Lyprinol Report. 1. Alpha-tocopherol (AT)

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1 Lyprinol Report 1. Alpha-tocopherol (AT) Two samples of neat Lyprinol were analysed by HPLC. The AT content was found to be 4300 and 2500 ug/ml (= 4.3 and 2.5 mg/ml). Question: Is AT added to Lyprinol at the point of manufacture by Flavex and again when the oil is encapsulated? If it is only added during the latter stage, then it is clear that the mussel oil contains quite a high endogenous level of AT. See the Table at the end of the report where tocopherol contents of various oils are shown. Lyprinol has between 250 and 430 mg AT/100 grams of oil which is substantially greater than all the common oils shown in the Table. Five samples of Lyprinol capsules were also analysed. These have a nominal AT content of mg per capsule (= 1.49 mg of D-alpha-tocopherol per gram of contents = 1.34 mg AT per ml; 1mL weighs about 0.9 gram). The AT content of Lyprinol capsules was found to range from 1500 to 3600 ug/ml (= 1.5 to 3.6 mg/ml). In other words, two of the samples had just above the level of AT added (1.34 mg/ml) while two of the other three samples had more than double the level added. Presumably this extra AT is derived from the endogenous AT content of Lyprinol. In summary, all Lyprinol capsules had more AT than was added at encapsulation and this included capsules made in Some capsules had more than double the AT level, presumably due to the endogenous AT content. These data should provide a level of comfort in terms of the presence of more than the minimum amount of AT in the capsules over a long period of storage. 13/10/03 1

2 Anti-oxidants Mussel 16/10/02 13/10/03 2

3 Fatty Acids Using a new method developed by Dr Tom Brenna, Cornell University, USA, a sample of neat Lyprinol was analysed to determine fatty acid identities using chemical ionization (CI) MS with acetonitrile as the CI reagent. Through CIMS, the chain length and number of double bonds were determined. In CIMS/MS with acetonitrile, the [M+54]+ ion is trapped which then forms two diagnostic ions used to determine the location of the double bond.(michaud, Diau et al. 2002) Percent composition and concentrations of fatty acids were determined through electron impact (EI) MS. Samples were run in triplicate and peaks were integrated using Varian Saturn GC/MS Workstation software version In these runs, different fatty acids had different ionization efficiencies, resulting in peak areas not proportional to their concentration. To compensate for this, an equal weight FAME mixture, 68A (NuCheck Prep; Elysian, MN), was run along with samples to generate instrumental response factors. Response factors for all fatty acids in the sample were based upon comparable fatty acids in the 68A mixture. For fatty acids not included in 68A, the response factor was interpolated from those directly calculated. Factors used in interpolation include: number of carbons, number of double bonds, and location of double bond relative to the end of the chain. All factors were normalized to 16:0. Tricosanoic Acid (23:0) was added as an internal standard at a concentration of 7.6 µg/µl. The raw peak area was multiplied by the appropriate response factor to generate peak areas proportional to the amount of fatty acid in the sample. The adjusted peak areas were used to determine the percent composition of the fatty acids in the sample. Concentrations were determined based upon the known concentration of the internal standard added to the original oil sample. Results The data in Table 1 show that there were 73 fatty acids identified in the sample. Previously, fatty acid analyses had confidently identified 17 fatty acids, however it was always known that there were many unidentified fatty acids in Lyprinol. These 17 fatty acids are shown in the table in bold/yellow. Of the 73 fatty acids, there were 11 saturated fatty acids, 9 omega-3 fatty acids, 4 omega-4 fatty acids, 3 omega-5 fatty acids, 5 omega-6 fatty acids, 5 omega-7 fatty acoids, 5 omega-9 fatty acids, 1 omega-10 fatty acid, 16 fatty acids reported as mixed fatty acids which means the identity was not sufficiently clear to assign it to one or the other of the mixture, and 14 fatty acids present in such low amount it was not possible to establish the identity. It is not known if any of the newly identified could contribute to the bioactivity of Lyprinol, however the absolute amounts of many of these new fatty acids is small. Reference Michaud, A. L., G. Y. Diau, et al. (2002). "Double bond localization in minor homoallylic fatty acid methyl esters using acetonitrile chemical ionization tandem mass spectrometry." Analytical Biochemistry 307(2): /10/03 3

4 Table 1. Fatty Acids Contained in the Lyprinol Sample Fatty Acid Weight % of Total conc.(mg/ml) 12: ± ± :0br ± ± :0br ± ± :0 8.4 ± ± 1 15:0 1.1 ± ± :0 18 ± ± 2 17:0br 0.13 ± ± : ± ± :0 3.3 ± ± 0 20: ± ± : ± ± :3n ± ± 0 18:4n ± ± 1 20:3n ± ± :5n-3 21 ± ± 3 21:5n ± ± :5n ± ± :6n-3 13 ± 0 87 ± 1 24:6n ± ± :8n ± ± :2n ± ± :2n ± ± :3n ± ± :4n ± ± :1n ± ± :1n ± ± :1n ± ± ± :2n ± :2n ± ± :3n ± ± :4n ± ± 1 22:4n ± ± :1n ± ± 1 18:1n ± ± :2n ± ± :1n ± ± :1n ± ± :1n ± ± /10/03 4

5 16:1n ± ± :1n ± ± :1n ± ± 1 22:1n ± ± :1n ± ± :1n : ± ± :0 / 15: ± ± :1n-6 / 16: ± ± :1n-9,11 / 16:2n ± ± :2n-6 / 17: ± ± :1n-7, ± ± :1n-7, ± ± :0 / 17:1n ± ± :3n-4 / 17:1n ± ± :1n-11, ± ± :1n-9,8 / 18:3n ± ± :2 / 19:4n ± ± :3 / 21: ± ± :5 / 20:4n ± ± :2 / 22: ± ± :5n-6 / 24: ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± : ± ± 0.2 Fatty Acids shown in yellow are those previously recognised in Lyprinol. 13/10/03 5

6 Appendix Table 2 How much vitamin E we take from oils? Tocopherols milligrams per 100 grams* Tocotrienols milligrams per 100 grams* Grand Total IU** Oil alpha beta gamm a delta Total alpha beta gamm a delt a Tota l mg/ 100g per 100g Soybean Corn Canola Sunflower Peanut Cottonseed Safflower Palm Coconut Olive Evening primrose Wheat Germ Rice Barley Oat Butter Lard Margarine * 100 grams of oil equals 0.54 cups. To get these numbers per cup of oil multiply by ** Note that in many oils the alpha-tocopherol is less than half of the grand total of all tocopherols and tocotrienols. 13/10/03 6

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