-3 activity resulted in DNA fragmentation and apoptosis. Combination of 2-ME with hypoxanthine and xanthine oxidase enhanced in vitro cytotoxicity. In vivo, 2-ME caused a slight inhibition of tumor growth, with no tumors cured. Combination of 2-ME treatment with localized 44 degrees C hyperthermia, respiratory hyperoxia and xanthine oxidase caused a tumor growth delay with 51% of tumors cured. These results suggest that amplifying the levels of reactive oxygen species within tumor tissue with substances such as 2-ME may prove to be a promising strategy for adjuvant treatment of solid tumors.
Abstract: Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.
Abstract: Endogenous antioxidants are decreased in skin and blood during UV exposure. Combined supplementation of beta-carotene, alpha-tocopherol and ascorbic acid in addition to topical sunscreens may help to lower the risk of sunburning. Acute UV erythema with sunburn reaction are the most important factors in conjunction with the cumulative life-long UV dose for inducing skin damage resulting in photoageing and precancerous and cancerous lesions. Therefore, a clinical, randomized, double-blind, parallel group, placebo-controlled study was conducted in healthy young female volunteers (skin type II) investigating the preventive, photoprotective effect of supplementation with Seresis, an antioxidative combination containing both lipid and water-soluble compounds: carotenoids (beta-carotene and lycopene), vitamins C and E, selenium and proanthocyanidins. In this study, the oral administration of Seresis appeared to be well tolerated. The preparation contains antioxidant compounds in quantities occurring at physiological levels and can therefore be used safely over a long period of time. Despite the fact that the assessment of the light sensitivity (minimal erythemal dose, chromametry) of the skin did not show any statistically significant differences between the Seresis and the placebo group, a clear statistical trend, however, could be demonstrated, i.e. Seresis was able to slow down the time of the development and grade of UVB-induced erythema. The primary efficacy parameter matrix metalloproteinases 1 (MMP-1) between treatment and placebo group following UV irradiation showed a significant difference (p < 0.05), which occurred due to the fact that after a 2-week UV irradiation, MMP-1 slightly increased (p < 0.03) in the placebo group and decreased (p < 0.044) in the treated group. The MMP-9 changes showed a clear tendency of decrease in the Seresis group (p < 1.393) and increase (p < 0.048) in the placebo group. These data emphasise that supplementation with Seresis decreases the UV-induced expression of MMP-1 and 9, which might be important in photoprotective processes. From our data, we thus finally draw the conclusion that by the combination of antioxidants, such as in the formulation of Seresis, a selective protection of the skin against irradiation can be achieved. This might be important for future recommendations for immediate suppression of the early phase of UV-induced erythema, that means pharmacological prevention of sunburn reaction as well as subsequent chronic skin damage.
Abstract: BACKGROUND: HFE knockout mice (C57BL/6 x 129/Ola strain) mimic the functional aberrations of human hereditary haemochromatosis (HH) in short-term experiments. The present study investigates functional and morphological long-term changes. METHODS: HFE(o/o), HFE(+/o) and HFE(+/+) mice were maintained on iron-rich and control diets for 2 weeks, 3, 12 and 18 months. Light microscopic tissue iron distribution, pathomorphological alterations, tissue iron content and oxidative stress were analysed in liver, pancreas, spleen, gastrointestinal tract, kidneys and myocardium. Additionally, duodenal 59Fe absorption and 59Fe whole body loss were measured. RESULTS: Iron distribution between organs and microscopic iron deposition in the tissues resembled the patterns described in HH. After 3 months of iron-rich feeding duodenal 59Fe absorption decreased to approximately 15% of iron-adequate controls but remained about twice as high in HFE(o/o) as in HFE(+/+) mice. Hepatic iron concentrations reached only half the values known to induce hepatic fibrosis in rats and humans, while whole body 59Fe loss was about twice as high. Consequently no hepatic fibrosis developed, although massive hepatocellular iron deposition and indication for oxidative stress were observed. CONCLUSION: C57BL/6 x 129/O1a HFE(o/o) mice mimic HH iron distribution and the regulation of intestinal iron absorption after long-term feeding. However, characteristic morphological late changes in untreated HH are not modelled.
Abstract: OBJECTIVE: To consider the relationship between nutrition and aging. To summarize existing knowledge and identify areas of ignorance. DESIGN: Experts from a range of relevant disciplines received and considered a series of questions related to aspects of the topic. SETTING: University of Hohenheim, Stuttgart, Germany. INTERVENTION: The experts met and discussed the questions and arrived at a consensus. CONCLUSION: Many specific conclusions were drawn that support the general view that as we age an inadequate nutrition contributes to the loss of function and the development and progression of disease. Nutritional status is influenced by a range of medical, physiological, psychological, social and situational variables. Adequate nutrition and physical activity are aspects of a health-promoting lifestyle. The encouraging of better nutrition and the taking of exercise is a cost-effective way of decreasing the incidence and progression of age-related disease. The earlier such interventions are introduced the better.
Abstract: Solar radiation is one of the most important environmental stress agents for human skin, causing sunburn, premature skin aging, and skin cancer. Beta-carotene is discussed to protect against photooxidative stress and thus prevent skin damage. Though beta-carotene has been successfully used against photosensitivity in patients with erythropoietic protoporphyria, its beneficial potential in normal skin is still uncertain. A number of experimental studies indicate protective effects of beta-carotene against acute and chronic manifestations of skin photodamage. However, most clinical studies have failed to convincingly demonstrate its beneficial effects so far. Nevertheless, intake of oral beta-carotene supplements before sun exposure has been recommended on a population-wide basis. Recent studies on skin cells in culture have revealed that beta-carotene acts not only as an antioxidant but also has unexpected prooxidant properties. At present, there is an ongoing debate on the protective or potentially harmful role of beta-carotene in human skin.
Abstract: Common assays for evaluation of antioxidative capacity of different compounds are usually performed in cell-free systems. By this approach, cell-specific regulatory mechanisms upon distinct stimuli are not taken into account. Therefore, there is a need to measure anti-oxidative capacity in a cellular setting. - We now developed a valid method that provides monitoring of anti-oxidative capacities of compounds in different cell types. Oxidative stress, induced by 100 microM H subset2O subset2 in human microvascular endothelial cells (HMEC-1), was quantified by the generation of oxidized, fluorescent C-DCF from C-H subset2DCF-DA/AM. As DCF-production could be almost completely blocked by diethyldithiocarbamate (DEDTC), which inhibits intracellular Cu/Zn superoxide dismutase (SOD), mainly intracellular production of C-DCF was assumed. Preincubation with alpha-tocopherol resulted in a dose-dependent reduction of both spontaneous and H subset2O subset2-induced C-DCF-production (maximal inhibition by 41.6% at 75 microM). A synergistic effect was observed with co-incubation with vitamin C (maximal inhibition 46.8% at 10 microM vitamin C and 50 microM alpha-tocopherol). In this way compounds with different modes of action and subcellular localization can be evaluated concomitantly in respect of their anti-oxidative capacities. As this method was established on 24- and 48-well plates in other cell lines (Caco-2, HFP-1), too, screening of a large array of antioxidative compounds in different cell lines can be performed.
Abstract: Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-alpha-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.
Abstract: Retinyl palmitate and its metabolites retinol and retinoic acid control growth and epithelial differentiation. Systemic or local vitamin A deficiency induced by malnutrition, continuous chemical irritation or locally induced by inflammation causes squamous metaplastic changes in the epithelium of mucous membranes. We demonstrate that in an animal model topically applied retinyl palmitate can be taken up by the mucosal cells independently from the systemic supply. Under in vivo conditions metabolic changes in vaginal epithelium of rats were shown to be reversed by treatment with topical retinyl palmitate. - After only two days treatment squamous metaplastic vaginal epithelium in rats shows a reversal of the epithelium into a normal phenotype which continues after cessation of the treatment for 7 to 11 days. Higher concentrations and longer retention times lead to a statistically significant (p = 0.025) increase in the protection time. These data demonstrate that squamous changes induced by vitamin A deficiency can be totally reversed with topically applied retinyl palmitate.
Abstract: The purpose of this study was a comparison of a commercial IRMA to an in-house ELISA method. Because IRMA methods are considered to be very sensitive and results of these assays are widely accepted, the ferritin levels of 378 school children were determined in parallel with a commercial IRMA kit (Becton Dickinson Co., Orangeburg, NY) and the ELISA. For organizing reasons plasma was used for the ELISA, whereas serum was used for the IRMA. Regression analysis of the pairs of values, taking the IRMA reading as the independent variable, yielded a coefficient of variation r=0,90 (Spearman, two-tailed, p=0,01) and the straight line y=0,99x+3,13. Using this ELISA technique described by DAKO, ferritin levels in plasma were, on average, 13,2% higher than in serum taken at the same time. The ELISA technique described was found to have good accuracy and the speed and ease with which it may be carried out makes it suitable for a large number of samples.
Abstract: beta-Carotene is discussed as an anti-oxidant micronutrient and singlet oxygen quencher in human skin, protecting against UV light-induced damage. However, we recently demonstrated that beta-carotene has a pro-oxidant potential in cultured human skin fibroblasts because it enhances the UVA induction of heme oxygenase-1 (HO-1). Herein, we further show that beta-carotene also strongly promotes the UVA induction of pro-inflammatory interleukin-6 (IL-6) in skin fibroblasts in vitro. Singlet oxygen quencher sodium azide abrogated up-regulation of IL-6, and likewise also of HO-1. In UVB-irradiated cells, beta-carotene did not modulate levels of IL-6 and HO-1. The observed effects might be relevant for UV-induced inflammatory processes.
Abstract: OBJECTIVE: Malnutrition and poor nutritional status among children are common problems in the Republic of Maldives, a small island nation in the Indian Ocean. The aim of this study was to determine possible macro- and micronutrient deficiencies in the traditional Maldivian diet. DESIGN: In five atolls, 333 women with children aged between 1 and 4 years who were no longer breast-fed were interviewed, using a 24-hour recall. Additionally, the weights and heights of both the women and children were measured, and blood samples from 15 women were collected for measurements of vitamins A and E, beta-carotene, homocysteine, cholesterol and haemoglobin. RESULTS: Of the women, 22% had a body mass index (BMI) below 18.5. Of the children, 41% were stunted, 14% were wasted and 51% were underweight. The women's and children's diets were sufficient in protein (14%) and carbohydrates (67%) but deficient in fat, which contributed only 19% to the total energy intake. Consumption of dietary substances that depend on vegetable and fruit intake (e.g. beta-carotene, vitamin C, dietary fibre and folic acid) was low. The low intake of beta-carotene was underlined by low plasma concentration. The estimated iron intake was low, although blood haemoglobin levels were normal. CONCLUSIONS: Marginal nutritional status and marginal malnutrition are due to low fat intake and selected micronutrient deficiency. Higher intakes of locally available vegetables and fruits and fat (especially for children) on a regular basis might reverse the deficits documented on the atolls.
Abstract: Vitamin A plays an important role in the pathogenesis and during the course of retinal degenerations. Following a brief overview of the metabolic pathway of Vitamin A from the entrance into the body to the arrival in retinal target structures, the most important retinal degenerations that are related to the Vitamin A-metabolism are presented. Ways of prevention or therapy of such diseases are discussed in the light of the still incomplete knowledge about basic mechanisms of retinol transport and metabolism.
Abstract: PURPOSE: The appearance of the cervical mucosa is regulated by different factors including retinoic acid. Hormone-dependent alteration of the cervix uteri mucosa is accompanied by a decrease or increase of cytoplasmatic retinoic-acid-binding protein (CRABP). To elucidate whether this hormone-dependent alteration of CRABP is preserved in the case of neoplasms of the cervix uteri, we measured the level of total and apo-CRABP in normal and neoplastically transformed cervical cells. METHODS: In a prospective pilot study, standardised biopsies of normal epithelium and cervical intra-epithelial neoplasm grade 3 (CIN III) were taken from 24 patients. A newly developed method was used to determine the intra-epithelial level of apo- and total CRABP. RESULTS: The concentration of total CRABP in normal squamous epithelium compared with that in intra-epithelial neoplasm grade 3 is very significantly lower in the CIN III areas (normal: 3.66 +/- 1.46 pmol/ mg wet weight +/- SD; CIN III 1.43 +/- 0.59 pmol/mg P < 0.01). In addition CRABP in the apo form is lower in normal than in neoplastic epithelium (Wilcoxon test for paired non-parametric values: P < 0.05; mean for all patients: normal: 1.65 + 0.82 pmol/mg; CIN III: 1.14 +/- 0.23 pmol/mg). CONCLUSION: From our results we conclude that, in neoplastically transformed cells, the hormone-dependent CRABP cycle is interrupted. Whether this has consequences for the further development of the neoplastic cells has to be elucidated.
Abstract: Employing a short, potassium hydroxide (KOH) saponification (25 min) and rapid extraction with n-hexane-toluene (1:1) and a <4-min retention time, a convenient, rapid, accurate and precise determination of vitamin A in breast milk was developed. The recovery rate was 102.3 +/- 2.5% and the small relative standard deviation (intra-assay 1.9%, inter-assay 2.3%) resulted in high precision. The amount of breast milk needed is very low (1 ml) which makes the method suitable for the use in free field studies with large sample sizes. The simple extraction and separation steps allow a high throughput screening under routine laboratory conditions.
Abstract: Many studies have implicated the role of oxidant stress in a wide range of human diseases and have led to the rapid expansion of research in this area. With many experimental approaches a direct detection of the production of reactive oxygen species (ROS) and free radicals is not possible. Free radicals are very reactive, short-lived and react in a non-specific way, so that ongoing oxidative damage is generally analyzed by measurement of secondary products e.g. H2O2, "oxidized" proteins, peroxidized lipids and their break-down products, "oxidized" DNA or by fluorographic analysis in combination with fluorescent dyes e.g. dichlorofluorescin (DCFH). The histochemical visualization of selected molecular markers for oxidative phenomena can often provide valuable information concerning the distribution of oxidative processes in vivo. A number of biochemical methods are available for the monitoring of almost all oxidant stress-related processes, although their applicability in vivo is limited. This review summarizes the biochemical methods currently available for histochemical detection and indirect visualization of an excess of free radicals and ROS. The cited methods are discussed and the results obtained from their application are critically evaluated.
Abstract: This study evaluated the effects of RRR-alpha-tocopherol (500 IU/day, 8 days) on in vivo cytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 +/- 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by RRR-alpha-tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. RRR-alpha-Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to RRR-alpha-tocopherol supplementation.
Abstract: Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.
Abstract: BC. Two days after supplementation with 5 microM BC in MbetaCD, cellular BC levels reached a maximum of 140+/-11 pmol/microg DNA, leveling off to 100+/-15 pmol/microg DNA until day 8. Incubation with BC dissolved in THF/DMSO resulted in a lower BC uptake of 105+/-14 pmol/microg DNA and 64+/-20 pmol/microg DNA respectively. No cytotoxic effects of these formulations were detected. The results show that the MbetaCD formulation is an improved method for investigations of carotenoids and other lipophilic compounds in in vitro test systems compared to methods using organic solvents.
Abstract: Experimental evidence suggests that carbohydrates and their corresponding receptors (endogenous lectins) decode biological information. Therefore, the expression of complex oligosaccharides--the potential ligand part of this recognition system--during chondrogenesis and osteogenesis was determined in the viscerocranium of fetal rats by mapping the staining patterns of exogenous lectins. Results were compared with the expression of bone- and/or cartilage-specific core proteins and the binding profiles of neoglycoconjugates. These synthetic tools make possible the localization of sugar-ligand-binding sites. The spatial and temporal distribution patterns of glycoconjugates were highly dynamic and demonstrated a clear correlation with characteristic morphological modifications. The glycobiological characterization of precartilage mesenchymal cells revealed distinct differences compared to prospective bone anlagen. Especially the binding of the exogenous lectin from Griffonia simplicifolia II, that selectively visualized prechondral aggregations, reveals that regulation of early chondral growth is at least phenomenologically correlated with a relatively atypical oligosaccharide composition terminating with N-acetylglucosamine.
Abstract: BACKGROUND: Two German sisters aged 14 and 17 y were admitted to the Tübingen eye hospital with a history of night blindness. In both siblings, plasma retinol binding protein (RBP) concentrations were below the limit of detection (<0.6 micromol/L) and plasma retinol concentrations were extremely low (0.19 micromol/L). Interestingly, intestinal absorption of retinyl esters was normal. In addition, other factors associated with low retinol concentrations (eg, low plasma transthyretin or zinc concentrations or mutations in the transthyretin gene) were not present. Neither sibling had a history of systemic disease. OBJECTIVE: Our aim was to investigate the cause of the retinol deficiency in these 2 siblings. DESIGN: The 2 siblings and their mother were examined clinically, including administration of the relative-dose-response test, DNA sequencing of the RBP gene, and routine laboratory testing. RESULTS: Genomic DNA sequence analysis revealed 2 point mutations in the RBP gene: a T-to-A substitution at nucleotide 1282 of exon 3 and a G-to-A substitution at nucleotide 1549 of exon 4. These mutations resulted in amino acid substitutions of asparagine for isoleucine at position 41 (Ile41-->Asn) and of aspartate for glycine at position 74 (Gly74-->Asp). Sequence analysis of cloned polymerase chain reaction products spanning exons 3 and 4 showed that these mutations were localized on different alleles. The genetic defect induced severe biochemical vitamin A deficiency but only mild clinical symptoms (night blindness and a modest retinal dystrophy without effects on growth). CONCLUSIONS: We conclude that the cellular supply of vitamin A to target tissues might be bypassed in these siblings via circulating retinyl esters, beta-carotene, or retinoic acid, thereby maintaining the health of peripheral tissues.
Abstract: PURPOSE: To describe the phenotype caused by a retinol deficiency in a family with compound heterozygous missense mutations (Ile41Asn and Gly75Asp) in the gene for serum retinol binding protein (RBP). METHODS: The two affected sisters, 17 (BR) and 13 (MR) years old, were examined clinically and with perimetry, color vision tests, dark adaptometry, rod- and cone-isolated electroretinograms (ERGs), multifocal ERGs, electrooculograms (EOGs), and laboratory tests. RESULTS: There were no complaints besides night vision problems and no history of systemic disease. Visual acuity was reduced to 20/40 (BR) and 20/25 (MR). Anterior segments were normal except for a discrete iris coloboma. Both patients showed a typical "fundus xerophthalmicus," featuring a progressed atrophy of the retinal pigment epithelium. Dark adaptation thresholds were elevated. In the scotopic ERG, only reduced mixed responses were recordable. The photopic ERG was reduced in BR and normal in MR; implicit times were highly (BR) to slightly (MR) elevated. There was no (BR) to little (MR) light reaction in the EOG. All-trans retinol levels were 0.19 microM and 0.18 microM (normal range, 0.7-1.5 microM) for BR and MR, respectively, and did not increase in a dose-response test. RBP was below detection threshold, and retinyl esters were normal. CONCLUSIONS: Both affected siblings had no detectable serum RBP, one sixth of normal retinol levels, and normal retinyl esters. The retinal pigment epithelium was severely affected, but besides acne there were no changes to other organs. This gives evidence for an alternative tissue source of vitamin A, presumably retinyl esters from chylomicron remnants. The normal retinol levels in the tear fluid explain the lack of xerophthalmia. However, considering the role of RBP in the tear fluid and, during development, in the yolk sac there is also evidence that there are organ-specific RBP forms not affected by the genetic defect.
Abstract: For determination of the vitamin status via mass screening, simple and rapid methods are required. Additionally, blood samples should be obtained using simple and low invasive sampling techniques. To fulfill this existing methods have been modified to analyze retinol, tocopherols, beta-carotene, vitamin C and homocysteine in 20 microliters plasma. Blood samples were obtained via skin punctures. HPLC measurements were carried out with isocratic separation and precolumn derivatization. Intra and interday variation coefficients were below 8% and regression coefficients better than 0.99 for all measurements. The difference between venous and capillary samples were < 5%. In conclusion, the methods employed proved satisfactory for the determination of important nutritional parameters in blood samples obtained via skin punctures. These methods are therefore well suited for mass screening, especially under field conditions in developing countries.
Abstract: beta-Carotene has often been discussed as a means to reduce the risk of skin photodamage. We studied the antioxidative potential of beta-carotene in human skin fibroblasts exposed to ultraviolet A light. Surprisingly, we found a pro-oxidative effect of beta-carotene. Using the induction of haem oxygenase-1 as a marker for oxidative stress, we found a strong enhancement of gene expression by beta-carotene in ultraviolet A-irradiated cells. This effect was clearly suppressed by concomitant addition of vitamin E but only moderately by vitamin C. The results show that beta-carotene has pro-oxidative properties in human skin fibroblasts exposed to ultraviolet-A light.
Abstract: This article is based on discussions of the lung cancer panel at the Hohenheim Consensus Meeting organized by the World Health Organization and the German Ministry of Health in November 1996. Panel members were international experts in the field of diet and cancer who discussed specific questions relating to lung cancer risk factors and prevention.
Abstract: The effects of respiratory hyperoxia (RH) and xanthine oxidase (XO) during localized hyperthermia (HT) were investigated by determining markers of oxidative damage to lipids and proteins and tumor growth. Anesthetized rats with s.c. DS-sarcomas underwent one of the following treatments: (a) localized saline-bath HT (60 min, 44 degrees C); (b) HT + RH (100% O2); and (c) HT + RH + XO (15 units/kg i.v.). Sham-treated animals served as controls. Tumors were investigated for: (a) thiobarbituric acid-reactive substance formation and protein-bound 4-hydroxynonenal, as indicators of lipid peroxidation; (b) reactive oxygen-mediated protein modifications; (c) apoptosis; and (d) tumor volume growth. Upon treatment, increases in thiobarbituric acid-reactive substances, protein-bound 4-hydroxynonenal, protein-associated carbonyl functions, and number of cells undergoing apoptosis were found in tumor tissue, together with an inhibition of tumor growth. When treatment groups were compared, effects in the group HT + RH + XO were generally most pronounced. These findings indicate that the antitumor effect of HT is at least partially mediated through the selective induction of lipid peroxidation and oxidative injury in tumor cells, leading to apoptosis. This effect was enhanced by adding RH or RH + XO, presumably due to enhanced tissue damage following an increased formation of reactive oxygen species, with higher levels of lipid peroxidation and protein oxidation.
Abstract: Although clinical evidence of improvement of human skin photodamage by all-trans-retinoic acid (atRA) has accumulated, evidence for its preventative effects against photodamage is limited. Here we studied human skin fibroblasts in vitro. For determination of atRA uptake and metabolism, cells were treated with 3 or 10 microM atRA and retinoids analyzed by HPLC. After 16h a peak of cellular retinoid levels was reached, mainly atRA and 13-cis-RA. At this time point cells were irradiated with UVA (1-20 J/cm2) or UVB (5-500 mJ/cm2). TBARS in medium supernatant were used as an indicator of lipid peroxidation; ornithine decarboxylase (ODC) activity served as a marker of the mutagenic and carcinogenic effects of UV light. 1 h post irradiation (p.i.) with 20 J/cm2 UVA, TBARS production was enhanced by a 3 microM atRA treatment (121+/-5% of vehicle treated cells) and decreased by a 10 microM atRA treatment (75+/-2% of vehicle treated cells), and not significantly altered in UVB irradiated cells. 24 h p.i. with 50 mJ/cm2 UVB, ODC activity peaked in vehicle treated cells at a 7.4+/-0.2-fold increase compared to sham irradiated control cells, and was reduced to a 4.9+/-0.2-fold increase by 3 microM atRA. Treatment with 10 microM atRA further decreased ODC activity (3.7+/-1.0-fold increase) and this delayed activity peak occurred at 36 h p.i. ODC activity was not significantly enhanced by UVA irradiation. These results suggest that in normal human skins fibroblast atRA and/or its metabolites influence the UVA-induced lipid peroxidation by at least two distinct antagonistic mechanisms, while the ODC response to UVB-induced DNA damage possibly involves a ROS-independent, retinoid-sensitive regulatory pathway.
Abstract: Oxidation is widely assumed to play a causal role in the pathogenesis of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) can be followed by analyzing the lag phase of the conjugated diene formation at 234 nm in LDL exposed to Cu2+. This procedure is restricted to isolated LDL fractions. To make this assay applicable to different biological systems, the present paper introduces a method to determine the time course of lipid peroxidation by measuring the EPR signal intensity and thereby the concentration of the radicals formed. Stable radical spin adducts were generated using the spin trap PBN (N-tert.-butyl-alpha-phenylnitrone) and were detected by EPR spectroscopy. Comparing the specific formation of radicals and the generation of conjugated dienes as measured by UV absorbance revealed analogous lag, propagation and decomposition phases.
Abstract: Numerous studies suggest that supplemental vitamin E prior to or during vast surgeries might diminish or even prevent ischemia/reperfusion-induced injuries. In the present placebo-controlled study male Sprague-Dawley rats were supplemented parenterally or orally with alpha-tocopherol for three consecutive days. The applied amount of alpha-tocopherol was 2.3 micromol per day for oral and 1.2 micromol per day for parenteral supplementation. The enrichment of vitamin E concentrations in plasma and tissue samples (aortic endothelium, liver, and lung) was determined by HPLC. The vitamin E level was elevated following intravenous supplementation in plasma (21.4 +/- 1.9 micromol/L vs. 10.2 +/- 1.7 micromol/L in parenteral control group), in aortic endothelium (1.1 +/- 0.2 pmol/mm2 vs. 0.5 +/- 0.1 pmol/mm2) and in liver and lung (41.3 +/- 7.5 pmol/mg vs. 22.9 +/- 6.5 pmol/mg and 75.6 +/- 13.6 pmol/mg vs. 51.7 +/- 5.9 pmol/mg, respectively). Oral supplementation for three days also led to an increased level in liver (38.2 +/- 7.7 pmol/mg vs. 22.9 +/- 6.6 pmol/mg in oral control group) and in lung (67.8 +/- 5.7 pmol/mg vs. 51.7 +/- 9.3 pmol/mg) but not in aortic endothelium or plasma (0.8 +/- 0.3 pmol/mm2 vs. 0.6 +/- 0.3 pmol/mm2 and 12.0 +/- 2.2 micromol/L vs. 10.7 +/- 2.6 micromol/L).
Abstract: To determine the influence of vitamin E on phylloquinone activity, one day-old chicks were raised on a masch diet supplemented with different amounts of vitamin E for 31 days. In chicks fed a diet high in vitamin E (4000 mg allrac-alpha-tocopheryl acetate/kg) but adequate in vitamin K (0.14 mg phylloquinone/kg) a threefold increase in prothrombin time and an increase in mortality rate (five out of twelve animals died from increased bleeding tendency) was observed. The inhibiting effect of high dietary vitamin E on procoagulant factors could be prevented by increasing dietary phylloquinone supplementation. Weight development, and feed utilization were insignificantly different in chicks fed different amounts and ratios of vitamins E and K1. Plasma and liver alpha-tocopherol levels correlated with dietary amounts of vitamin E. Increased phylloquinone levels in the diet did not significantly influence alpha-tocopherol concentrations in plasma and liver, but coagulopathy caused by high vitamin E intake could be reversed.
Abstract: We compared the ability of all-transretinoic acid (RA), all-trans-retinoyl-beta-D-glucuronide (RAGL), and all-trans-beta-carotene (BC) to inhibit growth and to induce differentiation of the human promyelocytic leukemia cell line HL-60 into morphologically mature granulocytes. BC was made water-soluble by the solutol-solvent-system. RA (1 microM) could induce differentiation of 85% of the HL-60 cells after a total incubation time of 180 hours, RAGL (5 microM) induced 64% of the cells, whereas 33% of the HL-60 cells were differentiated after incubation with BC (10 microM), which was determined by assessing cell functional capacity to reduce nitroblue tetrazolium dye in response to phorbolesters. The absence of RA in RAG and BC treated cells gives strong evidence that RAG and BC exert intrinsic biological effects.
Abstract: Bioavailability of orally administered vitamins, minerals, and trace elements is subject to a complex set of influences. Still, administrative regulation is necessary on how to quantify it. The most common approach to this problem is to determine the fraction of an oral dose that reaches the systemic circulation. For micronutrients, however, this approach has to consider the physiological plasma concentration as well as the mechanisms that regulate intestinal absorption and distribution of micronutrients between functional and storage compartments in response to the demand. The rate of exchange between these compartments has an impact on the delivery of such compounds into the plasma compartment as well as on the plasma clearance. Monitoring the area under the plasma concentration time curve after oral administration is an inadequate tool for bioavailability determination if there are substantial impacts of homeostatic mechanisms on the plasma concentration of a micronutrient. In nutritional science the term "bioavailability" encompasses the sum of impacts that may reduce or foster the metabolic utilisation of a nutrient. Bioavailability in this sense can be quantified by the rate by which deficiency symptoms are cured or by the weight gain during growth. both of these endpoints, again, are influenced by homeostatic mechanisms. To exemplify the scope of impacts on parameters that are commonly used to quantify the bioavailability of oral micronutrient preparations the basic traits of homeostatic regulation are summarised and compared for iron, magnesium, vitamin A, folic acid, and vitamin B12. The mechanisms that adapt absorption, distribution, and excretion of these five micronutrients to the demand differ to such an extent that no common approach can be derived to consider these impacts in bioavailability determination. In consequence, therefore, we recommend to define and regulate individual strategies for bioavailability testing for each micronutrient with regulated kinetics.
Abstract: The authors are aware that there is still a need for much research to elucidate the possible preventive effects of antioxidant vitamins with regard to degenerative and neoplastic diseases. There must also be vigorous examination of the evidence that antioxidant vitamins can play a crucial part in a large number of other disorders (Alzheimer's disease, Parkinson's disease, diabetes, chronic and acute inflammations, airway disorders, reperfusion syndrome). The aim of prevention-oriented medical research must be to develop suitable measures able to make a considerable contribution to the overall prevention policy. Although there is still uncertainty about the mode of action and the optimal dosage of antioxidant nutrients and dietary constituents, particularly because of the safety when the dosage is correct, more information must be provided about early prevention of an inappropriate, low antioxidant intake; intake in the diet should definitely be preferred to supplementation because of the other beneficial effects of the recommended foodstuffs.
Abstract: The widely used TBA assay for lipid peroxidation was modified to minimize artefactual oxidative degradation of lipids during the assay. Formation of the TBA-MDA condensation product was studied with and without exclusion of oxygen, and the concentration effect of BHT addition was examined. Oxygen was depleted from the reaction mixture by extensive argon gassing. Exclusion of oxygen resulted in decreased TBARS production in plasma but not in standard solutions. High BHT concentrations resulted in a similar effect. At concentrations higher than 3 mmol/l BHT exclusion of oxygen had no additional effect. By measuring n-butanol extracts in a multititer plate reader this modified method was made suitable as a preliminary screening assay of human body fluids for lipid peroxidation.
Abstract: Preruminant calves are regarded as a model for studying beta-carotene bioavailability in humans. The objectives of this trial were to determine the relationship between multiple beta-carotene doses and plasma steady-state concentration, accumulation in selected tissues, and vitamin A balance in liver. Seventy newborn Holstein calves in six treatments (n = 10/treatment) were fed a complete milk replacer diet low in vitamin A and supplemented with beta-carotene doses of 0, 0.23, 0.46, 0.92, 1.84 or 3.68 mumol/(kg body wt.d) for 28 d. Ten calves were killed on d 1. Plasma beta-carotene increased in relation to log transformations of dose and time (P < 0.05) in all supplemented calves and steady state was attained after 4 wk. For doses up to 0.92 mumol/(kg body wt.d), the dose-response relationship was linear. A dose-dependent accumulation of beta-carotene was found for liver, heart, lungs, adrenals and adipose tissue. All-trans-beta-carotene was the only isomer in plasma and adrenals and the predominant isomer in the remaining tissues. In liver, vitamin A increased with beta-carotene uptake. Hepatic balance between vitamin A accumulation and loss was achieved at beta-carotene intake of 0.36 mumol/(kg body wt.d) for a calf of 45 kg. It is concluded that preruminant calves within 1 mo of age utilize beta-carotene as a source of vitamin A, and that for testing bioavailability of beta-carotene sources, doses up to 0.92 mumol beta-carotene/(kg body wt.d) are most appropriate.
Abstract: Vitamin A deficiency results in typical morphological alterations of the respiratory mucosa (increase of goblet cells, decrease of ciliated cells, squamous metaplasia). This early effect of the deficiency is an explanation for the frequency, severity and persistence of the respiratory infectious diseases that have been responsible for high mortality in vitamin A-deficient children. Vitamin A supplementation leads to rapid reduction in morbidity and mortality. However, in many cases oral supplementation is not very effective due to gastrointestinal diseases with impaired absorption of the fat soluble vitamin. To overcome this problem we developed an inhalable vitamin A-ester and administered the vitamin through a tracheostoma in vitamin A-sufficient (n = 8) and vitamin A-deficient rats (n = 8). 15 minutes after inhalation the retinyl ester appeared in the blood and was detected (6 h after inhalation) in lung, testicle, liver, kidney and small intestine. The unphysiological retinyl ester (odd number of C-atoms) was rapidly hydrolysed and reesterified with physiological fatty acids (palmitate). The results of our study demonstrate that the inhalative application circumvents problems of oral administration and results in a sufficient supply to sensitive target tissues.
Abstract: We conducted a randomized placebo-controlled double-blind study in 20 healthy young female students (skin type II + III, body mass index 18-22) in order to evaluate the efficacy of 10 weeks of moderate dose (30 mg/d) beta-carotene (BC) on plasma and skin beta-carotene levels during 12 days of time and intensity controlled sunlight exposure at sea level (30 degrees latitude, Red Sea, Eilath, Israel). After 12 days of controlled sun exposure (total UV dose of about 10.000J/cm2), plasma beta-carotene decreased in the placebo (p < 0.01) and beta-carotene group (not significant). In addition cutaneous beta-carotene decreased significantly in both groups. Plasma alpha-tocopherol decreased significantly (p < 0.01) during exposure time in both groups. In the supplemented group, however, the decrease of a-tocopherol was significantly greater (p < 0.01) than in the placebo group. We conclude that sunlight influences the beta-carotene and alpha-tocopherol content of blood and tissues.
Abstract: Different cell culture and organ systems are used to evaluate the physiological responses of the airways to the effects of carcinogenic [e.g., benzo(a)pyrene] and anticarcinogenic (e.g., retinoids) compounds on cellular growth and differentiation. However, in contrast to in vivo conditions dissociated epithelial cells or tracheal ring cultures are covered with medium. Therefore, we developed an ex vivo perfusion model enabling evaluation of morphology and metabolism of different compounds under near-physiological conditions. The trachea was surrounded with culture medium and perfused with air by means of a small animal respirator. To test the viability of the system under various experimental conditions tracheal probes were incubated with either retinoids (retinol 10(-5) mol/l; retinyl palmitate 10(-5) mol/l) or benzo(a)pyrene (10(-7) mol/l) for up to 7 days. At the end of the incubation period metabolites in the trachea and in the medium were measured by means of high-performance liquid chromatography. Samples were examined by light microscopy, and by scanning and transmission electron microscopy for cell morphology. Glycoconjugate expression was assessed by lectin histochemistry. Specimens incubated in a retinoid-supplemented medium revealed no alterations in the distribution of cell types and characteristics of the epithelial layer compared with tracheal biopsies assessed immediately after removal from the animals. Glycoconjugate patterns especially remained intact. Histological changes after incubation with benzo(a)pyrene resembled in vivo morphology of vitamin A-deficient rats. An important advantage of this in vitro model compared with common cell or organ cultures is the preservation of the original phenotype and environment of the tracheobronchial surface. In addition, carcinogenic substances, such as benzo(a)pyrene, can easily be applied by airway or through the medium.
Abstract: The aim of this study was to determine the influence of sustained marginal vitamin A deficiency on the morphology of glycoconjugate expression in the tracheobronchial epithelium of guinea pigs. The distribution of oligosaccharide chains was investigated by applying a panel of 24 lectins. Glycosaminoglycans were detected by histochemical techniques. Number as well as morphology of ciliated cells showed no significant alterations in hypovitaminosis A. In contrast, the quantity of goblet cells was constantly decreased. A considerable reduction of secretory granules was also observed in these cells. Cytomembranes of ciliated cells (especially in the area of ciliar extensions) showed constant alterations in the patterns of lectin binding in vitamin A-depleted guinea pigs. Our results demonstrate a significant augmentation of accessibility of fucosyl molecules in proximal domains of glycoconjugates of ciliary membranes, whereas the presence of mannose structures seemed unchanged. In distal bronchioli, terminal N-acetylgalactosamine molecules were expressed. During marginal vitamin A deficiency, ciliary cells were specially labelled by GSA I(B), indicating presentation of terminal galactose molecules in alpha-position. Additionally, the cytoplasm of epithelial cells demonstrated enhanced concentrations of polyantennary oligosaccharide core structures. Staining of epithelial cells by VVA was restricted to control specimens. Abundance of N-acetylglucosamine residues on the non-reducing terminus of oligosaccharides was significantly enhanced in the connective tissue of depleted animals as demonstrated by the binding patterns of GSA II. We suggest that altered oligosaccharide patterns may contribute to enhanced predisposition to tracheobronchial infection in marginal vitamin A deficiency.
Abstract: A rat model was used to investigate whether high oral doses of vitamin A lead to fetal malformations and to what extent retinyl esters (RES) are transferred from the mother to the fetuses. Retinol and RES concentrations in plasma behave similarly in rats and humans. When high concentrations of vitamin A are administered, plasma retinol concentrations remain relatively constant, whereas plasma RES increased in parallel with the dose. To achieve an elevation from approximately 150 to > 1525 nmol x L(-1) in the experimental group before mating, female Ibm: RORO (spf) rats were fed a maintenance diet enriched with 15.2 x 10(3) retinol equivalents (RE) x kg(-1) at the start and increased stepwise to 52.5 x 10(3) for a total of 8 mo. A parallel subgroup was maintained to measure progress in experimental rats without interference by blood taking. Rats of the control group received the basal diet analyzed to contain 4.5 x 10(3) RE x kg(-1). Before mating the mean body weights of experimental and control rats were not significantly different. All-trans, 13-cis, 4-oxo-all-trans and 5,6-epoxy-all-trans retinoic acid (RA) concentrations were determined in maternal and fetal plasma. With high vitamin A intake, 4-oxo- and 5,6-epoxy RA concentrations were significantly higher in the fetuses than in their mothers. Although these high intakes of vitamin A by the rat dams resulted in high maternal and fetal plasma concentrations of vitamin A and its metabolites, fetal malformations were not observed. This may be due to the fact that circulating RES are not teratogenic and that after crossing the placental barrier, they are stored mainly in fetal liver.
Abstract: In 34 patients with HVP-infection of the cervix and in 40 patients with CIN III standardised biopsies were taken from the involved area and normal cervical epithelium for determination of the local concentration of retinylester. In all cases diagnosis was confirmed colposcopically, cytologically and by histology. HPV infection was confirmed by in situ hybridisation. Determination of retinylester was performed by HPLC. No significant difference of local retinyl-plamitate concentration was detectable in HPV infected versus normal tissue. Retinyl-palmitate concentration was extremely lower in CIN III compared with normal cervical epithelium and HPV-infected tissue. The determination of plasma level of retinol showed no significant difference between the two groups. So it can be presumed that the reduction of retinyl-palmitat in CIN III is a local process and a local supplementation of Vitamin A might contribute to the prevention of cervical neoplasia.
Abstract: The staining patterns of 24 biotinylated lectins were analyzed in serial sections of the mandible of 13- to 21-day-old rat embryos by means of the avidin-biotin-peroxidase method. A ubiquitous distribution of binding sites was demonstrated after incubation with Con A (Canavalia ensiformis), DSL (Datura stramonium; except bone matrix), and WGA (Triticum vulgare). ECL (Erythrina cristagalli), GSL I (Griffonia simplicifolia), SJA (Saphora japonica), VVL (Vicia villosa), DBA (Dolichus biflorus), UEA I (Ulex europeus), and LTA (Lotus tetragonobolus) were constantly negative. In early stages of development, GSL II (Griffonia simplicifolia II) was a selective marker of prechondral blastema. In contrast, PNA (Arachis hypogaea) did not stain condensing mesenchyme. During chondrogenesis of Meckels's cartilage a general decrease of lectin binding was observed. Mature cartilage matrix was constantly negative. Chondrocytes were marked by the lectins PSA (Pisum sativum), WGA, PHA-E, and PHA-L (Phaseolus vulgaris E and L). A strong GSL II binding was restricted to the mesial-superior region of the perichondrium. In later stages, several lectins revealed significant differences between preskeletal ("central") areas and the remaining ("peripheral") mesenchyme. A clear binding reaction was noted in central regions by applying LEA (Lycopersicon esculentum) and STL (Solanum tuberosum), while the peripheral tissue was only faintly stained. Developing bone was specifically marked by succinylated WGA (sWGA). The lectins LCA (Lens culinaris) and RCA (Ricinus communis) bound to fibers and extracellular matrix of the connective tissue. Jacalin (Artocarpus integrifolia) and SBA (Glycine max) binding sites were found in macrophages. Affinity of VAA (Viscum album) increased parallel with maturation of endothelial cells. Specific lectin-binding patterns revealed no correlation with the distribution of glycosaminoglycans. The results demonstrate a general reduction of oligosaccharide structures during development of Meckel's cartilage. From our observations we conclude that intralaminar glucose and/or mannose sequences as well as terminal sialic acid molecules are ubiquitously distributed, while terminal alpha-fucose was constantly negative. Lectin-binding patterns of macrophages may reflect the presence of specifically linked terminal galactose. Our findings indicate that oligosaccharides terminating in N-acetylglucosamine are bone-specific. The significance of the restricted staining of the perichondrium by GSL II remains to be elucidated.
Abstract: Free radicals and reactive oxygen species can damage cells and tissues of biological organisms. Due to the fact that these compounds are generated continuously in living cells defense mechanisms must exist. This so-called antioxidative system ensures that the formation of free radicals during different physiological processes does not result in cellular damage. Free radicals (oxidants) are produced form the immune system. The purpose of this immune cell products is to destroy invading organisms and damaged tissue. Oxidants enhance IL-1, IL-8 and TNF production in response to inflammatory stimuli. Sophisticated antioxidant defense systems like enzymes or vitamins protect directly and indirectly the host against the damaging influence of oxidants. While endogenous systems can hardly be influenced, exogenous antioxidants, delivered by the diet, can be upregulated in the body. By this way the pro-/antioxidative capacity can be balanced or even unbalanced.
Abstract: Alterations in the expression of glycoconjugate structures during cartilage development in the chondrocranium, nasal skeleton, Meckel's cartilage, limb buds, vertebral bodies and ribs were investigated comparatively in 13 to 21-d-old rat embryos. The binding patterns of 24 biotinylated lectins were analysed in serial sections and compared with results obtained using histochemical methods. Proteoglycan distribution, assessed by conventional staining procedures, was not associated with lectin binding sites. During early fetal development, hyaluronate concentrations were enhanced in areas of prospective chondrogenesis. With few exceptions, the lectins showed a general increase in intensity of binding to mesenchymal structures. Con A (Canavalia ensiformis), DSL (Datura stramonium), and WGA (Triticum vulgare) displayed a ubiquitous distribution of binding sites. After incubation with LCA (Lens culinaris), PSA (Pisum sativum), STL (Solanum tuberosum), and VAA (Viscum album), characteristic differences in binding intensity between focal areas of developing mesenchyme were seen. DBA (Dolichus biflorus), ECL (Erythrina cristagalli), GSL I (Griffonia simplicifolia), LTA (Lotus tetragonobolus), SJA (Saphora japonica), UEA I (Ulex europaeus) and VVL (Vicia villosa) consistently failed to bind. During chondrogenesis a general reduction of lectin staining was detected. In early stages of development GSL II (Griffonia simplicifolia) was a specific marker of the prechondral blastema in the viscerocranium. PNA (Arachis hypogaea) selectively labelled the prevertebral blastema. In contrast, condensing mesenchyme of limb buds and viscerocranium was not stained. Using RCA (Ricinus communis), it was possible to distinguish chondroblasts from mature cells. All chondrocytes were stained by PSA, PHA-E, PHA-L (Phaseolus vulgaris E and L), and WGA, whereas Con A, LCA, and GSL II detected distinct differences between cartilage with different localisations. Cartilage matrix was constantly negative. Applying GSL II it was possible to distinguish specific segments of the perichondrium. From our results we conclude that especially high mannose oligosaccharides are amplified during development. Terminal sialic acid molecules, branched intralaminar glucose and/or mannose, respectively, internal galactose-(beta 1,4)-N-acetylglucosamine sequences as well as galactose-(beta 1,3)-N-acetylgalactosamine sequences in a preterminal position are diffusely distributed in mesenchymal tissue. In contrast, no evidence for the presence of terminal GlcNAc(beta 1,4)GlcNAc sequences and terminal alpha-fucosyl residues in (1,2) or (1,3)-linkage was obtained. Chondrogenesis appears to be correlated with a general reduction in the extent of expression of oligosaccharide structures. No proof of terminal N-acetylgalactosamine and alpha-galactose moieties was found, whereas our staining results document the expression of terminal beta-galactose structures in restricted areas of the developing mesenchyme.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: The clonal rat rhabdomyosarcoma cell line BA-HAN-1C is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. This cell line has been shown to be susceptible to differentiation induction with all-trans retinoic acid (RA). Since it is still unknown whether exclusively all-trans RA itself or also its metabolites can act as inductive compounds in our cell line, we exposed BA-HAN-1C cells to the metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA. Exposure to these RA metabolites resulted in a significant inhibition of proliferation (P < 0.001) and induction of cellular differentiation, as evidenced by a significant increase in the number of myotube-like giant cells (P < 0.05) and a significant increase in creatine kinase activity (P < 0.05). However, differences in the inductive potency of these RA metabolites became apparent. Furthermore, RA metabolites exhibited a significantly weaker (P < 0.05) inductive activity when compared to all-trans RA. Summarizing our results we could demonstrate that the endogenous metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA are not merely deactivated cellular excretion products of all-trans RA, but potent inducers of differentiation and inhibitors of proliferation, possibly contributing to the complex physiological actions of retinoic acid.
Abstract: The distribution of complex carbohydrate structures during the embryonic development of the rat palate was analysed by examining lectin-binding patterns in serial paraffin and cryostat sections. With few exceptions, the binding patterns showed a general increase in lectin receptors in the more developed stages of palatogenesis. High mannose oligosaccharides were especially amplified during development. Terminal fucose molecules were not expressed. In contrast, terminal sialic acid molecules were ubiquitously distributed in epithelial and mesenchymal tissues. Non-sialylated terminal N-acetylglucosamine was specifically restricted to evolving bone matrix. Before palatal fusion, quantitative but not qualitative differences were detected between oral, nasal, and medial-edge epithelial surfaces. The only exception was LCA, which specifically marked epithelial cells at the tip of palatal shelves. A very selective affinity for Jacalin was demonstrated in the oral epithelium of the palate after day 16, suggesting the presence of sialylated terminal galactose-(beta-1,3)-N-acetylgalactosamine. PNA specifically marked the basal lamina of the oral side of palatal processes. The binding patterns of DBA, GSL IA, SBA, and VVA indicated that the epithelium of the tongue is characterized by terminal alpha- and beta-galactose residues, whereas palatine cells possess only molecules with beta-anomery. During palatogenesis, glycosaminoglycans patterns were significantly modified. Our data suggest that alteration of complex carbohydrate structures may play a central role in modulating cell-cell and cell-matrix interactions. The significance of these findings, however, remains to be elucidated.
Abstract: Antiproliferative activities of all-trans-retinoic acid (RA, CAS 302-79-4) and some retinoid derivatives (all-trans-retinyl-beta-D-glucuronide (RYG), methyl-(1-O-retinoyl-beta-D-glucopyranoside) uronate (MRG), all-trans-retinoic acid beta-D-galactopyranosyl ester (RGA), and all-trans-retinoic acid beta-D-glucopyranosyl ester (RGU)) were determined by microculture tetrazolium assay (MTT assay) and cell counting by Coulter Counter (CC) in breast (MCF7, MDA MB 231) and colon (SW 948) cancer cell lines of human origin. RA, MRG, RGU, and RGA (5, 25 mumol/l) were significantly more growth-inhibitory in MCF7 cells, which are known to be cellular retinoic acid-binding protein (cRABP) and cellular retinol binding protein (cRBP) positive, than in MDA MB 231 cells which are cRABP and cRBP negative. RYG was active only in MDA MB 231 cells. RA, MRG, RGA and RGU (25 mumol/l) stimulated the proliferation of SW 948 cells as determined by CC, whereas the MTT assay indicated an inhibition of cell growth.
Abstract: Excessive postprandial triglyceride (TG) responses despite normal fasting TG levels have been described in single cases within small groups of healthy subjects and in patients with obesity or precocious atherosclerosis, known to be associated with high insulin fasting levels. To clarify this association, fasting and postprandial TG and insulin levels were studied in 113 healthy young (25.7 +/- 2.6 years), normal weight (body mass index 20.8 +/- 2.3 kg/m2) male subjects who were selected from among 117 subjects on the basis of TG fasting levels < 200 mg/dl. After a 12-hour fast a standardized liquid lipid load was administered containing 58 g mainly saturated fat and 1,017 kcal energy. Both fasting TG values and postprandial TG peak values showed bimodal frequency distributions. Statistical analysis of fasting TG discriminated two groups: a low fasting TG group with normally distributed values < 150 mg/dl (mean +/- SEM: 79.5 +/- 2.7 mg/dl; n = 104) and a high fasting TG group > 150 mg/dl (194.5 +/- 7.2 mg/dl; n = 13). Likewise, two groups could be differentiated according to their maximal postprandial TG response (TG max) to the lipid load: (1) normal responders with TG max < 260 mg/dl (mean +/- SEM: 123 +/- 4.8 mg/dl; n = 96) and (2) high responders with TG max > 260 mg/dl (272.5 +/- 20.5 mg/dl; n = 17). Fasting TG and TG max were highly correlated (r = 0.745; p < 0.0001). However, 9 of 17 (53%) high responders had fasting TG < 150 mg/dl, which means that the prediction of high response is only 47.0% based on fasting TG values. Fasting insulin levels were significantly higher in high responders than in normal responders, whereas they did not differ between the low and high fasting TG group. In conclusion, the bimodal frequency distribution of TG max after a lipid load permitted the differentiation of two groups, normal responders and high responders, with higher fasting insulin levels, which might indicate a link to the metabolic syndrome.
Abstract: The aim of the study was to find out the influence of marginal vitamin A deficiency on morphological structures in the tracheobronchial epithelium in guinea pigs. The tracheobronchial epithelium of animals with vitamin A deficiency (n = 15) and control animals (n = 7), kept under optimal laboratory conditions, was evaluated by light and electron microscopy. The cellular ultrastructure was morphometrically analyzed. The height of the respiratory epithelium was slightly increased. The basal cells were arranged in a loose cell band of three to four layers. The quantity of cytofilaments in their cytoplasm was enhanced. Goblet cells were significantly reduced in vitamin A deficiency. There was also a significant decrease in their secretory granules. The number of ciliated cells was almost unchanged. They showed a significant reduction in mitochondria. The kinocilia often contained an atypical structure of the microtubules. Our findings confirm multiple ultrastructural dysplasias in early vitamin A deficiency which may lead to a disturbance of mucociliary clearance.
Abstract: In 13 healthy, male nonsmokers (mean age: 25.7 +/- 2.4 years) with normal fasting triglycerides we investigated postprandial changes of triglycerides in several lipoprotein fractions. After a 12-hour overnight fast they ingested a standardized lipid load (1,017 kcal) including 30,000 IU retinyl palmitate. Postprandially, total triglycerides increased significantly (p < 0.001) to a peak value of 221 +/- 81 mg/dl at 5 h. Two subjects had an exceptionally strong triglyceride response (peak values: 363 and 390 mg/dl). They had the highest levels of retinyl palmitate in the chylomicron and the nonchylomicron fraction, and one of them showed elevated intermediate-density lipoprotein values throughout the test period. In addition, they showed an altered early and an increased late postprandial insulin response. Thus, our data provide evidence that an exaggerated postprandial triglyceride response may point to an increased atherogenic risk even in healthy subjects with normal fasting triglycerides.
Abstract: The influence of all-trans-retinoic acid-beta-D-glucopyranosylester, all-trans-retinoic acid-beta-D-galactopyranosylester, methyl-(1-O-retinoyl-beta-D-glucopyranoside)uronate and all trans-retinyl-beta-D-glucuronide were investigated on the celle line BA-HAN-1C. This clonal cell line was derived from a dimethylbenzanthracene induced rhabdomyosarcoma in the rat. The tumor cells were incubated for 5 days with medium which was supplemented with various concentrations of the different compounds. The action of the retinoids were measured by comparing the cellular growth and the creatine kinase activity (as differentiation marker) with an supplemented cell line. The retinoids which are based on all-trans-retinoic acid (all-trans-retinoic acid-beta-D-glucopyranosylester, all-trans-retinoic acid-beta-D-galactopyranosylester, methyl-(1-O-retinoyl-beta-D-glucopyranoide)uronate and their chemical precursors) showed similar biological effects as all-trans-retinoic acid and could be used in higher concentrations than retinoic acid without the appearance of toxic effects. The all-trans-retinyl-beta-D-glucuronide derivatives did not show any influence on the cell growth and their creatinine kinase activity. With respect to the effects of the compounds two hypothesis about their function were possible: They act as a whole molecule, or: they are bound to a receptor where the really effective substance, all-trans-retinoic acid is released from the molecule by hydrolytic cleavage as required. Investigations with the carbohydrates D-glucose, D-galactose and D-uronic acid disproved the second theorie because these substances enormously support the growth of the tumor cells. The effectively of the free all-trans-retinoic acid would have been diminished by these components. However, this effect did not appear if hydrolysis is considered.
Abstract: We demonstrate that the cells of the sponge Geodia cydonium are equipped with the basic elements required for a retinoic acid (RA)-dependent response pathway; RA was identified and quantitated, the cellular RA-binding protein (CRABP) was detected and the nuclear RA receptor (RAR) was found. In the isolated cell system the level of CRABP, but not of RAR, is strongly induced after incubating the cells for 10h with the homologous aggregation factor. In induced cells incubation with 0.3 microM RA results in a strong down-regulation of the c-myb (or c-myb-related) proto-oncogene (M(r) 63,000; mRNA 3.3 kb). We postulate that this pathway is also functionally active and that RA acts as a natural morphogen.
Abstract: The effect of vitamin A-deficiency on jejunal Paneth cells in rats was investigated. Crystalloid particles were observed in secretion granules of Paneth cells from 6 out of 8 rats with vitamin A-deficiency. The particles were similar to those found in Paneth cells under other experimental conditions. Using an immuno-electron-microscopic technique we demonstrated a clear lysozyme immunoreactivity of these particles. In 2 vitamin A-deficient rats tubular structures have been detected in addition to the crystalloid particles. Crystalloid particles or tubular structures were not detectable in a control group of 8 vitamin A-supplemented rats. The morphological alterations of Paneth cells may be correlated to an impaired local immunity of the intestine during vitamin A-deficiency.
Abstract: Retinoic acid causes a significant inhibition of cell growth of the tumor cell line BA-HAN-1C. This growth inhibition is the same whether the cells are treated with a pulse dose of retinoic acid (RA) or continuously expand to RA. The determination of RA and its degradation products within the culture medium and in the cells showed that after 24 hours 13-cis-RA was the major retinoid in all cells (96 ng/10(6) cells); all-trans-RA represented 56 ng/10(6) cells. After 48 hours 4-hydroxy-RA and a small amount of 5,6-epoxy-RA was found in the cells and also in the culture medium. 4-hydroxy-RA increased up to 96 hours, whereas 13-cis- and all-trans-RA were not detectable in the cells after 96 hours. We conclude that the BA-HAN-1C cells take up and metabolize RA. Nonlinear fit analysis of the time behavior of the RA concentration in medium demonstrates that the RA uptake unexpectedly follows a mono-exponential time function. Discussion of the experimental results in connection with a proper compartment model shows that uptake and metabolism of RA cannot be described really by a first order kinetics. The mathematical analysis leads to a more complicated kinetic model with certain restrictions for the corresponding rate constants.
Abstract: The available data of members of Weight Watchers groups were statistically evaluated and their efficiency is discussed with respect to the underlying therapeutic concept. As a result of the data evaluation and the discussion of the literature it can be concluded: 1. The time of active membership in the group correlates with the weight loss. 2. Dropouts can be expected at the beginning of the therapy and have usually a greater obesity than other members. An early motivation has to consider the slower success in those people with a high degree of obesity. 3. A continuous motivation and re-motivation is of greatest importance to prevent any weight gain after the end of group therapy. This re-motivation program should be established within the group; members who succeeded in losing weight should return for meetings at regular intervals.
Abstract: The effect of vitamin A deficiency in guinea pigs on noise-induced temporary threshold shift (TTS) was evaluated after short (15 min) acoustic overstimulation with a moderate (90 dB) broad-band white noise. Some guinea pigs were fed ad libitum a purified diet deficient in vitamin A (VAD group) until biochemical signs of deficiency occurred. A second, control group (VA group) received the same diet as well as 100 IU vitamin A daily by pharyngeal tube. Cochlear potentials were recorded by special computerized equipment using implanted electrodes. Before acoustic stimulation, a baseline value was determined with a test stimulus [90 dBA (A-filter according to usual DIN instructions)] corresponding to that for TTS measurements. Noise-induced changes were determined by calculating the changes in latency and amplitude of the N1-signal of the compound action potential (CAP) at various times (1, 3, 5, 7, 11 min) after termination of acoustic stimulation in comparison with baseline values. Statistical analysis of the CAP data showed that the VAD group had significantly smaller amplitudes and increased latency of the N1-potential after acoustic stimulation and that the VA group did not show a significant change in amplitude or latency. The reduction in N1-amplitude and N1-latency in the VAD group reflects changes in inner ear hair cell activity. We conclude that vitamin A deficiency increases the sensitivity of the inner ear to noise and that this increased sensitivity increases the probability of noise-induced hearing loss.
Abstract: A sensitive method for the determination of retinyl esters, including their geometric isomers, by isocratic adsorption HPLC is described. The development of a special recycling system allows the separation of all-trans-, 13-cis, 11-cis- and 9-cis-retinyl palmitate, -stearate, -oleate, -palmitoleate and -linoleate with short retention times and high sensitivity. Extraction of the retinyl esters from various organs with mobile phase avoids additional evaporation steps. The method was evaluated by the determination of retinyl esters in small tissue samples such as tongue, trachea and inner ear. This is the first report of the detection of retinyl esters in these tissues, which are known to depend on an adequate vitamin A supply. The main ester in all cases was retinyl palmitate, followed by retinyl stearate and oleate.
Abstract: We report on the establishment of a model for differentiation induction in sarcomas, using the clonal rhabdomyosarcoma cell line BA-HAN-1C. This rhabdomyosarcoma cell line is composed of morphologically undifferentiated mononuclear stem cells, some of which spontaneously fuse to form terminally differentiated multinuclear myotube-like giant cells. The deprivation of fetal calf serum (FCS) or the exposure to retinoic acid, respectively, resulted in a significant inhibition of proliferation (P less than 0.001) and a marked increase in cellular differentiation as shown by a significant increase in the number of myotube-like giant cells (P less than 0.001) and in the creatine kinase activity (P less than 0.05) used as a biochemical marker of differentiation. Furthermore, after exposure to retinoic acid about 30% of the mononuclear tumour cells exhibited morphological features of rhabdomyogenic differentiation, such as bundles of thick and thin myofilaments, which had never been observed in the mononuclear cells of untreated cultures. These results confirm that the inverse linkage between proliferation and differentiation known from embryonic myogenesis is preserved in our rhabdomyosarcoma cell line. The failure to induce terminal differentiation by exposure to retinoic acid in all the cells of our clonal cell line indicates that some tumour cells might epigenetically be blocked from responding to retinoic acid. The temporary growth retardation observed after FCS-deprivation suggests that autocrine stimulation of proliferation may be operating in our cell line, too.
Abstract: Due to the rapid development of biochemical analyses in the last 10 years different substances like vitamin A, with an apparent clarified metabolism and action, were re-estimated. As a result, new knowledge was presented which could be essential for human health. Some details and consequences are reviewed in this paper. Marginal deficiency, which also may occur in industrialized nations, cannot be determined with certainty by usual blood analyses. The reasons for marginal deficiency are either different diseases or unbalanced nutrition. From epidemiological research it is argued that low vitamin A intake is associated with a higher incidence of cancer in different tissues. However, vitamin A may lead by over-dosing to toxic side effects. There exists a possibility that vitamin A is teratogenic also in humans. Thus, for safety reasons, woman who can become pregnant should not be advised to supplement the vitamin more than twice the RDA of the US Food and Nutrition Board for pregnant women (10,000 I.U./day) if there is no clear-cut indication. On the other hand there are indications that malformations may also caused by vitamin A deficiency.
Abstract: As the title implies, any assessment of the toxic effects of vitamin A derivatives must distinguish between vitamin A in the truest sense, i.e. retinol, and retinoic acid and its synthetic derivatives. Just as no single description is universally applicable to the mode of action of vitamin A derivatives, so too do their toxic effects defy generalization. The recommendation made in 1982 by IUPAC [Eur. J. Biochem., 129 (1989) 1] to designate all derivatives with the typical structure of the vitamin as being retinoids may be chemically logical and correct but, when it comes to describing the effects and side-effects of vitamin A derivatives, it leads to misunderstandings. Retinol, which is frequently used as synonym for vitamin A, can eliminate all symptoms of vitamin A deficiency if it is taken in sufficient quantity with the diet. The term retinol will therefore be used here as a synonym for vitamin A whereas retinoic acid and its derivatives--including the synthetic ones--will be referred to as retinoids because they do not cover the whole spectrum of effects exerted by retinol and because they also vary markedly in their side-effects. In contrast to the nomenclature proposed by IUPAC, this system provides a clear and logical distinction for describing biological processes. Other authors have favoured it in recent times [Chytil, F., J. Am. Acad. Dermatol., 15 (1986) 741; Olson, J.A., Semin. Oncol., x (3) (1983) 290; Olson, J.A., Am. J. Clin. Nutr., 45 (1987) 704; Zbinden, G., Acta Dermatovener., 74 (1975) 36]. By vitamin A, therefore, is meant all derivatives that can possibly originate from retinol in the organism. This also covers the small quantities of retinoic acid formed from retinol. On the other hand, by retinoids is meant the natural retinoic acid derivatives and their synthetic forms in their special modes of action. Since retinoic acid cannot be reduced to retinol in the organism, this nomenclature provides a clear demarcation within the biological system. Vitamin A is essential to the growth and development of higher life forms and functions in many different ways within the organism. Although vitamin A was one of the first vitamins to be described, even today there is still some uncertainty as to its mode of action, with the exception of that of retinal (vitamin A aldehyde) in vision.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Three clonal subpopulations (A, B, C) isolated from the same rhabdomyosarcoma of the rat were tested and compared for their susceptibility to differentiation induction using retinoic acid (RA), dimethylformamide (DMF), and N-monomethylformamide (NMF). These subpopulations differ in that a block to spontaneous differentiation is imposed at different stages which are characteristic for each subpopulation. Whereas tumor cell proliferation was significantly inhibited (P less than 0.001) in all three subpopulations, the effects of RA, DMF, and NMF on tumor cell differentiation were strikingly heterogeneous. The response was most marked in subpopulation C, as evidenced by a significant increase in the number of terminally differentiated myotube-like giant cells (P less than 0.001) and in biochemical differentiation, as indicated by the creatine kinase activity (P less than 0.05). Between 5% (DMF and NMF) and 30% (RA) of the mononuclear cells in subpopulation C exhibited thick and thin myofilaments, which were never observed in the mononuclear cells of the control. In contrast, subpopulation A and B responded to RA, DMF, and NMF quite heterogeneously with an increase in biochemical differentiation, whereas terminally differentiated myotube-like giant cells were never observed. These results demonstrate that the therapeutic potential of differentiation induction in malignant tumors may be impaired by tumor heterogeneity.
Abstract: Short-term parenteral application of vitamin A was examined in rats. Retinyl margarinate, which is chemically similar to physiological retinyl esters, was used in vitamin A-depleted rats to study uptake, distribution, and storage of retinyl esters in tissues. Vitamin A-depleted and Vitamin A-sufficient rats were infused with a micellar suspension of retinyl margarinate for 7 h and then killed at different times. Retinyl margarinate was directly taken up by all tissues examined. It appears that infusion of retinyl esters in micellar form provides a direct way to supply vitamin A to peripheral, vitamin A-dependent tissues. Therefore, a short-term infusion of retinyl esters with an emulsifier may be an effective means of preventing development of vitamin A-deficiency during long-term application of TPN, particularly in cases of liver dysfunction.
Abstract: Fish oil capsules are increasingly used by self-medicating patients. We studied 22 commercial fish oil and menhaden oil preparations in respect to accompanying substances that could be harmful. The substances measured were: cholesterol as determined by gas liquid chromatography, heavy metals measured by atomic absorption, and vitamin A as determined by high-performance liquid chromatography (HPLC). The contents of cholesterol and heavy metals were in ranges which can be regarded as negligible; the content of vitamin A in menhaden oils, however, was found in amounts which warrant that pregnant women do not exceed the dosage as recommended by the manufacturers.
Abstract: As demonstrated in the literature on vitamin A metabolism and homeostasis of retinol in serum, the concentration of retinol in serum is regulated very exactly if the liver stores are within the physiological range (20-300 micrograms/g liver). Therefore, the serum level indicates the status of vitamin A storage only if there is an extreme depletion or overconsumption of vitamin A. At marginal depletion, however, there is damage to peripheral tissue before changes in the vitamin A level in serum occur. At the beginning of hypervitaminosis A, changes in the level of vitamin A in serum also occur later. Therefore, the determination of vitamin A in serum gives no information on the adequacy of liver reserves for judging the necessity of a substitution.
Abstract: In mammals, the indolealkylamine melatonin is synthesized mainly in the pineal gland and to a lesser degree in a number of extrapineal sites. To obtain more information on its distribution, melatonin was measured in the membranous cochlea of the guinea pig. Moreover, melatonin and other serotonin derivatives were determined in organ cultures after incubation with 14C-labelled serotonin. The results show that melatonin is detectable in the organ of Corti and the basilar membrane, and to a lesser degree also in the cochlear nerve and stria vascularis, including the spiral ligament. In vitro studies reveal the occurrence of a number of radiolabelled serotonin derivatives indicating that two of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) are present in membranous cochlea. It is concluded that melatonin is synthesized in the guinea pig cochlea. Its role as modulator of hair cell function is dissolved.
Abstract: BA-HAN-1C is a clonal rat rhabdomyosarcoma cell line composed of proliferating mononuclear cells, which partly fuse to terminally differentiated postmitotic myotube-like giant cells. The exposure to retinoic acid in vitro resulted in time- and dose-dependent changes of both cell differentiation and cell growth. The mononuclear cells revealed bundles of newly formed thick and thin myofilaments, never observed in untreated cultures, and exhibited signs of contact inhibition. In addition, there was a statistically significant increase (P less than 0.001) in the number of terminally differentiated postmitotic myotube-like giant cells and in the creatine kinase activity (P less than 0.05) which was used as a biochemical differentiation marker. At the same time cell growth was significantly inhibited (P less than 0.001) in vitro and a decrease in plating efficiency, as well as in saturation density, was observed. These data demonstrate that retinoic acid can suppress cell growth and simultaneously initiate differentiation in a malignant mesenchymal tumor cell line. However, despite the clonal nature of BA-HAN-1C, the complete status of terminal differentiation was not achieved by all tumor cells. The reason why not all tumor cells responded to retinoic acid is unknown at the present time and will have to be the subject of further studies.
Abstract: To estimate the role of vitamin A on ciliated cells we investigated whether ciliated cells undergo any alteration during vitamin A deficiency. The epithelia examined include the ciliated cells of the respiratory tract and the ciliated sensory cells of the inner ear, the tongue, and the olfactory cells. This part of the paper will describe the ciliated epithelium of the tracheobronchial tract and its relation to vitamin A status. During vitamin A deficiency a partial loss of ciliae can be observed before any squamous metaplasia (which usually occurs during longer lasting vitamin A deficiency) develops. The scanning electron microscopic data illustrate the altered surface of the epithelium during vitamin A deficiency better than transmission electron microscopy.
Abstract: Morphological investigation of tongue, olfactory epithelia, trachea and inner ear in vitamin A deficiency are reported. The results support assumptions concerning the loss of sensory function as been at least a secondary effect of alterations of the neighbourhood of the sensory cells caused by vitamin A deficiency. Taste buds are hindered in function by a dense layer of squamous cells and olfaction is decreased by atrophy of the surrounding respiratory epithelium. Inner ear functionality seems to be affected by vitamin A status via a stabilizing effect on the endolymph-perilymph barrier.
Abstract: The uptake and chemical identification of 14C-retinyl acetate in the inner ear of the guinea-pig after oral administration is reported. For methodological reasons the experiment was carried out in vitamin A-deficient guinea-pigs. In the sensory tissues a time-dependent distribution was found similar to that in other organs. The chemical identification shows that the orally administered labeled retinyl acetate can be detected as retinyl palmitate in the membranous structures of the inner ear. This may be an indication for the ability of the inner ear tissues to esterize and probably store the transport form of vitamin A, retinol.
Abstract: The estimation of vitamin A in serum of patients suffering from different diseases (M. Crohn, Hypothyroidism, Hyperthyroidism, Liver cirrhosis, Renal insufficiency, Carcinoma of Prostate, ENT-Carcinomas) and healthy controls by means of a recent developed method (HPLC) is reported. Decreased and increased vitamin A serum levels had been reported in literature during different diseases but we could not reveal identical results in all cases. Significantly lowered values were only estimated in patients suffering from liver cirrhosis whereas increased vitamin A serum levels were determined during renal insufficiency. In hypo- or hyperthyroidism there was no difference from healthy persons. In patients with Crohn's disease the distribution of vitamin A concentrations in serum was bimodal, probably depending on extension and localization of the process. Patients with carcinoma of the prostate had only minor deviations from the normal value, whereas patients with tumors of the larynx had in part very low vitamin A concentrations with a bimodal distribution. Causes for the deviations and consequences for the assessment of the vitamin A status of patients under intravenous alimentation are discussed.
Abstract: Chemical separation of different vitamin A derivates in inner ear extracts was performed by means of high pressure liquid chromatography (HPLC) with fluorescence and UV detection. For more accurate site identification the membranous cochlea was dissected and the basilar membrane, the spiral ligament and the stria vascularis were isolated for analysis. Retinyl esters were found in the basilar membrane at a concentration of 11.4 +/- 5.9 micrograms/g dry weight, a high concentration compared with that in other tissues of other organs. The spiral ligament contained retinyl esters at a lesser concentration and no retinyl esters were found in the stria vascularis. Retinol was not detected in the spiral ligament or basilar membrane but was found in the stria vascularis at a concentration of 1.2 +/- 0.8 micrograms/g. Retinyl palmitate and retinyl stearate were identified. This investigation shows that vitamin A forms retinyl esters with long-chain fatty acids in the basilar membrane in high concentrations relative to other organs and in greater quantity than in other sites in the inner ear.
Abstract: Since the first characterization and description of vitamin A this is used in otolaryngologic therapy for different forms of hearing disorders, and its relation to the inner ear is subject of investigation. Animal experiments and clinical studies were done to clarify the significance of vitamin A for the function of hearing. Besides this there were a lot of observations describing correlations between vitamin A metabolism and hearing loss. Recent investigations showed that vitamin A is present in high concentrations in the inner ear and stored there. Morphological experiments revealed different and in some way contradictory results, but they showed that vitamin A seems to be essential for inner-ear morphogenesis.
Abstract: A simple, fast, and specific HPLC-method to estimate retinol (vitamin A) in serum is reported. In the presence of small amounts of acetonitrile retinol is extracted quantitatively with n-hexane from serum. Neither the use of an internal standard nor evaporation to concentrate the extract is necessary. The HPLC-method avoids interferences of substances like phytofluenes, carotinoids or retinyl palmitate which is present in serum to a fraction of about 2-5%. The HPLC-method was applied to healthy adults and healthy children. The method allows about 100 determinations per day and operator.
Abstract: A modified method for the diagnosis of the Pendred Syndrome in children by the Perchlorate discharge test using 123I is described. The older child, who has the Pendred Syndrome and the obligatory hearing deficit, frequently has neither a goitre nor hypothyroidism, but other investigations (bone growth, scars and function tests) can also show changes. However a more certain diagnosis of this disorder in children is possible by the Perchlorate discharge test using 123I. The test using 131I is no longer justified in the face of more recent radiobiological knowledge.
Abstract: Retinol and Retinol-binding protein are estimated in the plasma of 91 children with hearing impairments of different etiology and 35 normal children of similar age. The concentrations of Retinol and Retinol-binding protein are age-dependent but in a given age class the concentrations of these substances are found to be similar in both groups. It is obvious, however that the concentrations in children with Alport-syndrome lie mainly in the upper range, while these with Pendred-syndrome have values in the lower range of the total distribution curve.
