Sunday, January 10, 2016

DNA Barcode Testing: Do You think It Is The Right Answer For Natural Health Products?”

DNA Barcode Testing: Do You think It Is The Right Answer For Natural Health Products?”


Recently, the United States Food and Drug Administration (FDA) announced the creation of an "Office" of Dietary supplements. This move which was anticipated and even welcomed by the industry gives the Federal Government more oversight of the manufacturers, distributors and retailers who are involved in the industry. 

There is no question, even by those who are involved in the industry, that many manufacturers of natural health products, dietary supplements, nutraceuticals etc... make false claims regarding the ingredients in their formulations. 

To tackle this problem, many consumer advocates, as well as those in the industry, have turned to a classification technique called DNA barcoding to help counter this problem. 

But the question remains "When it comes to Natural Health Products is DNA barcoding the right answer? Does the technique solve the current problem of manufacturers falsifying claims of product quality?

To help answer this question and without getting too technical in respect to the scientific process itself, we have taken 3 articles and have presented them simultaneously. 

The first article gives the reader a good, yet non technical introduction of what DNA barcoding is, the reason it was developed, and the many uses that this technique offers to the scientific community. The next article is written by Constitutional Lawyer Shawn Buckley who questions an investigative news story by the "Fifth Estate" which examined the whole question of "barcoding in the natural health industry." And the third article is written Ashleigh Thurston who questions the validity of DNA barcoding when it comes to the question of identifying "extracts" within the process.


Using DNA Barcodes to Identify and Classify Living Things

An Introduction 

The science of classifying living things according to shared features, has always been a part of human society. Carl Linneas formalized biological classification with his system of binomial nomenclature that assigns each organism a genus and species name.Identifying organisms has grown in importance as we monitor the biological effects of global climate change and attempt to preserve species diversity in the face of accelerating habitat destruction. We know very little about the diversity of plants and animals – let alone microbes – living in many unique ecosystems on earth. Less than two million of the estimated 5-50 million plant and animal species have been identified. Scientists agree that the yearly rate of extinction has increased from about one species per million to 100-1,000 per million. This means that thousands of plants and animals are lost each year. Most of these have not yet been identified.
Classical taxonomy falls short in this race to catalog biological diversity before it disappears. Specimens must be carefully collected and handled to preserve their distinguishing features. Differentiating subtle anatomical differences between closely related species requires the subjective judgment of a highly trained specialist – and few are being produced in colleges today.
Now, DNA barcodes allow non-experts to objectively identify species – even from small, damaged, or industrially processed material. Just as the unique pattern of bars in a universal product code (UPC) identifies each consumer product, a “DNA barcode” is a unique pattern of DNA sequence that identifies each living thing. Short DNA barcodes, about 700 nucleotides in length, can be quickly processed from thousands of specimens and unambiguously analyzed by computer programs.
The International Barcode of Life (iBOL) organizes collaborators from more than 150 countries to participate in a variety of “campaigns” to census diversity among plant and animal groups – including ants, bees, butterflies, fish, birds, mammals, fungi, and flowering plants – and within ecosystems – including the seas, poles, rain forests, kelp forests, and coral reefs. The 10-year Census of Marine Life, completed in 2010, provided the first comprehensive list of more than 190,000 marine species and identified 6,000 potentially new species.
There is a surprising level of biological diversity, literally in front of our eyes. For example, DNA barcodes showed that a well-known skipper butterfly (Astraptes fulgerator), identified in 1775, is actually ten distinct species. DNA barcodes have revolutionized the classification of orchids, a complex and widespread plant family with an estimated 20,000 members. The urban environment is also unexpectedly diverse; DNA barcodes were used to catalogue 54 species of bees and 24 species of butterflies in community gardens in New York City.
DNA barcodes are also used to detect food fraud and products taken from conserved species. Working with researchers from Rockefeller University and the American Museum of Natural History, students from Trinity High School found that 25% of 60 seafood items purchased in grocery stores and restaurants in New York City were mislabeled as more expensive species. One mislabeled fish was the endangered species, Acadian redfish. Another group identified three protected whale species as the source of sushi sold in California and Korea. However, using DNA barcodes to identify potential biological contraband among products seized by customs is still in its infancy.
Barcoding relies on short, highly variably regions of the genome. With thousands of copies per cell, mitochondrial and chloroplast sequences are readily amplified by polymerase chain reaction (PCR), even from very small or degraded specimens. A region of the chloroplast gene rbcL – RuBisCo large subunit – is used for barcoding plants. The most abundant protein on earth, RuBisCo (Ribulose-1,5-bisphosphate carboxylase oxygenase) catalyzes the first step of carbon fixation. A region of the mitochondrial gene COI(cytochrome c oxidase subunit I) is used for barcoding animals. Cytochrome c oxidase is involved in the electron transport phase of respiration. Thus, the genes used for barcoding are involved in the key reactions of life: storing energy in carbohydrates and releasing it to form ATP. COI in fungi is difficult to amplify, insufficiently variable, and some fungal groups lack mitochondria. Instead, the nuclear internal transcribed spacer (ITS), a variable region that surrounds the 5.8s ribosomal RNA gene, is targeted. Like organelle genes, there are many copies of ITS per genome, and the variability in fungi allows for their identification.
This laboratory uses DNA barcoding to identify plants, fungi, or animals – or products made from them. First, a sample of tissue is collected, preserving the specimen whenever possible and noting its geographical location and local environment. A small leaf disc, a whole insect, or samples of muscle are suitable sources. DNA is extracted from the tissue sample, and the barcode portion of the rbcL, COI and ITSgene is amplified by PCR. The amplified sequence (amplicon) is submitted for sequencing in one or both directions.
The sequencing results are then used to search a DNA database. A close match quickly identifies a species that is already represented in the database. However, some barcodes will be entirely new, and identification may rely on placing the unknown species in a phylogenetic tree with near relatives. Novel DNA barcodes can be submitted to GenBank® (http://www.ncbi.nlm.nih.gov).

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THE FIFTH ESTATE EPISODE “MAGIC PILLS” HAS BEEN GENERATING SOME CONCERN AND I HAVE BEEN ASKED TO SHARE MY THOUGHTS ON THE PROGRAM. 

Shawn Buckley,

For those who did not see the program, it was largely based on a dated 2013 publication called DNA barcoding detects contamination and substitution in North American herbal products, published in the open access journal BMC Medicine (see BMC Medicine 2013, 11:222).

The first question that came to mind when watching the episode was: why is a 2013 study news now? After I read the study my main question became why would the Fifth Estate rely so heavily upon this publication? The publication was interesting for the questions it raised for me. The authors took a very small sample of 44 single ingredient products from 12 companies. They tested these products using PCR amplification which is a technique used to “amplify” DNA in a sample. This was done to try to identify the plant species in the product. The authors reported 59% of the products (which the Fifth Estate reported as 60%) contained plant DNA of a species different than the plant on the label.  They also reported that a third of the products contained contaminants and fillers not on the label. The message from the study and the Fifth Estate’s dramatization of the study findings to me was:

-  60% of natural health products are adulterated with ingredients not on the label, and

-  1/3 of natural health product s have contaminants and fillers not on the label.

This message is truly shocking. It was meant to be shocking. However, what truly shocked me was how misleading this message seemed to be.

For starters you need to be aware of how sensitive PRC amplification is. It is designed to find trace-amounts of DNA and amplify them so that they can be identified. So that you have an appreciation of how sensitive this is, I will share an example from a law file. The police wanted to get a DNA profile of a person of interests. They followed this person who went into a restaurant for a meal. When the person left the police seized a glass the person had drank from. The glass went to the lab which used PCR amplification with the intention of getting this person’s DNA profile. As it turned out this exercise was not helpful for the police as the PCR amplification identified three separate human DNA profiles on the glass. For greater clarity, on a clean glass used by one person in a restaurant three human DNA profiles were found. 

Turning to plants, if I grew a field of parsley beside a field of alfalfa, do you think it is possible to do PCR amplification on the parsley and not find alfalfa DNA? Perhaps, but I venture to guess it is not likely. Similarly, despite very appropriate and well done cleaning of processing equipment between the manufacture of different plant based natural health products, do we really expect we would not find through PCR amplification traces of DNA from previous products? Judging by my restaurant glass example, I would venture to guess it would not be unlikely to find other DNA. Yet according to the study and to the Fifth Estate:

-  finding DNA from a plant species not listed on the label is “contamination” or adulteration, and
-  Finding DNA from common natural health product ingredients not listed on the label means there are unlisted fillers (the implication being you are being defrauded).

Nobody at the Fifth Estate seems to have asked themselves if we should expect genetic purity of food ingredients. Natural health products usually include plant and/or animal ingredients used for health purposes. The same parsley you can buy for cooking in your grocery store can be used as a natural health product ingredient. The only difference is that when it is put in a natural health product it is tested for things like microbial contamination. We wouldn’t consider our food parsley to be adulterated or contaminated if PCR amplification could identify trace amounts of other plant DNA such as alfalfa. When we consider that much of the protein in common flour comes from the insect particles in it, I wonder how many distinct DNA profiles could be found in flour (plant, insect and animal). We don’t consider flour to be adulterated. 
I could go on with issues I see with the study such as whether their DNA profile bank has learned enough to recognize differences between strains of plants within a species, but I think my point is made. This DNA barcoding technique is a novel approach for ingredient identification of plants. It may develop into a useful tool for NHP manufacturers to even better identify their ingredients. However, the way the study was written, and the way the Fifth Estate presented the study, were in my opinion misleading and likely deliberately calculated to generate fear.

The same could be said about the rest of the Fifth Estate show starting with the title “Magic Pills” which connotes fraud. Other highlights I thought were meant to create fear and concern about taking natural health products included:

-  an old story about a New York contract manufacturer that apparently manufactured vitamins with anabolic steroids;
-  concerns we can take “too much” of a supplement;
-  citing an editorial (yes, a personal opinion piece) to give us the message that the case is closed: supplements may be harmful;
-  saying too much vitamin D is harmful;
-  saying 83% of fish oils in New Zealand were found to have high oxidation levels;
-  that people are being encouraged to take supplement amounts above Health Canada’s Recommended Daily Allowances (“RDAs”);
-  that 40% of complaints to Health Canada’s inspectorate are about natural health products.

This is not an exhaustive list but you get the message. I want to discuss three of these topics which I believe to be exceptionally funny or misleading by omission.

The funny example was one of the themes the Fifth Estate used to suggest we may be taking too high of doses of nutrients in supplements. They compared the amounts of nutrients in foods to the amounts in supplements. One example was you can take a supplement with 1000 mg of vitamin C but to get this amount of vitamin C from cantaloupes you would have to eat 8 of them. You cannot eat 8 cantaloupes at once – the message being that 1000 mg of vitamin C must be too much. I am smiling as I write this as it is truly humorous that this cantaloupe example can in any way inform us as to how much vitamin C is too much. I thank the Fifth Estate for their humour.
The second example I wanted to discuss was the Fifth Estate’s use of Health Canada’s recommended daily allowances for vitamins and minerals as a guide for what is too much. The implication I was left with was that exceeding the amount of these RDAs may be dangerous. Now I accept that taking too much of anything can be dangerous, but I am skeptical that exceeding a Health Canada RDA for a vitamin or mineral in any way informs us as to how much is too much. My understanding is that RDAs are not even meant to tell us what an optimal amount of a vitamin or mineral is for good health. I am open for correction on this, but I thought RDAs were developed in the second world war for D-day. The concern was that after the invasion it would be difficult to re-supply the invading soldiers and they had to determine the minimum amount of vitamins and minerals necessary to enable a soldier to continue to function for roughly two weeks. These guidelines later morphed into our RDAs. The RDA amounts are extremely conservative and are meant to be extremely conservative. 

The last fear meme I wanted to address was the report that 40% of complains to Health Canada’s inspectorate are about natural health products (without any indication of the number of complaints this represents which is necessary for it to be meaningful).  Part of my law practice involves assisting people and companies who make natural health products deal with Health Canada when there is a complaint.  In my experience most complaints to Health Canada about natural health products are from professional complainers.  Years ago I was defending three different companies in three different Provinces with charges that began with complaints to Health Canada.  If my recollection is correct all three Health Canada investigations were begun by complaints from the same person whom I am told (but have not verified) has ties to a pharmaceutical company. 

Health Canada tries to never disclose the identity of the complainer to the person or company being complained about, despite a Court decision saying they should.  Despite this, in another file I had it became apparent that the complainer was from a skeptics group that was clearly antagonistic to natural health products.  Although I have been dealing with complaints to Health Canada for almost two decades, as I write this I am not certain I have ever had to deal with a complaint from more than one actual consumer, although I might have but don’t know because of Health Canada’s policy of not disclosing the identity of the complainer (or I cannot recall more than one).  My point being, however, that to say 40% of complaints to Health Canada are about natural health products does not inform us as to whether there are many at all, or as to whether these are complaints about product quality or adverse events.  I should also add that some complaints I have dealt with are complaints by one natural health product company against another natural health product company.  Some of these complaints I have interpreted as an attempt to use Health Canada to shut down or cripple a competitor for economic gain. 

Finally, the majority of complaints I have recently dealt with concerns claims made by natural health product companies. When NHP’s become licensed, the license contains a “label claim” that the company can use and is expected to put on their label.  Apart from some specific prohibitions, I can say that overall there is no prohibition from making other claims, providing they are not fraudulent. However, despite there being no clear legislative authority, Health Canada takes the position that only the authorized label claim is allowed. This leads Health Canada to try to censor other claims, even if they are truthful.  This puts natural health product companies in an awkward situation. They may have evidence a product can truly help people but cannot share it without risking Health Canada’s wrath.  And it is not an answer to say they could apply for the claim as overwhelmingly Health Canada restricts label claims to what they call structure function claims in the U.S. (although the Natural Health Product Regulations do not have this restriction). Where I am going with this is to point out that advertising complaints are not necessarily a safety issue, and often the complainer risks creating a safety issue by taking truthful information away from consumers. 

The Fifth Estate episode repeatedly brought to my mind that a core message was also that natural health products are poorly regulated and that we need stronger regulation.  There was no discussion about the risks stronger regulations would bring, let alone the risks our current regulations have caused by removing some NHPs. I am passionate about protecting our access to NHPs because I have run across person after person whose very lives have depended on them, when the chemical drug model failed them.  I have called some of these as witnesses in Court. The number whose quality of life is dramatically enhanced by NHPs is even larger. We cannot have a rational and helpful discussion of how to best regulate NHPs without also looking at the benefits and considering the risks of removing or restricting them.

In my opinion the Fifth Estate did not bring this balance to their program. When I was receiving the message: “we need more regulation”, I thought maybe we do: of mainstream media programs which may be influencing people’s health decisions.

The Fifth Estate is a widely watched television show with credibility. In delivering this program, they are likely influencing peoples’ health by affecting their attitude towards natural health products. If I did not have my extensive background to draw on, this episode would have strongly discouraged me from taking natural health products.

I strongly hope that in the future they will be as balanced as they can on these issues. Everyone wants the best regulatory environment possible, but in the issue of health nothing is simple or clear cut.  Creating a demand for stricture regulation without balance can ironically lead to poor health outcomes.
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Hot Topic: Fake Supplements in 2015 
Ashleigh Thurston

This past year saw a much heated debate about the sale of ‘fake supplements’ in the US and Canada. In the early part of the year, the New York Attorney General (NYAG), A.G. Schneiderman, accused several large US retailers of selling poor quality herbal supplements based on the use of a technique called DNA barcoding. Just so we are all on the same page, DNA barcoding is an analytical method used to validate the genetic identity of plant material. While this seems like a home-run approach to validating the manufacturing methods used by so many companies, there are some limitations that the NYAG may not have considered. DNA barcode testing can be used to confirm the genetic content of whole plant material, such as the Fenugreek found in Swiss Natural Fenugreek tablets, but is not a reliable method to confirm the identity of any extracted herbal ingredient, such as that in the garlic extract present in Jamieson Odorless Garlic. The reason why this test is not accurate in all situations is based on the fact that the processing of extracts produces an ingredient that contains little to no high quality DNA. Without intact DNA, a genetic analysis would make it seem like the product contains little to none of the ingredient(s) listed on the label. While this isn’t to say that all manufacturers are fully compliant, it does suggest that this may not be the correct test to use for all herbal products across the board.

This whole situation in the US was prompted by a single study conducted out of Ontario where a researcher tested 44 herbal products purchased from the US and Canada and found evidence of fraudulent ingredient claims. It is important to note that in the concluding remarks, the author of the study states that DNA barcoding should be voluntarily used to authenticate raw materials, but does not confirm the acceptability of the test for the evaluation of finished products or extracted materials.

Furthermore, all this buzz about fraudulent supplements this year has prompted CBC Marketplace to prepare their own piece on supplements. It was scheduled to air in November, but has now been pushed to a later date. If you plan on tuning in, I want you to think of this article and how some test methods may not be the tests of choice when confirming the quality of your products. The current tests accepted by Health Canada have been widely used by the herbal industry for years and are considered acceptable to yield high quality herbal products. While the introduction of new technologies, such as DNA barcoding, can assist to further verify herbal identity, it is not currently considered the gold-standard approach to verifying all herbal products

Whew! That was a long blurb! Just to summarize if you glazed over the science-y part: what you hear in the media is not always correct and there are manufacturers of herbal products which are dedicated to producing high quality products. So don’t fret the next time you are browsing for your herbal products and if you are still somewhat concerned, feel free to review my previous articles on how to choose the best natural health products & dietary supplements here and here.  


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Sources

Using DNA Barcodes To Help and Classify Living Things, Cold Springs Harbor Laboratory 

The 5th Estate episode "Magic Pills" Has Been Generating Some Concern and I have been asked to share my thoughts on the program... Shawn Buckley

Hot Topic: Fake Supplements in 2015, Ashleigh Thurston





















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