Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

Microcystins (MCs) comprise a group of cyclic heptapeptide toxins that share a common backbone and have two variable l-amino acids that yield at least 21 known analogs of varying potency. These hepatotoxins and potential tumor promoters are produced by certain cyanobacteria, including Microcystis aeruginosa. The cyanobacterium M. aeruginosa blooms in freshwater lakes and can potentially co-occur with other species such as Aphanizomenon flos-aquae, which is targeted and harvested for the production of dietary supplements known as blue-green algae (BGA). BGA supplements are currently marketed in the U.S. and internationally as a product that may elevate mood, increase energy, and alleviate attention deficit hyperactivity disorder. However, the potential for BGA dietary supplements to be contaminated with MCs is of concern, and there are currently no validated methods for detection of MCs in these products. This research focused on establishing screening methods for toxic Microcystis and MCs in BGA supplements. A DNA-based method employing polymerase chain reaction (PCR) was used as a prescreening tool to evaluate the dietary supplements and to detect the presence of toxin genes (i.e., presence of toxic Microcystis). A rapid, sensitive surface plasmon resonance (SPR) biosensor, directed towards recognition of all MC forms, was also developed and validated. This improved SPR biosensor incorporates a commercial Adda-group antibody (Ab) that has the capacity for broader recognition of MCs than previously developed sensors for BGA supplements that rely solely on an arginine-reactive Ab and can quantitate MC levels down to 0.24 ng/mL (equivalent to 0.24 μg per gram of BGA supplement) in less than 10 min. Such a rapid, quantitative screening method may allow for further surveillance of BGA products to assist risk assessment efforts, establishment of regulatory guidance levels, and response to potential consumer complaints related to BGA products. The PCR technique and SPR biosensor may be used in concert as prescreening and screening tools, respectively or individually, thereby limiting the number of samples that must be evaluated with confirmatory methods.     

For my wellness, not just my illness: North Americans’ use of dietary supplements

Passage of the DSHEA in 1994 created a new “liminal” category for the FDA: dietary supplements are regulated as neither food nor drugs. However, there appears to be a significant disconnect between the “official” discourse surrounding dietary supplements and supplement users’ actual practices. Despite this discrepancy, and the inadequacy of surveys to capture the dynamics of pharmaceutical practice, there is little ethnographic information available on the ways that Americans think about or use dietary supplements. We offer some preliminary observations from a pilot ethnographic study of Americans’ use of dietary supplements in which we consider not only the reasons why people are using supplements, but how they are using them, and how their experimentation has been influenced by the information they seek and receive from a variety of sources. We illustrate how anthropological studies of supplement related practice can help us better understand Americans’ attraction to and use of dietary supplements, and suggest that anthropology can contribute to a more balanced perspective on supplement use—one that moves the study of supplements beyond surveys and randomized controlled studies of efficacy to considerations of patterns of use in context, user expectations, and measures of perceived effectiveness.     

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.     

Clinical and Analytical Toxicology of Dietary Supplements: A Case Study and a Review of the Literature

The use of dietary supplements has grown dramatically in the last decade. A large number of dietary and herbal supplements escape regulatory and quality control; components of these preparations are poisonous and may contain, among other toxins, heavy metals. Uncontrolled use of dietary and herbal supplements by special populations, such as the military, may therefore pose a health risk. Clinical symptoms are not always properly attributed to dietary supplements; patients often do not mention supplement use to their health care provider. Therefore, a health risk estimate is hard to make on either the individual or the population level. The literature on this issue was reviewed and discussed in the light of a representative clinical-chemical case study. This case study was performed on a host of preparations that were used by one single individual in the military. Both essential (chromium, copper, zinc, and iron) and poisonous (arsenic, lead, and nickel) trace elements were determined using inductively coupled plasma combined with optical emission spectrometry (ICP-OES) or with mass spectrometry (ICP-MS). Arsenic and lead were detected at exposure levels associated with health risks. These health risks were detected predominantly in hormone-containing supplements and the herbs and botanicals used for performance enhancement. To the extent that this is a representative sample, there is an underestimation of supplement use and supplement risk in the US military, if not in the general population. Since clinical symptoms may be attributed to other causes and, unless patients are specifically asked, health care providers may not be aware of their patients' use of dietary supplements, a strong support of laboratory diagnostics, such as a toxicological screening of blood or urine, is required. In addition, screening of the preparations themselves may be advised.     

Botanicals authentication in food,food supplements and herbal medicinal products

Nowadays, there is a constant increase of health products (food supplements, medical devices and so on), in the market, despite the crisis that involves a lot of nations at the international level. The plants and their derivatives are present in high percentage in these products and moreover people usually have great trust in plants and in natural products in general. The above-mentioned products are available in the market in the form of tablets, pills, lozenges, syrups and other forms that supplement our diet (food or dietary supplements) or act with a therapeutic effect (herbal medicines). In Italy, about 50% of the sold products contain plants and/or plant extracts, better known as botanicals according to the European Food Safety Authority settlement. It is therefore understandable how the botanicals adulteration and/or the sophistication can heavily weigh on the quality of the finished products, not only from an economic point of view but also in guaranteeing the safety of consumers.     

Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

Risk Associated with the Use of Selected Ingredients in Food Supplements

This review focuses on four new product categories of food supplements: pre-workout, fat burner/thermogenic, brain/cognitive booster, and hormone/testosterone booster. Many food supplements have been shown to be contaminated with unauthorized substances. In some cases, the ingredients in the new categories of dietary supplements were medicinal products or new synthetic compounds added without performing clinical trials. Some of the new ingredients in dietary supplements are plant materials that are registered in the pharmacopoeia as herbal medicines. In other cases, dietary supplements may contain plant materials that have no history of human use and are often used as materials to ‘camouflage’ stimulants. In the European Union, new ingredients of dietary supplements, according to European Food Safety Authority or unauthorized novel food. Furthermore, selected ingredients in dietary supplements may be prohibited in sports and are recognized as doping agents by World Anti-Doping Agency.     

Gluten screening of several dietary supplements by immunochromatographic assay

Celiac disease (CD) is a chronic intestinal disorder of public health concern caused by gluten ingestion in sensitive individuals. Gluten is a protein found not only in gluten-containing food but also as normal component of drugs and dietary supplements. Detection of gluten in dietary supplements is a very important task required for establishing their gluten status, which is highly important for the safety of products consumed by CD and gluten-sensitive patients. In this paper, we investigated the presence of gluten in twenty one common dietary supplements from the national market using the immunochromatographic assay. This visual assay proved to be an efficient rapid tool for gluten screening as an alternative to the ELISA techniques. The results have shown the presence of gluten in 23.8% of the investigated samples (vitamins, minerals, plant extracts, probiotics supplements, lactoferrin, propolis supplements). The results provide information which may contribute to the completion of the existing lists of gluten-free pharmaceuticals. It is known that for CD patients obtaining accurate information about the gluten content of a particular item is a difficult and time-consuming process.     

Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR

Arthrospira species, under the name ‘Spirulina’, are used as food supplement for its protein, vitamins, and minerals which have several health benefits. Cyanobacterial toxins including microcystins can possibly contaminate these dietary supplements causing hepatotoxicity, tumour formation, and other disorders. The safe use of dietary supplements necessitates the need to assess such toxins in the algal food supplement. The methods which evaluate these dietary supplements should be highly sensitive, cost-effective, and rapid. In this study, multiplex HRM qPCR analysis was used to detect microcystin (MC)-producing cyanobacteria in Spirulina dietary supplements. The multiplex HRM qPCR detection limit was found to be 25 ag of mcyB spiked in a standard concentration of pcb (25 pg). Two distinct melt curves characteristic of pcb (Tm 82.8 ± 0.07 °C) and mcyB (Tm 77.9 ± 0.05 °C) were observed. Microcystin contamination was detected only in the fish food supplements and not in human dietary supplements of Spirulina. Liquid chromatography–high-resolution mass spectrometry analysis further confirmed the presence of the congeners of microcystin in the identified positive samples.     

Effects of dietary factors on oxidation of low-density lipoprotein particles

Oxidized low-density lipoproteins (ox-LDLs) appear to play a significant role in atherogenesis. In fact, circulating ox-LDL concentrations have been recognized as a risk factor for cardiovascular disease (CVD). A higher intake of some nutrients and specific food compounds such as monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and flavonoids have also been associated with a lower risk of CVD. These dietary factors could be associated to a lower risk of CVD through a reduction of the atherogenicity of LDL particles through limited oxidation. Therefore, the purpose of this article is to review human clinical studies that evaluated effects of dietary antioxidant vitamins, fatty acids (MUFA, PUFA) and specific flavonoid-rich foods on LDL particle oxidation and describe potential mechanisms by which dietary factors may prevent oxidation of LDL particles. Antioxidant vitamin supplements such as alpha-tocopherol and ascorbic acid as well as beta-carotene and fish-oil supplements have not been clearly demonstrated to prevent oxidation of LDL particles. Moreover, inconsistent documented effects of flavonoid-rich food such as olive oil, tea, red wine and soy on LDL particle oxidizability may be explained by difference in variety and quantity of flavonoid compounds used among studies. However, a healthy food pattern such as the Mediterranean diet, which includes a combination of antioxidant compounds and flavonoid-rich foods, appears effective to decrease LDL particle oxidizability, which may give some insight of the cardiovascular benefits associated with the Mediterranean diet.     

Production of good manufacturing practice-grade cytotoxic T lymphocytes specific for Epstein-Barr virus, cytomegalovirus and adenovirus to prevent or treat viral infections post-allogeneic hematopoietic stem cell transplant

Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.     

Quantitative analysis on the characteristics of targets with FDA approved drugs

Accumulated knowledge of genomic information, systems biology, and disease mechanisms provide an unprecedented opportunity to elucidate the genetic basis of diseases, and to discover new and novel therapeutic targets from the wealth of genomic data. With hundreds to a few thousand potential targets available in the human genome alone, target selection and validation has become a critical component of drug discovery process. The explorations on quantitative characteristics of the currently explored targets (those without any marketed drug) and successful targets (targeted by at least one marketed drug) could help discern simple rules for selecting a putative successful target. Here we use integrative in silico (computational) approaches to quantitatively analyze the characteristics of 133 targets with FDA approved drugs and 3120 human disease genes (therapeutic targets) not targeted by FDA approved drugs. This is the first attempt to comparatively analyze targets with FDA approved drugs and targets with no FDA approved drug or no drugs available for them. Our results show that proteins with 5 or fewer number of homologs outside their own family, proteins with single-exon gene architecture and proteins interacting with more than 3 partners are more likely to be targetable. These quantitative characteristics could serve as criteria to search for promising targetable disease genes.     

Vitamin D in foods and as supplements

Numerous studies have shown that the vitamin D status is far from optimal in many countries all over the world. The main reason for this is lack of sunshine. Only a limited number of foods naturally contain vitamin D. Good sources of vitamin D(3) are fish (not only fatty fish), egg yolk, and offal such as liver. Some foods such as milk are fortified with vitamin D in some countries. Dietary vitamin D intake is low in many countries, especially as the dietary sources are limited. The use of supplements is important and seems to be high in some countries. Current dietary intake recommendations are too low to preserve/reach optimal S-25-OHD concentrations, when UVB radiation is not available. We suggest that the recommendations should be increased to at least 10 microg per day in all age groups when solar UVB is scarce. The elderly may need a daily vitamin D intake of 25 microg. If dietary intake of vitamin D is to be increased, food habits will have to change. From a public health point of view it is better to increase the potential sources of vitamin D by fortifying specific products that are consumed commonly in a whole population, or if necessary by especially vulnerable groups. Supplement use is probably the right alternative for vulnerable groups such as infants and inactive elderly in whom this is more easily implemented.     

Regulatory issues concerning the safety, efficacy and quality of herbal remedies

Herbal remedies and alternative medicines are used throughout the world, and in the past herbs were often the original sources of most drugs. Today we are witnessing an increase in herbal remedy use throughout the Western world raising the question as to how safe are these preparations for the unborn fetus? Many women use herbal products during pregnancy. The dilemma facing most regulatory authorities is that the public considers these products as either traditional medicines or natural food supplements. The user sees no reason for regulation. Most countries have laws concerning foods, drugs, and cosmetics, the details of which seldom clearly define to what section of the law and regulations alternative remedies belong. In most countries alternative remedies are regulated as foods, provided that no medicinal claim is made on the label. The global regulatory sector, however, is changing rapidly. The Therapeutic Goods Administration (TGA) in Australia created a Complimentary Medicines Evaluation Committee in late 1997 to address this issue, and Canada has created a new Natural Health Products Directorate in the realigned Therapeutic Products and Foods Branch in 2000. In parallel, the European Agency for the Evaluation of Medicinal Products has drafted test procedures and acceptance criteria for herbal drug preparations and herbal medicinal products. In the US, the Food and Drug Administration classifies these natural products as dietary supplements. Manufacturers must label a dietary supplement thus: “this statement has not been evaluated by the FDA [, and] this product is not intended to diagnose, treat, cure or prevent any disease.” Whether these products are foods or drugs is undecided. To add complexity to this issue, most of the potential deleterious effects of natural products on the unborn may be related to hormonal effects (e.g., phytoestrogens) and nutriceutical drug interactions (e.g., St. John's Wort and antidepressants), rather than direct embryotoxicity per se. We suggest that ensuring quality of herbal products should receive immediate attention by regulatory authorities, before embarking on the more arduous tasks of safety and efficacy. Birth Defects Res B 68:505–510, 2003. © 2003 Wiley‐Liss, Inc.     

Regulation of the insulin antagonistic protein tyrosine phosphatase 1B by dietary Se studied in growing rats

Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme in the counterregulation of insulin signaling, and its physiological modulation depends on H2O2 and glutathione (GSH). Se via GSH peroxidases (GPxs) and its specific metabolism is involved in the removal of H2O2 and in the regulation of GSH metabolism. Recent results from animal trials and epidemiological studies with humans have shown that a high GPx1 activity or a permanent surplus of Se may promote the development of obesity and diabetes. Our nutrition physiological study with 7 x 7 growing rats was carried out to examine if PTP1B is modulated by Se supplements and, thus, may represent one trigger mediating these undesirable metabolic effects of Se. One group of rats was fed an Se-deficient diet for 8 weeks. The diets of the other six groups contained Se as selenite or selenate according to the recommendations (0.20 mg/kg diet) and at two supranutritional levels (1.00 and 2.00 mg/kg diet). All Se-supplemented animals featured a significantly higher body weight (6-14%) compared to their Se-deficient companions. Expression and activity of GPx1 in the liver of Se supplemented animals was 10- and 70-fold higher compared to Se deficiency. The detailed study of PTP1B regulation using an enzymatic assay and Western Blot analysis with an antibody against protein glutathionylation revealed that PTP1B was significantly up-regulated by both a maximization of GPx1 activity and by increasing dietary Se supply, reducing its inhibition via glutathionylation. Selenate effected a stronger PTP activation compared to selenite. In conclusion, our results suggest that the modulation of PTP1B activity may represent one plausible mechanism by which a long-term intake of Se supplements exceeding the requirements can promote the development of obesity and diabetes and needs further intensive investigation.     

High-performance liquid chromatographic analysis of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol in ginger-containing dietary supplements, spices, teas, and beverages

Ginger root powder is widely used as a dietary supplement as well as a spice and flavoring agent in foods and beverages. In this study, we developed a high-performance liquid chromatographic (HPLC) method that is suitable for the analysis of 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol in a wide variety of ginger-containing dietary supplements, spices, teas, mints, and beverages. 6-Gingerol, 6-shogaol, 8-gingerol, and 10-gingerol were extracted from various ginger-containing products with ethyl acetate and analyzed by HPLC on a C-8 reversed phase column at 282 nm. The recoveries of 6-, 8-, and 10-gingerol, and 6-shogaol from the ginger dietary supplements and ginger-containing products were 94.7+/-4.1, 93.6+/-3.4, 94.9+/-4.0, 97.1+/-3.8%, respectively. The within-day coefficients of variation for 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol standards at 50.0 microg/mL were 2.54, 2.38, 2.55, and 2.31%, respectively. The lower limit of quantitation was 25 ng injected. The standard curves for 6-, 8-, and 10-gingerol and 6-shogaol were linear from 10.0 to 1000 microg/mL. The variation (CV's) in the 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol concentrations of nine different ginger root dietary supplements were 115.2, 45.7, 72.3, and 141.7%, respectively. The gingerol composition of various ginger-containing spices, teas, and beverages also were found to vary widely. The proposed method can be used for the analysis and standardization of 6-, 8-, and 10-gingerol in ginger-containing dietary supplements, spices, food products and beverages and as a method for determining the amounts of 6-shogaol as a marker for 6-gingerol stability.