Matt Pratt-Hyatt PhD

The Importance of Testing for Glyphosate: The World’s Most Widely Used Herbicide

by William Shaw, PhD, and Matthew Pratt-Hyatt, PhD
Director and Associate Director of The Great Plains Laboratory, Inc. 

Published in the January 2017 issue of Townsend Letter

Glyphosate is the world’s most widely produced herbicide and is the primary toxic chemical in Roundup™, as well as in many other herbicides. In addition, it is a broad-spectrum herbicide that is used in more than 700 different products from agriculture and forestry to home use. Glyphosate was introduced in the 1970s to kill weeds by targeting the enzymes that produce the amino acids tyrosine, tryptophan, and phenylalanine. This pathway (called the Shikimate Pathway) is also how bacteria, algae, and fungi produce the same amino acids. This pathway is not present in humans, so manufacturers of glyphosate claim this compound is “non-toxic” to humans. However, evidence shows there are indeed human consequences to the widespread use of this product when we consume plants that have been treated with it and animals who’ve also consumed food treated with it. 

Usage of glyphosate amplified after the introduction of genetically modified (GMO) glyphosate-resistant crops that can grow well in the presence of this chemical in soil. In addition, toxicity of the surfactant commonly mixed with glyphosate, polyoxyethyleneamine (POEA), is greater than the toxicity of glyphosate alone.1 In 2014, Enlist Duo™, a herbicide product which contains a 2,4-dichlorophenoxyacetic acid (2,4- D) salt and glyphosate, was approved in Canada and the US for use on genetically modified soybeans and genetically modified maize, both of which were modified to be resistant to both 2,4-D and glyphosate. 2,4-D, which has many known toxic effects of its own is perhaps better known as a component of Agent Orange, an herbicide used by the United States during the Vietnam War to increase aerial visibility from war planes by destroying plant growth and crops. 

Glyphosate and Chronic Health Conditions

Recent studies have discovered glyphosate exposure to be a cause of many chronic health problems. One specific scientific paper listed Roundup™ as one of the most toxic herbicides or insecticides tested.2 Exposure to glyphosate has been linked to autism, Alzheimer’s, anxiety, cancer, depression, fatigue, gluten sensitivity, inflammation, and Parkinson’s.3-4 A 54-year-old man who accidentally sprayed himself with glyphosate developed disseminated skin lesions six hours after the accident.6 One month later, he developed a symmetrical parkinsonian syndrome. Figure 1 shows the correlation between glyphosate usage and rates of autism, tracking services received by autistic children under the Individuals with Disabilities Education Act (IDEA). This data was originally collected by Dr. Nancy Swanson, along with similar data for many other chronic disorders.14 The causes for these disorders have been linked to glyphosate’s impact on gut bacteria, metal chelation, and P450 inactivation.5-6 It can enter the body by direct absorption through the skin, by eating foods treated with glyphosate, or by drinking water contaminated with glyphosate. A recent study stated that a coherent body of evidence indicates that glyphosate could be toxic below the regulatory lowest observed adverse effect level for chronic toxic effects, and that it has teratogenic, tumorigenic and hepatorenal effects that can be explained by endocrine disruption and oxidative stress, causing metabolic alterations, depending on dose and exposure time.7 

Glyphosate, Cancer, and the Microbiome

The World Health Organization International Agency for Research on Cancer published a summary in March 2015 that classified glyphosate as a probable carcinogen in humans.8 Possible cancers linked to glyphosate exposure include non- Hodgkin lymphoma, renal tubule carcinoma, pancreatic islet-cell adenoma, and skin tumors.. Studies have also indicated that glyphosate disrupts the microbiome in the intestine, causing a decrease in the ratio of beneficial to harmful bacteria.9 Thus, highly pathogenic bacteria such as Salmonella entritidis, Salmonella gallinarum, Salmonella typhimurium, Clostridium perfringens, and Clostridium botulinum are highly resistant to glyphosate, but most beneficial bacteria such as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis, and Lactobacillus spp. were found to be moderately to highly susceptible. The relationship between the microbiome of the intestine and overall human health is still unclear, but current research indicates that disruption of the microbiome could cause diseases such as metabolic disorder, diabetes, depression, autism, cardiovascular disease, and autoimmune disease. 

Glyphosate and Chelation

Another study found that glyphosate accumulated in bones. Considering the strong chelating ability of glyphosate for calcium, accumulation in bones is not surprising. Other results showed that glyphosate is detectable in intestine, liver, muscle, spleen and kidney tissue. 5 The chelating ability of glyphosate also extends to toxic metals.10 The high incidence of kidney disease of unknown etiology (renal tubular nephropathy) has reached epidemic proportions among young male farm workers in sub-regions of the Pacific coasts of the Central American countries of El Salvador, Nicaragua, Costa Rica, India, and Sri Lanka.11 The researchers propose that glyphosate forms stable chelates with a variety of toxic metals that are then ingested in the food and water or, in the case of rice paddy workers, may be absorbed through the skin. These glyphosate-heavy metal chelates reach the kidney where the toxic metals damage the kidney. These authors also propose that these chelates accumulate in hard water and clay soils and persist for years, compared to much shorter periods of persistence for non-chelated glyphosate. Furthermore, these chelates may not be detected by common analytical chemistry methods that only detect free glyphosate, thus dramatically reducing estimates of glyphosate persistence in the environment when metals are high (for example, in clay soil or hard water). 

Testing for Glyphosate

Because glyphosate has been linked with many chronic health conditions, testing for glyphosate exposure and particularly the level of exposure is important. The lower limit of quantification (LLOQ) for The Great Plains Laboratory’s Glyphosate Test is 0.38 μg/g of creatinine. The Great Plains Laboratory is the only CLIA certified lab currently performing a test for glyphosate in urine. Our Glyphosate Test can be performed on the same urine sample as for some of our other comprehensive tests, including the Organic Acid Test (OAT) or GPL-TOX (Toxic Non-Metal Chemical Profile). See Figure 2 for an example of our Glyphosate Test report. 

As previously mentioned, glyphosate works by inhibiting the synthesis of tryptophan, phenylalanine, and tyrosine in plants. Humans need to obtain these amino acids from food sources. When food sources have scarce amounts of these amino acids due to glyphosate use, humans are at risk for deficiency too. Humans also require bacteria to maintain a healthy immune system. Research indicates that glyphosate decreases the amount of good bacteria in the gut such as bifidobacteria and lactobacilli and allows for the overgrowth of harmful bacteria such as campylobacter and C. difficile.12 Our lab has observed this in patients. We had a female patient who was suffering from depression who did a Glyphosate Test and an Organic Acids Test. Her glyphosate results were 2.99, which was over the 95th percentile and can be seen in Figure 3

Figure 2

Figure 2

Figure 4

Figure 4

Figure 3

Figure 3

Figure 5

Figure 5

Upon analyzing her OAT we noticed two things. The first was that her 4-cresol was extremely high. This increased 4-cresol can be seen in Figure 4. As stated earlier, glyphosate exposure decreases the good bacteria and allows C. difficile to invade. C. difficile produces a toxin called 4-cresol, which we measure in the OAT. Research has shown that 4-cresol inhibits dopamine beta-hydroxylase.13 Dopamine beta-hydroxylase converts dopamine to norepinephrine. In the OAT we measure both homovanillic acid (dopamine metabolite) and vanillylmandelic acid (norepinephrine metabolite). We have observed patients with a high 4-cresol value have elevated homovanillic acid, which indicates an inability to convert dopamine to norepinephrine. The results from our aforementioned patient were consistent with these other results and can be seen in Figure 5. The recommendations for this patient were to treat her glyphosate exposure and to treat her C. difficile infection. 

The results from these tests are indicative of why using the Organic Acids Test and Glyphosate Test together is so valuable and can help you provide more focused treatment for your patients. Treatment of glyphosate toxicity should be centered on determining the route of introduction and avoiding future exposure. Eating organic, non-GMO (genetically modified organism) foods and drinking reverse osmosis water are two of the best ways to avoid glyphosate. A recent study showed that people eating organic food had considerably lower concentrations of glyphosate in the urine.7 Drinking extra water may also be beneficial since glyphosate is water soluble, but that water should be filtered to remove pesticides or, ideally, be treated by reverse osmosis. More than 90% of corn and soy used are now of the GMO type. In addition, non-GMO wheat is commonly treated with glyphosate as a drying procedure. Glyphosate is somewhat volatile and a high percentage of rain samples also contained glyphosate.7 

Summary

High correlations exist between glyphosate usage and numerous chronic illnesses, including autism14. Other disease incidences with high correlations include hypertension, stroke, diabetes, obesity, lipoprotein metabolism disorder, Alzheimer’s, senile dementia, Parkinson’s, multiple sclerosis, inflammatory bowel disease, intestinal infections, end stage renal disease, acute kidney failure, cancers of the thyroid, liver, bladder, pancreas, kidney, and myeloid leukemia.14 Correlations are not causations, yet they raise concern over the use of a chemical to which all life on earth appears to be exposed. Testing for glyphosate along with specific markers in the Organic Acids Test can both help determine the level of exposure to glyphosate and guide you toward the most optimal treatment plans for your patients. 


References

1. Bradberry SM, Proudfoot AT, Vale JA. Glyphosate poisoning. Toxicol Rev. 2004;23(3):159-67. 

2. Mesnage R et al. Major pesticides are more toxic to human cells than their declared active principles. Biomed Res Int. 2014: 179691 

3. Samsel A, Seneff S. Glyphosate, pathways to modern diseases II: Celiac sprue and gluten intolerance. Interdiscip Toxicol. 2013;6:159-184. 

4. Samsel A, Seneff S. Glyphosate, pathways to modern diseases III: Manganese, neurological diseases, and associated pathologies. Surg Neurol Int. 2015; 6: 45. 

5. Krüger M, Schledorn P, Schrödl W, Hoppe HW, Lutz W, Shehata AA. Detection of Glyphosate Residues in Animals and Humans. J Environ Anal Toxicol. 2014. 4:2 http://dx.doi.org/10.4172/2161- 0525.1000210 

6. Barbosa ER, Leiros da Costa MD, Bacheschi LA, Scaff M, Leite CC. Parkinsonism after glycine-derivative exposure. Mov Disord. 2001. 16: 565-568. 

7. Mesnage R, Defarge N, Spiroux de Vendômois J, Séralini GE. Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem Toxicol. 2015 Oct;84:133-53. 

8. Guyton KZ, Loomis D, Grosse Y et al. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. Lancet Oncol. 2015 May;16(5):490-1 

9. Shehata AA, Schrödl W, Aldin AA, Hafez HM, Krüger M. The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Curr Microbiol. 2013 Apr;66(4):350-8. 

10. Jayasumana C, Gunatilake S, Siribaddana S. Simultaneous exposure to multiple heavy metals and glyphosate may contribute to Sri Lankan agricultural nephropathy. BMC Nephrology 2015;16:103. doi 10.1186/s12882-015-0109-2 

11. Jayasumana C, Gunatilake S, Senanayake P. Glyphosate, hard water and nephrotoxic metals: Are they the culprits behind the epidemic of chronic kidney disease of unknown etiology in Sri Lanka? Int. J. Environ. Res. Public Health 2014;11:2125-2147. 

12. Clair E et al. Effects of Roundup® and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus. Curr Microbiol. 2012;64: 486-491. 

13. DeWolf WE Jr. Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides. Biochemistry. 1988;27: 9093-9101. 

14. Swanson NL, Leu A, Abrahamson J, and Wallet B. Genetically engineered crops, glyphosate and the deterioration of health in the United States of America. Journal of Organic Systems. 2014; 9(2):6- 37. 

15. Environmental Protection Agency. Pesticides Industry Sales & Usage. 2006 and 2007 Market Estimates. Available at https://www.epa.gov/sites/production/files/2015-10/documents/market_ estimates2007.pdf. Accessed July 15, 2015. 

16. Shehata AA et al. The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Curr. Microbiol. 2013;66: 350-358. 

17. Larsen K et al. Effects of sublethal exposure to a glyphosate-based herbicide formulation on metabolic activities of different xenobiotic-metabolizing enzymes in rats. Int J Toxicol. 2014;33: 307-318. 


William Shaw, PhD, is board certified in the fields of clinical chemistry and toxicology by the American Board of Clinical Chemistry. Before he founded the Great Plains Laboratory Inc., Dr. Shaw worked for the Centers for Disease Control and Prevention (CDC), Children’s Mercy Hospital, University of Missouri at Kansas City School of Medicine, and Smith Kline Laboratories. He is the author of Biological Treatments for Autism and PDD, originally published in 1998, and Autism: Beyond the Basics, published in 2009. He is also a frequent speaker at conferences worldwide. 

He is the stepfather of a child with autism and has helped thousands of patients and medical practitioners to successfully improve the lives of people with autism, AD(H)D, Alzheimer’s disease, arthritis, bipolar disorder, chronic fatigue, depression, fibromyalgia, immune deficiencies, multiple sclerosis, OCD, Parkinson’s disease, seizure disorders, tic disorders, Tourette syndrome, and other serious conditions. 

Matthew Pratt-Hyatt, PhD, received his PhD in cellular and molecular biology from the University of Michigan. He has trained under Dr. Paul Hollenberg, a prominent researcher on drug metabolism, and Dr. Curtis Klaassen, one of the world’s leading toxicologists. He has over a dozen publications in well-known research journals such as the PNAS and Cell Metabolism. He is currently associate laboratory director at the Great Plains Laboratory Inc. in Lenexa, Kansas, focused on diagnosis and treatment of mitochondrial disorders, neurological diseases, chronic immune diseases, and more. He specializes in developing tools that examine factors at the interface between genetics and toxicology. His work is bringing new insight into how genes and toxicants interact and how that may lead to mental health disorders, chronic health issues, and metabolism disorders. 

Genetic Testing – The Key to Truly Personalized Medicine

Matthew Pratt-Hyatt, PhD

Personalized medicine has been called the future of medicine since the inception of the Human Genome Project (HGP) in the early 90s, which was a project set up by the United States government to sequence the complete human genome.  The HGP was completed in 2003.(1)   This new wealth of knowledge allowed scientist to develop tests that sequence the 3 billion base pairs and the 20-25 thousand genes in the human genome.(2) Over those 25 thousand genes there are over 80 million variants in the human genome.(3)   These variations include single nucleotide polymorphisms (SNPs) as well as small deletions and insertions throughout the genome and many of those variants play a significant role in patient health.  The dream of personalized healthcare is to use genetic testing to understand a patient’s predisposition for developing different conditions, and then undergo molecular diagnostic tests to determine how the environment is interacting with these genes. 

At The Great Plains Laboratory, Inc., we have been primarily focused on looking at the second half of this equation -- finding the root cause of patient symptoms in a wide variety of chronic disorders.  We have developed tests that look at hundreds of different analytes and have worked with doctors to help them interpret how these data can be used to personalize treatment for patients. Even though traditional medicine has mostly followed the philosophy that one size fits most, functional medicine says that each person is unique and deserves unique care.  That is why we have developed our new genetic test, GPL-SNP1000, which now allows us to have a more complete picture of what contributes to a patient’s health status.

The first generation of genetic sequencing was first published in 1977 by Frederick Sanger.  This technology first used radiolabeling and then later fluorescent labeling for sequencing reactions.  This technology uses these labeled nucleotides and the length of the copied DNA in order to arrange the nucleotide sequence. The Sanger method  is good for sequencing short (300-1000 nucleotides long) amounts of DNA in a single reaction.(4) There are some benefits and drawbacks to this type of sequencing.  The Sanger technology allowed scientists to sequence one stretch of DNA and then compare it to a database and look for differences. This technology was useful if you had a suspected mutation in a known gene, because you could sequence the whole gene in a small number of reactions.  However, there are also drawbacks to this technology, such as only being able to sequence a low number of both genes and patients at one time. 

The next major advance in genotyping technology was the advent of the TaqMan Allelic Discrimination assay.  This assay uses a fluorescent reporter that is generated during the Polymerase chain reaction (PCR).(5)  The TaqMan assay uses DNA probes that differ at the polymorphic SNP site.  One set of probes is complementary to the wild-type allele and another set is complementary to the variant allele. These probes only bond to sequences of DNA that are 100% complementary. These probes, which are bonded to fluorescent reporter dyes, are also bonded to quencher dyes.  The quencher dye prevents the reporter from becoming fluorescent when both are attached to the reporter. The probes hybridize to the complementary strands.  When DNA is copied during the PCR reaction by Taq polymerase the probe is degraded and the dyes are released.  The DNA is then genotyped by determining the signal intensity ratio of the dyes bonded to the wild-type probe and the mutant variant.(6)

 The most recent advance in sequencing technology has been the advent of Next Generation Sequencing (NGS).  There are several companies that use different means to accomplish this, but NGS machines are able to monitor what nucleotide is added at each place during the DNA chain prolongation reaction.  This principle has been labeled “sequencing-by-synthesis.”  This new technique allows for sequencing to move from about 1000 nucleotides long to about 1000 billion bases per run.  This gives researchers the ability to perform a very in-depth sequence for one patient, or sequence several dozen patients at a time using more pinpointed analysis.(7)

Using NGS, our scientists at The Great Plains Laboratory, Inc., in partnership with the genetic company Courtagen, have developed what we think will be the next great tool for personalized medicine.  Our new test GPL-SNP1000 is a genetic screen that covers 1048 SNPs over 144 different genes.  These genes are broken up into nine different groups, which are: DNA methylation, mental health, drug metabolism/chemical detoxification, autism risk, oxalate metabolism, cholesterol metabolism, acetaminophen toxicity, and the transporter genes.

The GPL-SNP1000 test report (see figure 1) is programmed to only depict the SNPs that are mutated.  We are including the gene symbol, the RS number (or reference SNP number), which indicates which SNP is mutated (so that you can look up new research on that mutation), a pathogenicity number (we look at all available research on each SNP and predict how severe a mutation at that SNP would be) genotype (what is the change in nucleotide), phenotype (whether the patient is heterozygous or homozygous [one of two mutated copies], and the disease(s) associated with that mutation (we have listed the most common conditions associated with every SNP in our assay).  The report also has interpretations that are auto-generated for genes that are found to be mutated in the assay. One additional feature our report has is hyperlinks to the references on Pubmed used to make the interpretations.  This allows both patients and healthcare practitioners to review the literature about those particular mutations, without having to search the Internet for these articles. 

Figure 1

We were also very strategic about selecting the nine specific groups of genes and SNPs that our test evaluates. We talked to dozens of functional medicine professionals and asked them what groups of genes would help them the most in their practices. The top answer was the DNA methylation pathway, which was not surprising because the most utilized genetic tests on the market are currently the MTHFR tests.  The MTHFR pathway is a process by which carbons are added onto folic acid from amino acids and redistributed onto other compounds throughout the body.  This process is responsible for the formation of methionine, S-Adenosyl methionine (SAMe), and thymidylate monophosphate (dTMP).  These compounds play critical roles in nucleotide synthesis, neurotransmitter function, detoxification, and numerous other processes.(8)  We believed that we could provide better coverage of these genes than previously done by other genetic tests. We knew that no other test had more than 35 SNPs in their assay for the MTHFR gene, so we redesigned our existing DNA Methylation Profile by increasing the number of SNPs from 32 to 105.    One reason why this test is so popular is the very common occurrence of one of the more serious SNPs of the MTHFR gene, rs1801133 (C667T).  This mutation has mutant allele frequency of 39%  for the heterozygous genotype and a 17% frequency for the homozygous mutant.  It can decrease the enzyme’s functionality by 90%, causing patients to have an increased risk of developmental delay, mental retardation, vascular disease, and stroke.(9) 

Our second most requested group of genes was those that correlate with mental health.  Mutations to these genes can predispose patients to a variety of ailments including depression, schizophrenia, anxiety, and bipolar disorder.  We designed this group to include the nine genes and 53 SNPs that are most commonly the cause of mental disorders.    One of the more important genes in this group is the catechol-o-methyltransferase (COMT) gene.  This enzyme is responsible for the degradation of catecholamines, which include dopamine, epinephrine, and norepinephrine.  Mutations to COMT can lead to bipolar disorder, anxiety, obsessive compulsive disorder, and attention deficit disorder.  One of the more common mutates of COMT is the Val108Met mutation (rs4680), which can cause a heightened risk of developing anxiety.(10)

The next gene group we focus on is the group for drug metabolism/chemical detoxification.  These enzymes include the cytochrome P450s, sulfur transferases, glutathioine transferases, and the methyltransferases.  The P450s are important for multiple molecular functions including drug metabolism, hormone production, toxicant detoxification, and more. The P450s are expressed throughout the body, but primarily in the liver.  There are 57 different genes for the cytochrome P450 enzymes, however eight are responsible for most of the drug metabolism done by the body.  The P450 enzymes are responsible for 75% of all drug metabolism.(11)  Mutations to P450s can cause changes in the rate of metabolism of some medications, causing decreased effectiveness and other dangerous complications.  Some medications known to be affected by drug mutations include but are certainly not limited to warfarin, Diazepam, antiarrhythmic drugs, antidepressants, and antipsychotics.(12-13)  P450s that are known to have alleles in the population that dramatically affect drug metabolism include CYP2C9, CYP2C19, and CYP2D6.(14)  Besides the P450s,which are considered phase I detoxification, GPL-SNP1000 covers phase II detoxification enzymes that include glutathione S-transferse, Sulfotranferase 1a1, betaine-homocysteine methyltransferase 2, and UDP glucuronosyltransferease 1A1 .

The next group of genes we analyze tells parents if they or their children may have a mutation that is commonly found in autistic patients.  It has been reported that the prevalence of autism has increased dramatically in the last two decades.(15)  We looked at many different studies to determine what mutations are more commonly found in autistic patients, but not found in the neurotypical, non-autistic public.  Three large studies that were done using over 3000 participants were very useful in developing this panel.(16-18)  We selected 252 SNPs that cover 33 genes that were found in these three studies.  These genes cover many different pathways including glucose metabolism, ion and calcium channels, DNA transcription regulation, and nervous system genes.

Next, we included a group of genes that are involved with oxalate metabolism.  Oxalate and its acidic form, oxalic acid, are formed from diet, human metabolism, and yeast/fungal.  Oxalates are known to combine with calcium to form crystals that can cause kidney stones.  These crystals may also form in the bones, joints, blood vessels, lungs, and even the brain.(19)  The oxalate group from our test analyzes 32 SNPs that cover five different genes.  One of these genes is Alanine-glyoxylate aminotransferase (AGXT).  Mutations to AGXT can lead to kidney stones and primary hyperoxaluria.(20)       

In addition to these groups of genes, our new test also looks at genes for cholesterol metabolism, as well as transporters.  Both of these pathways are important for the body to regulate itself properly.  Cholesterol is important because it is critical for producing cellular membranes, hormones, and bile acids.  There are numerous recent articles discussing the importance of these cholesterol-produced molecules that regulate sugar metabolism and our metabolic rate.  Transporters are also necessary because they move large molecules and other chemicals into and out of the cell, which are not able to move across cellular membranes without assistance.  Without transporters, cells are not able to attain the proper building blocks necessary for optimum functionality or dispose of toxic cellular waste.

Truly personalized medicine may not be a reality today; however I believe the recent developments in genetic testing are the biggest leaps we’ve had in a long time.  GPL-SNP1000 helps healthcare professionals know what problems their patients may have now or in the future due to genetic mutations, as well as what specific treatments may be beneficial.  The Great Plains Laboratory, Inc. offers cutting-edge diagnostic tools that help identify underlying causes of many chronic conditions and provides recommendations for treatment based on test results.  In addition to our new genetic test, we offer other comprehensive biomedical testing, including our Organic Acids Test (OAT), IgG Food Allergy Test, GPL-TOX (our Toxic Organic Chemical Profile), and many more.  Utilizing a combination of our genetic and molecular diagnostics, we can now see a more complete picture of a patient’s overall health, both at present and potential problems for the future, which can all be addressed now.  I think the sun is rising on a new horizon of health.


References

1. Biello D, Harmon K. Tools for Life. Sci Am. 2010;303:17-18.

2. Marian AJ. Sequencing your genome: what does it mean? Methodist Debakey Cardiovasc J. 2014;10(1):3-6.

3. McCarthy DJ, Humburg P, Kanapin A, et al. Choice of transcripts and software has a large effect on variant annotation. Genome Med. 2014;6(3):26.

4. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74(12):5463-5467.

5. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 1995;4(6):357-362.

6. Shi MM, Myrand SP, Bleavins MR, de la Iglesia FA. High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes. Mol Pathol. 1999;52(5):295-299.

7. Lin B, Wang J, Cheng Y. Recent Patents and Advances in the Next-Generation Sequencing Technologies. Recent Pat Biomed Eng. 2008;2008(1):60-67.

8. Wiemels JL, Smith RN, Taylor GM, et al. Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia. Proc Natl Acad Sci U S A. 2001;98(7):4004-4009.

9. Deloughery TG, Evans A, Sadeghi A, et al. Common mutation in methylenetetrahydrofolate reductase. Correlation with homocysteine metabolism and late-onset vascular disease. Circulation. 1996;94(12):3074-3078.

10. Craddock N, Owen MJ, O'Donovan MC. The catechol-O-methyl transferase (COMT) gene as a candidate for psychiatric phenotypes: evidence and lessons. Mol Psychiatry. 2006;11(5):446-458.

11. Guengerich FP. Mechanisms of drug toxicity and relevance to pharmaceutical development. Drug Metab Pharmacokinet. 2011;26(1):3-14.

12. Ingelman-Sundberg M. Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenomics J. 2005;5(1):6-13.

13. Ingelman-Sundberg M. Genetic susceptibility to adverse effects of drugs and environmental toxicants. The role of the CYP family of enzymes. Mutat Res. 2001;482(1-2):11-19.

14. Kalra BS. Cytochrome P450 enzyme isoforms and their therapeutic implications: an update. Indian J Med Sci. 2007;61(2):102-116.

15. Rutter M. Incidence of autism spectrum disorders: changes over time and their meaning. Acta Paediatr. 2005;94(1):2-15.

16. Sanders SJ, He X, Willsey AJ, et al. Insights into Autism Spectrum Disorder Genomic Architecture and Biology from 71 Risk Loci. Neuron. 2015;87(6):1215-1233.

17. Iossifov I, O'Roak BJ, Sanders SJ, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515(7526):216-221.

18. De Rubeis S, He X, Goldberg AP, et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature. 2014;515(7526):209-215.

19. Hall BM, Walsh JC, Horvath JS, Lytton DG. Peripheral neuropathy complicating primary hyperoxaluria. J Neurol Sci. 1976;29(2-4):343-349.

20. Poore RE, Hurst CH, Assimos DG, Holmes RP. Pathways of hepatic oxalate synthesis and their regulation. Am J Physiol. 1997;272(1 Pt 1):C289-294.

The Importance of Genetic Testing in Mental Health

Matthew Pratt-Hyatt, PhD
Associate Laboratory Director, The Great Plains Laboratory, Inc.

Personalized medicine has been called the future of medicine since the inception of the Human
Genome Project (HGP) in the early 90s. The dream of personalized healthcare is to use genetic
testing to understand a patient’s predisposition for developing different conditions, and then
undergo molecular diagnostic tests to determine how the environment is interacting with these genes. Genetic testing has become a much more economical tool with the advent of Next Generation Sequencing (NGS) technology. Even though traditional medicine has mostly
followed the philosophy that one size fits most, functional medicine says that each person is
unique and deserves unique care. That is why The Great Plains Laboratory, Inc. has developed
our new genetic test, GPL‐SNP1000, which now allows us to have a more complete picture of what contributes to a patient’s health status, including mental health.

GPL‐SNP1000 looks for mutations in over 140 genes and over 1,000 different SNPs (singlenucleotide polymorphisms) and is a very useful tool for everyone working in the fields of functional and integrative medicine. GPL‐SNP1000 looks at genes and SNPs in nine specific
pathways that we believe are most important to integrative medicine. Three of the nine gene
groups we analyze ‐‐ the mental health group, the autism risk group, and the drug metabolism
group are particularly invaluable for those working in the mental health field, helping guide
practitioners in both diagnoses and more personalized treatment.

For mental health, we analyze 88 SNPs across 14 different genes. The mental health genes are
CaMkk, ELOVL6, MAOA, COMT, DAOA, SHMT1, AHCY, GAMT, MAT2B, MAT1A, MTRR, MUT, and
MTR. Some of the important mutations in these genes are:

COMT: Catechol‐O‐methltransferase (COMT) is present in the body in two different
forms. The short form is called soluble catechol‐O‐methyltransferase (S‐COMT). The
longer form is called membrane‐bound catechol‐O‐methyltransferase (MB‐COMT). MBCOMT is mainly present in the nerves of the brain, while S‐COMT is located in the liver, kidney, and blood. In the brain, MB‐COMT is responsible for degrading
neurotransmitters called catecholamines, which include dopamine, epinephrine, and
norepinephrine. GPL‐SNP1000 analyzes six different SNPs for COMT. Conditions
associated with these mutations include OCD, depression, and schizophrenia.

MAOA: Monoamine oxidase A is important for the metabolism of biogenic amines such
as the neurotransmitters dopamine, norepinephrine, and serotonin. Patients with
mutations in this gene can have Norrie disease (an eye disease that causes blindness in
males at birth or soon after), severe intellectual disability, autistic behaviors, and
seizures. Mutations to this gene have also been linked to depression, borderline
personality disorder, and bipolar disorder.

The autism risk genes are another group that would be important to practitioners of mental
health and integrative medicine, especially those who focus on pediatrics. We looked at many
different studies to determine which mutations are more commonly found in autistic patients,
but not found in the neurotypical, non‐autistic public. We selected 252 SNPs in 33 genes that
cover many different pathways including glucose metabolism, ion and calcium channels, DNA
transcription regulation, and autoimmune system genes. If a patient has one of these
mutations, it does not mean that he/she will develop Autism Spectrum Disorder, but their risk
for developing ASD may be higher than that of the general public.

The other group of genes that could be of great use in mental health is the cytochrome P450
drug metabolizers. Even though many functional practitioners are trying to move away from
using pharmaceuticals, antidepressants, neuroleptics, and beta‐blockers are still some of the
most commonly used medications. The P450 enzymes metabolize 75% of all medications.
However, many of these enzymes have possible mutations that could affect their efficacy and
safety. Over 100,000 hospitalizations occur annually because of adverse drug reactions. GPLSNP1000
looks at 241 SNPs covering all of the major mutations that could cause a decrease in
drug efficacy and safety. A recent study indicated that genetic tests could reduce the adverse
drug reactions for some medications by as much as 66%.

The new genetic test from The Great Plains Laboratory, Inc. will be a great tool for all healthcare practitioners, but especially those practicing in the mental health field. We hope that you’ll make great use of it to deliver more personalized diagnoses and treatments for your patients. For more information about GPL‐SNP1000, please visit our website or contact us and ask to speak with one of our lab scientists or consultants. www.GreatPlainsLaboratory.com


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Great Plains Announces New Test for Phospholipase A2 (PLA2)

William Shaw, Ph.D., Lab Director and Matt Pratt-Hyatt, Ph.D., Associate Lab Director

The Great Plains Laboratory is excited to announce a new test for PLA2 activity, and we are the only commercial lab currently offering this particular test in urine. PLA2 is elevated in a wide range of inflammatory disorders from multiple sclerosis to cancer. This test can be easily added to the organic acids test to provide powerful new clinical insights and treatments for a variety of serious illnesses.

PLA2 Overview:

Chronic diseases are caused by many different biological imbalances, but they almost all create and have inflammation as a cornerstone. Inflammation plays a major part in most of the disorders that we spend billions of dollars to combat, searching for relief from the pain, swelling, and other symptoms that inflammation causes. Inflammation is the immune system's natural response to infection and injury. Phospholipase A2 (PLA2) is one of the key biochemical factors produced in the inflammation response. It is commonly found in human tissues, as well as insect and snake venom. In normal amounts, PLA2 is involved in remodeling cell membranes and changing cell architecture. In infections, PLA2 can break down the phospholipids in the membranes of bacteria, fungi, and parasites leading to their death. However, inflammation, like many other biological processes often has negative effects. The same phospholipase that attacks infectious agents may also attack the cell membranes of the human host, damaging or killing those cells. In addition, the products of the PLA2 reaction, lysolecithins and free fatty acids (Figure 1) are powerful detergents that have the ability to denature proteins and destroy their biological functions. The lysolecithins produced by PLA2 initiate the pain response.

The most common free fatty acid produced by PLA2 is arachidonic acid which can increase the production of powerful mediators of inflammation called prostaglandins, leukotrienes, and thromboxanes, collectively known as eicosanoids. These mediators play an important role in the generation and maintenance of inflammation in neural cells. In addition, arachidonic acid can be converted to 4-hydroxynonenal (4-NE), which can be very toxic due to covalent modification of important biomolecules including proteins, DNA, and phospholipids containing amino groups. In addition to PLA2 causing local damage, it may be transported by the blood vessels to other parts of the body, causing widespread tissue damage.

 

Diseases Associated with PLA2

Increased levels of PLA2 have been observed in most systemic inflammatory diseases. Studies have linked elevated PLA2 activity with multiple sclerosis, rheumatoid arthritis, Crohn's disease, pancreatitis, ulcerative colitis, allergies, atherosclerosis and cardiovascular disease, lung, prostate, small intestine, and large intestine cancers, with increased susceptibility to metastases, Candida infection, asthma, autism, chronic pulmonary obstructive disorder (COPD), and sepsis.

What Causes Elevated PLA2?

Phospholipase A2 is produced by the pancreas and released into the small intestine following a fatty meal. Infection or trauma of the pancreas may result in the release of phospholipase into the circulation, causing widespread damage or even death. Activation by viruses of proenzymes of PLA2 within the pancreas instead of, as normally, in the intestine, may cause pancreatitis. Phosholipase may be produced by cells of the immune system in response to bacterial antigens, especially those containing certain lipopolysaccharides (LPS). Allergies, especially those to house dust and cats, have been implicated as a trigger for PLA2 synthesis and release. Venoms from snakes, spiders, and bees contain high amounts of PLA2, which is responsible for much of the toxicity of these venoms. In addition, microorganisms such as Candida albicans and certain Clostridia species produce PLA2 which increases the ability of the microorganism to infect the host. Trauma may also cause significant increases in PLA2 and result in brain injury.

PLA2 and Inflammatory Disease

Research has implicated PLA2 in the pathophysiology of neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer's disease (AD). Multiple sclerosis involves both antigen-specific mechanisms and components of the innate immune system that result in inflammatory response. Elevated PLA2 activity was found to be ongoing among MS patients, with the highest levels measured in patients with progressive disease. In the development of Alzheimer's disease, the abnormal PLA2 levels appear to be related to oxidative signaling pathways involving NADPH oxidase and production of ROS species that lead to impairment and destruction of neurons and inflammation of glial cells.

Inflammation is the hallmark of rheumatoid arthritis (RA), a joint-destructive autoimmune disease. PLA2 is found in synovial fluid of RA-affected individuals and in the cartilage of RA patients as compared to cartilage from osteoarthritic and normal individuals.

Measurement of PLA2 is emerging as an important tool for evaluating the chance of cardiovascular disease (CVD), including future stroke, myocardial infarction, heart failure, and other vascular events. PLA2 appears to be more specific than hsCRP for CVD risk and may also have a pivotal role as a mediator of cardiovascular pathology. In atherosclerosis, PLA2 not only activates macrophages and formation of foam cells, but it also hydrolyzes LDL and HDL, spawning increased numbers of pro-atherogenic small LDL particles, and impairing anti-atherogenic HDL. PLA2 activity may even precipitate bleeding from atherosclerotic plaques.

PLA2 is expressed normally in pancreatic, gall bladder, and GI epithelial cells, but is significantly increased in inflammatory gastrointestinal disorders. In ulcerative colitis and Crohn's disease, all intestinal cell types increase expression of PLA2, which increases gut permeability and may actually contribute to infectivity.

PLA2 and Cancer

Elevations of PLA2 have been found in gastrointestinal cancers including colonic adenomas and carcinomas and pancreatic ductogenic carcinomas, among others. Patients with lung tumors positive for PLA2 had a greatly increased tumor growth rate and a markedly reduced survival rate. Patients with lung cancer also had higher plasma levels of PLA2 than patients with benign nodules. A similar pattern has been observed in prostate cancer, although metastatic tumors expressed lower PLA2 than primary tumors. As PLA2 releases arachidonic acid and other fatty acids from cell membranes, they initiate downstream production of tumor-promoting eicosanoids. In cancer, the spread of tumor cells from a primary tumor to the secondary sites within the body is a complicated process involving cell proliferation and migration, degradation of basement membranes, invasion, adhesion, and angiogenesis. Continued research on PLA2 expression in cancer will certainly reveal valuable new insights.

What lowers PLA2?

There has been a great deal of research done by both academia and pharmaceutical companies to find chemical inhibitors to PLA2. However, there has also been research on more natural methods for inhibiting PLA2. Glucocorticoids such as the natural hormone cortisol and pharmaceutical agents such as dexamethasone inhibit the production of phospholipase, decreasing harm caused by the enzyme but also decreasing the benefits of the enzyme in killing harmful microorganisms. Thus, excess glucocorticoids can reduce inflammation in a patient with tuberculosis while reducing the effects of PLA2 against the bacteria resulting in spread of the illness. Lithium at pharmacological doses, carbamazepine, and the antimalarial drug chloroquine are all PLA2inhibitors. Vitamin E is also an inhibitor of PLA2. In addition, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) (belonging to the omega-3 class of fatty acids) inhibit PLA2. Analysis has shown that treatment with supplements of Cytidine 5'-Diphosphocholine (CDP-choline) can limit the ability of PLA2 to promote inflammation. CDP-choline is a precursor in the formation of phospholipids and has been used as a nutritional supplement at doses ranging from 500-4000 mg per day in the treatment of patients with a variety of disorders including Parkinson's disease, memory disorders, vascular cognitive impairment, vascular dementia, senile dementia, schizophrenia, Alzheimer's disease (especially effective in those with the epsilon-4 apolipoprotein E genotype), head trauma, and ischemic stroke. A trial in patients with Alzheimer's disease indicated that CDP-choline (1,000 mg/day) is well tolerated and improves cognitive performance, cerebral blood perfusion, and the brain bioelectrical activity pattern. No side effects were noticed at the lower doses of CDP-choline and only some mild gastrointestinal symptoms were found using higher doses. No abnormal blood chemistry or hematology values were found after the use of CDP-choline.

Testing for PLA2

Because PLA2 is a relatively small enzyme (about 14 KD), it is able to be excreted in urine. 10 mL of the first morning urine before food or drink is suggested for testing. There are no dietary restrictions. This test is convenient to include with other urine tests such as organic acids, amino acids, and peptides. Since chelating agents might interfere with the test, they should not be used for at least 48 hours prior to testing. PLA2 testing is recommended for the following disorders:

  • Multiple sclerosis
  • Rheumatoid arthritis
  • Crohn's disease
  • Pancreatitis
  • Ulcerative colitis
  • Allergies
  • Cardiovascular disease including atherosclerosis
  • Neurodegenerative diseases
  • Schizophrenia
  • Bipolar depression, subtype with psychosis
  • Candida infection
  • Sepsis
  • Long term depression
  • Asthma
  • Chronic obstructive pulmonary disease (COPD)

Inflammation plays such a key role in so many diseases, and we believe this new PLA2 test will be a valuable tool in the treatment of patients suffering from numerous disorders. The test is now available and we hope you will integrate it into your practice. For more information about PLA2 and possible treatments, please see the references below.

Clinical References:

  • Green, .J.A., Smith, G.M., Buchta, R., et al. (1991) Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis. Inflammation 15: 355-367
  • Lilja, I., Smedh, K., Olaison, G., et al. (1995) Phospholipase A2 gene expression and activity in histologically normal ileal mucosa and in Crohn's ileitis. Gut. 37: 380-385.
  • Mounier, C.M., Wendum, D., Greenspan, E., et al. (2008) Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate. Br J Cancer 98: 587-595.
  • Nicolas, J.P., Lin, Y., Lambeau, G., et al. (1997). Localization of structural elements of bee venom phospholipase A2 involved in N-type receptor binding and neurotoxicity. J Biol Chem. 272: 7173-7181.
  • Pinto, F., Brenner, T., Dan, P., et al. (2003) Extracellular phospholipase A2 inhibitors suppress central nervous system inflammation. Glia 44(3):275-282.
  • Pruzanski. W., Scott, K., Smith, G., et al. (1992) Enzymatic activity and immunoreactivity of extracellular phospholipase A2 in inflammatory synovial fluids. Inflammation 16: 451-457.
  • Sawada, H., Murakami, M., Enomoto, A., et al. (1999) Regulation of type V phospholipase A2 expression and function by proinflammatory stimuli. Eur J Biochem 263:826–835.
  • Adibhatla, R.M. and Hatccher, J.F. (2005). Cytidine 5'-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders. Neurochemical Research. 30: 15-23.
  • Shaw, W. Possible synergistic effects of nonesterified fatty acids and lysolecithins, a toxic methionine metabolite, and ammonia in the production of hepatic encephalopathy and schizophrenia. Orthomolecular Medicine. 1988.3: 87.
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  • Rosenson, R.S. and Gelb, M.H. Secretory phospholipase A2: A multifaceted family of proatherogenic enzymes. Current Cardiology Reports 2009, 11:445–451
  • Farooqui, A.A., Ong, W., and Horrocks, L.A. Inhibitors of brain phospholipase A2 activity: Their neuropharmacological effects and therapeutic importance for the treatment of neurologic disorders. Pharmacol Rev 58:591–620, 2006
  • Wang, M., Hao, F.Y., J.G. Wang, and Xiao, W. Group IIa secretory phospholipase A2 (sPLA2IIa)and progression in patients with lung cancer. European Review for Medical and Pharmacological Sciences 2014; 18: 2648-2654
  • Pniewska, E., Sokolowska, M., Kupry-Lipinska, I., et al. The step further to understand the role of cytosolic phospholipase A2 alpha and group X secretory phospholipase A2 in allergic Inflammation: pilot study. Biomed Red Int. Volume 2014, Article ID 670814, 9 pages
  • Touqui, L. and Alaoui-El-Azher, M. Mammalian secreted phospholipases A2 and their pathophysiological significance in inflammatory diseases. Current Molecular Medicine 2001, 1, 739-754 739
  • Putignano, S., Gareri, P., Castagna, A., et al. Retrospective and observational study to assess the efficacy of citicoline(CDP-choline) in elderly patients suffering from stupor related to complex geriatric syndrome. Clinical Interventions in Aging 2012:7 113–118.
  • Farooqui, A.A. and Horrocks, L.A. Phospholipase A2-generated lipid mediators in the brain: The good, the bad, and the ugly. The Neuroscientist, Vol. 12, No. 3, 245-260 (2006)
  • Mahmoudabadi, A.Z., Zarrin, M., and Miry, S. Phospholipase activity of Candida albicans isolated from vagina and urine samples. Jundishapur J Microbiol. 2010; 3(4): 169-73.
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Toxic Crumb Rubber: Nothing to Play Around With

Matt Pratt-Hyatt, Ph.D.

 

As a parent of two young children I understand that parents have a long list of things to worry about. We parents worry if our children are eating right, getting enough sleep, or if they're making friends. Unfortunately, we now also have to worry about the toxic environment to which our children may be exposed, be it the toys they are playing with or the cups they use for drinking. The latest data shows that the playgrounds and artificial turf fields they play on may be quite toxic and hazardous to their health.

In the last two decades, many playgrounds, soccer fields, and football fields have been replacing their natural surfaces with a synthetic surface of rubber granules made up of ground up tires. Despite the popularity of these types of surfaces many different activist groups have expressed concern that these synthetic materials may be a toxic burden on our children.

In 2006 a commentary was written in Environmental Health Perspectives detailing how little we knew about the material we are having our children play on (Anderson et al, 2006). In the years since, there have been some insightful studies performed that give clues into how harmful prolonged exposure to these playing fields may be. In 2007, a study from the nonprofit organization Environment and Human Health, Inc. and the Department of Analytical Chemistry at the Connecticut Agricultural Experimental Station produced one of the first reports about chemicals found leaching from artificial surfaces made from rubber tires. This report indicated that benzothiazole, butylated hydroxyanisole, n-hexadecane, 4-(t-oxtyl) phenol, and zinc were found leaching from the tires. These chemicals are known carcinogens and neurotoxicants (Brown et al., 2007).

A second report in 2008 in the Journal of Exposure Science and Environmental Epidemiology provided some additional data on the chemicals that could affect children. The report indicated that the rubber granules have a much higher amount of polycyclic aromatic hydrocarbons (PAHs) than soil. Zinc and chromium were also found to be much higher in the artificial surfaces than in soil. The report also stated that although lead was not found to be much higher than in soil the bioaccessibility was much higher (Zhang et al, 2008). PAHs are known neurotoxic chemicals which have been found in air pollution from fossil fuel combustion. A recent study published in PLOS One from the University of Columbia discovered a link between PAH exposure and the development of attention deficit and hyperactivity problems (Perera et al., 2014).

In the last several years many alternatives to crumb rubber have emerged. One drawback to these alternatives is that they will add cost to the play area project. However, these costs do not calculate the damage these surfaces may be inflicting on our children. In light of the new data, any new playground or school field should reconsider the use of crumb rubber.

Vitamins E, C and Age‐Related Diseases

Matt Pratt‐Hyatt, Ph.D.

Age‐related diseases are becoming more commonplace as the population's average age increases. These age‐related diseases include macular degeneration (AMD), hearing loss, and dementia. Many people believe that development of these diseases is inevitable, and that nothing can be done to control their occurrence; however studies have shown that treatment with antioxidant vitamins prevents and sometimes reverses the onset of these age‐related diseases.

As our population's average age increases, the incidence of dementia as well as hearing and vision loss has also increased. The Eye Diseases Prevalence Research Group projects that the rate of age‐related macular degeneration (AMD) in the United States would double from 2004 to 2020. The Better Hearing Institute reports that 3 in 10 people over the age of 60 have hearing loss. The development of vision and hearing loss can be a very stressful situation for patients. Difficulty with hearing can cause stress in social situations due to the production of muffled sounds, required frequent repetition of others speaking, and ringing in the ears. Loss of vision can also create its own difficulties such as struggles with reading and the decreased ability to drive over time.

Recent studies have found that antioxidant intake can have beneficial effects in the alleviation of these age‐related diseases. In 2001 the National Institutes of Health published a report in the Journal of the American Medical Association Ophthalmology of a study with 3,640 participants that indicated that supplementation with vitamins C and E, beta carotene, and zinc decreased AMD and vision loss. In 2013 a joint study between the University of Michigan, University of Toronto, and Seoul National University of Medicine found that patients that took supplements of vitamin C and magnesium had significantly better hearing at high frequencies. Finally a 2010 study of 5,395 participants who were 55 years and older found that patients that took vitamin E supplements were 25% less likely to develop dementia. This study collaborates an earlier longitudinal study in 2000 of 3,385 men that suggested that vitamin E and C supplements may protect against dementia.

These studies raises the question of how much vitamin E and C someone worried about age‐related disease should be taking. The consensus of these studies is that 500 mg of vitamin C and 400 international units (I.U.) of Vitamin E were sufficient to obtain these results. However, patients should consult with their medical practitioner before starting a vitamin regiment.

Clinical References:

  • 1. Kochkin S. (2001). MarkeTrak VI: The VA and direct mail sales spark growth in hearing aid market. The Hearing Review, 8 (12): 16‐24,63‐65.
  • 2. Age‐Related Eye Disease Study Research Group. (2001) A Randomized, Placebo‐Controlled, Clinical Trial of High‐Dose Supplementation With Vitamins C and E, Beta Carotene, and Zinc for Age‐Related Macular Degeneration and Vision Loss: AREDS Report No. 8. Arch Ophthalmol. 2001;119(10):1417‐1436.
  • 3. Choi Y et al. (2014) Antioxidant vitamins and magnesium and the risk of hearing loss in the US general population. Am J Clin Nutr. 99 (1) 148‐55.
  • 4. Devore EE et al. (2010) Dietary antioxidants and long‐term risk of dementia. Arch Neurol. 67 (7) 819‐825.
  • 5. Masaki KH et al. (2000) Association of Vitamin E and C supplement use with cognitive function and dementia in elderly men. Neurology 54 (6): 1265‐72.