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People have caught and consumed sharks for hundreds of years but global demand for shark products increased over 40% from 2000 to 2012. Why the sudden hike in demand? Many claim it was popular media boasting that shark cartilage is a natural anti-carcinogenic and anti-inflammatory substance, with people rushing to buy it regardless of these false claims. And while the total declared value of world trade in shark products is close to (and may have well by now surpassed) $1 billion USD traded per year, little is known about this increasingly globalized market. Due to the unsubstantiated belief that shark cartilage can be used to prevent or treat cancer and/or arthritis, consumers are at-risk of using these products in place of properly treating their ailments, possibly resulting in their own death. In fact, a recent study showed 48.3% of the shark cartilage samples had at least one instance of noncompliance with USA labeling regulations, which shows how misleading these products can be.
Mislabeling these products also has ecological impacts. Previous research discovered mislabeling of the contents in shark cartilage supplements where 35 commercial shark products were collected for DNA barcoding and 23 of the products tested positive for near-threatened, vulnerable and/or endangered species. Elasmobranch (shark and ray) identification in processed products is important if we wish to conserve them but identification of these species in these products can be difficult. One of the most popular medicinal shark products is shark cartilage pills, used by many for an array of medical conditions like arthritis and cancer, and can contain numerous species in one capsule or tablet.
But where does the cartilage come from in sharks? Their skeletons! Unlike the skeletons of bony fish and terrestrial vertebrates, elasmobranchs have skeletons made of cartilage and connective tissue which is less dense than bone. Shark cartilage products are made by separating the cartilage from the meat of the shark and drying it into a powder for use. According to previous research, it contains a substance that strongly inhibits the growth of new blood vessels toward solid tumors (thereby restricting tumor growth) and that is one of the reasons why many believe they are immune to cancer and that by ingesting the cartilage they too will be immune to the disease. Several types of cartilage extracts are made from shark cartilage including squalamine lactate, AE-941, and U-995.
Identification of elasmobranch species in shark cartilage pills has proven difficult using the methods that currently exist. Currently scientists use DNA barcoding, a sequencing-based method for species identification, to determine what species are in these capsules. DNA barcoding uses universal primer sets to target a short, standardized region of the genome while an alternative approach, DNA mini-barcoding, targets shorter regions of the full-length barcode. DNA barcoding of shark cartilage supplements has proven challenging due to factors such as DNA degradation during processing, the presence of PCR inhibitors, and the possibility of multiple species in one product. But a new study conducted by Rowena Zahn, developed a DNA mini-barcoding protocol for species identification in shark cartilage pills.
“Previous protocols based on DNA mini-barcoding had been developed for the identification of species in other shark products, such as shark fins,” explained one of the authors Dr. Rosalee S. Hellberg, Assistant Professor in the Food Science Program at Chapman University. “When we applied these protocols to shark cartilage pills, we were only able to identify the species in a portion of the products. Therefore, we decided to develop an optimized protocol specifically for use with shark cartilage pills. We examined a number of steps in the previous protocol to determine where we could optimize the methodology and we made several improvements. We incorporated an extra purification step following DNA extraction and we optimized the amplification of the DNA target by developing novel PCR primers. The optimized method allowed for identification of 82% of the shark cartilage pills. This was a significant improvement over the previous method, which only allowed for identification of 36% of the same set of pills.”
A total of 22 shark cartilage products underwent DNA extraction. “In order to extract DNA from the products, we first removed three shark cartilage pills from each product. The three pills were used to make a composite sample. We ground any pills in tablet form using a mortar and pestle. Capsules were twisted open to remove the contents. Once we had a composite sample, we then mixed a portion of it with some lysis buffer from a commercial DNA extraction kit made by Qiagen, called the DNeasy Blood and Tissue Kit. The sample was then heated at 56 degrees Celsius for 2 hours. During this time, the lysis buffer caused the cells to lyse and break open, releasing the DNA inside,” said Hellberg. “Next, we performed a series of wash steps to remove compounds such as proteins and carbohydrates from the sample. Finally, we obtained a purified sample of DNA that could be used for amplification and sequencing.”
When the results for all three primer sets were combined, 18 of the 22 shark cartilage products were identified to the species or genus level. On an individual basis, the best-performing primer set identified 16 of the 22 products to the species or genus level. “The new protocol was developed specifically for use with shark cartilage pills and greatly improves our ability to identify spark species in these products,” commented Hellberg. In fact, the protocol developed in this study increased the identification rate for elasmobranchs in cartilage products by more than 2-fold as compared to previous research. A surprising finding that came out of their combined studies with shark cartilage pills was the discovery of a non-shark species, winter skate (Leucoraja ocellate), in two products labeled as containing shark cartilage. The winter skate is considered an endangered species by the IUCN Red List, but specimens that are ‘wild caught’ in the United States is considered to be sustainably managed and responsibly harvested, according to NOAA FishWatch.
This new testing method isn’t perfect, of course. While the species were identified in the majority of samples in the current study, standard DNA barcoding and mini-barcoding don’t yet allow for simultaneous identification of multiple species in a single sample. “We hope that this new protocol can be used to identify species used in shark cartilage pills sold on the international market,” remarked Hellberg. “Because this protocol increases the ability to identify threatened and endangered shark species in shark cartilage products, we believe it will facilitate conservation efforts and monitoring of international trade.”