Effectiveness of whole dried Dunaliella salina marine microalgae in the chelating and detoxification of toxic minerals and heavy metals.

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1 Effectiveness of whole dried Dunaliella salina marine microalgae in the chelating and detoxification of toxic minerals and heavy metals. Zac Bobrov, Ian Tracton, Kathrine Taunton, Dr Megan Mathews March 6, 2008 Background The microalgae Dunaliella salina (DS) is an edible, nutrient rich, single cell, marine phytoplankton (micromarine algae). Dunaliella salina is one of the most ancient life forms on earth and is considered to be an extremophile as it lives in an extreme saline environments where it is commonly exposed to severe UV radiation. As a response to these environments Dunaliella salina produces high levels of antioxidants and Dunaliella salina has recently been developed as a novel food source/nutraceutical with high levels of carotenoid antioxidants, minerals and trace elements with whole dried Dunaliella salina being listed on the Australian Register of Therapeutic Goods (AUSTL ) and available as an over-the-counter nutritional supplement sold under the trade name Algotene. Dunaliella salina is one of the highest natural sources of dietary carotenoids. It is richest in beta-carotene. Natural beta-carotene consists of several isomers, with the two main isomers 9-cis and all-trans having different functions. The 9-cis isomer is known as one of nature s most powerful antioxidants. The all-trans isomer is a major pro-vitamin A nutrient, but has little antioxidant activity. In contrast to synthetic beta-carotene, which only contains the alltrans isomer, natural (all-trans) beta-carotene is found together with other carotenoids. Both synthetic and natural forms of all-trans beta-carotene are readily converted by the body into vitamin A and thus play an essential role in vision, growth, reproduction and regulation of the immune system and in helping maintain the health and integrity of the skin and mucous membranes. (Braun 2005). However, while high doses of vitamin A can be toxic, betacarotene is only converted to vitamin A by the body as required, thus making it non-toxic even when given at doses of up to 180mg/day for long periods of time. (Mathews-Roth 1990). Studies involving microalgae, such as chlorella, have suggested that dried algae consumed in sufficient quantities may be beneficial in the removal of toxic elements from the body. Whole Dunaliella salina cells contain particularly high levels of carotenoids, minerals and other phytonutrients making this algae potentially useful in assisting with elimination of toxic elements and heavy metals. To date most of the clinical research on Dunaliella salina has been on beta- carotene containing extracts of Dunaliella salina rather than the whole organism. Human studies suggest that Dunaliella salina extracts can protect against exercise-induced asthma (Neuman, Nahum et al. 1999; Moreira, Moreira et al. 2004), normalise high LDL oxidation in patients with diabetes (Levy, Zaltsberg et al. 2000) and male hyperlipidaemic smokers (Chao, Huang et al. 2002). Dunaliella salina also protects against radiation damage, as demonstrated by its use in children exposed to the Chernobyl disaster (Ben-Amotz, Yatziv et al. 1998). It protects the skin from sun damage by providing double the skin s protection against sunburn. This was shown when 24mg of beta-carotene derived from Dunaliella salina was taken daily for more than 10 weeks (Heinrich, Gartner et al. 2003). Animals studies further suggest that natural beta-carotene from Dunaliella salina can protect against

2 gastrointestinal inflammation (Lavy, Naveh et al. 2003), water immersion stress (Takenaka, Takahashi et al. 1993), whole body irradiation (Ben-Amotz, Rachmilevich et al. 1996) and CNS oxygen toxicity (Bitterman, Melamed et al. 1994). The purpose of the study is to determine the bioavailability of the carotenoids in whole dried Dunaliella salina cells and its effect on mineral stores when given over a 14 week period. The study also aimed to assess the effect of Dunaliella salina supplementation on the heavy metal content of hair, which is recognized by the World Health Organization, the International Atomic Energy Agency, and the US Environmental Protection Agency as being an indicator of heavy metal contamination. It was hypothesized that the supplementation with Dunaliella salina will: Increase the body s beta-carotene stores in a dose dependent fashion. Improve the bodies mineral stores and balances Produce a noticeable alteration in skin colour. Increase serum beta-carotene that will reach a plateau within Materials and Methods Subjects recruited were healthy male and female adults with normal diets, able to provide informed consent and attend the study site for required tests. Blood serum carotenoid levels were monitored at regular intervals weeks 0 (baseline), 1, 3, 6, 9, 12 and 14. Hair mineral levels were assessed at baseline and week 14. These assessments included analysis of nutrient and trace minerals as well as heavy metals such as Pb, Cd, Hg, As, Be and Al using established ICP-Mass Spectrometry analytical techniques. Subjects were also given a questionnaire asking about demographic data, smoking status, skin appearance, side effects and general health. Three grams of whole dried Dunaliella salina were administered to participants in capsule form as three 500mg capsules (Algotene ) taken twice daily over a fourteen week period. The product used consisted of encapsulated whole dried Dunaliella salina cells which contained around 1.5 beta-carotene. The active product all came from the same batch. An analysis of that batch including a precise breakdown of carotenoids, antioxidants and minerals was performed. Serum Carotenoid Testing Methods Samples of 1.0ml serum were analysed using the Shimadzu HPCL (High performance liquid chromatography ) system. The method used for serum carotenoid testing involved the precipitation of plasma proteins followed by extraction of compounds in an organic phase, separation on a reverse phase HPLC column and measurement using a programmed variable wavelength UV-visible detector. The reference range was established by the testing laboratory with a normal Range for beta- Carotene of µmol/L with a detection limit of 0.1µmol/L.

3 Tissue Mineral Analysis Procedure Hair specimens were obtained by normal and accepted collection protocol, i.e. approximately 150 mg. of untreated hair obtained from four to five different locations of the posterior vertex and posterior temporal regions of the scalp, not exceeding one and one-half inches in length from the scalp. Excess distal hair was discarded prior to sending the specimen to the lab. In the trace element laboratory, the hair specimen was cut into hair strands less than 3mm in length and mixed to allow a representative sub-sampling. After cutting, each specimen was weighed accurately (+/ g) then transferred into a 15 ml acid-washed polypropylene centrifuge tube to which ultra-pure trace-metal grade HNO3 was added. The specimen tubes containing the HNO3 and hair were then introduced into a CEM Mars 5 Plus Microwave Digestion apparatus. Under precise microprocessor control, the hair specimen was subject to the HNO3 acid and a uniform high temperature digestion via a twostage temperature ramping sequence, utilizing a fibre optic temperature probe placed into a representative specimen tube within the microwave digestion tray. In the first stage, the samples were taken to 70 degrees centigrade and held for 20 minutes. After 20 minutes, the temperature was slowly increased (ramped) to 115 degrees centrigrade and held for an additional 15 minutes. After digestion, the samples were cooled, uncapped and then accurately diluted to a set volume with a deionized water/gold solution, recapped and placed on a vortexer for thorough mixing of each hydrated specimen. All measurements were performed on a Perkin-Elmer Mass Spectrometer (Sciex Elan 6100 or 9000). Instrument calibration for all analytes was performed with the linear regression method by means of a four-point calibration curve using commercially available (NIST sourced) stock standard solutions. Accuracy of study calibration and entire methodology was verified by the appropriate use of; reagent blanks, independent calibration verification, standard check solutions, and the use of pooled hair specimen, pooled hair aqueous solutions and other hair material with established reference ranges. Verification of calibration linearity and recalibration of the mass spectrometer was performed after every 24 specimens. Results Carotenoid levels Table 1 presents the results of vitamin A and carotenoid testing at each time point. All participants showed a marked increase in serum carotenoid levels, with larger increases seen among the non smokers. The greatest increase during the 14 week study was 767 from baseline, with other non smoking participants showing increases of 247, 290 and 511. In the two smokers, blood serum carotenoids increased by 111 and 193. The participant who reported having the highest intake of fruit and vegetables in the questionnaire also had the highest baseline carotenoid level at the beginning of the test. The increase in serum carotenoid level at week 14 for this participant was in the mid-range of results observed (290). Responses to the questionnaire showed at least one participant noticed an increase in skin tone and colour, believing it was an improvement. While no patients reported any adverse reactions, one patient reported cold and flu symptoms during the study.

4 Table 1 Serum carotenoid and Vitamin A analysis at baseline and TEST SUBJECT No. WEEKS DATE VITAMIN A ( umol/l)* Carotene ( umol/l)* Participant 1 4/04/ year old, male 1 19/04/ non-smoker, 3 28/04/ high consumption 6 17/05/ of fruit & vegetables 9 6/06/ /06/ /07/ Participant 2 5/04/ year old, female 1 11/04/ non-smoker, 3 26/04/ low-moderate consumption 6 10/05/ of fruit & vegetables 9 30/05/ /06/ /07/ Participant 3 9/04/ year old, female 1 20/04/ smoker, 3 3/05/ moderate consumption 6 15/05/ of fruit & vegetables 9 9/06/ /06/ /07/ Participant 4 13/04/ year old, male 1 20/04/ non smoker, 3 5/05/ moderate consumption 6 22/05/ of fruit & vegetables 9 9/06/ /06/ /07/ Participant 5 6/04/ year old, female 1 13/04/ non-smoker, 3 28/04/ moderate consumption 6 11/05/ of fruit & vegetables 9 1/06/ /06/ /07/ Participant 6 7/04/ year old, male 1 20/04/ smoker; 3 8/05/ high consumption of 6 26/05/ fruit & vegetables 9 16/06/ /07/ /07/

5 * Reference ranges established by the testing laboratory Tissue Mineral Analysis Results Results showed significant reductions in the levels of heavy metals and toxic elements across the majority of participants in particular Arsenic, Lead Cadmium and Copper. Table 2 presents the results of the Hair Tissue Mineral Analysis testing. Arsenic (As) Arsenic levels were consistently lower across all participants at week 14 with the largest reduction being 77 and the smallest reduction 23 of baseline levels. Cadmium (Cd) Four out of six sets of results showed reductions in levels of cadmium. The maximum reduction was shown at 75. One participant with the highest baseline level of cadmium showed a 93 increase in their results along with increased levels of mercury and lead. Lead (Pb) Five out of six participants showed a notable reduction from the baseline levels with the highest reduction being 78. Copper (Cu) Copper levels were reduced in five out of six participants. One participant who was well below the reference range at baseline testing showed an increase in copper of 18 placing the result closer to the recommended reference range for copper. The participant who showed the most dramatic reduction in copper levels also had the highest baseline copper level. This was the same participant with the highest baseline levels of mercury, cadmium and lead. Nickel (Ni) Four out of six participants showed notable nickel reductions. Tin levels were increased in one of the participants, again in the same individual who had shown the highest levels in other metals. Tin (Sn) Four out six participants showed a reduction in tin levels. One patient s level remained static. Mercury (Hg) Two of the six participants showed slightly elevated mercury and both these showed a 25 and 8 reduction respectively. The other patients showed statistically low levels of mercury at the lowest end of the reference scale, with variations at this level of little statistical significance. Aluminium (Al) All participants registered as below the reference scale for aluminium at baseline testing, however five out of six participants showed further reductions. Beryllium (Be) There were no changes in any participants beryllium levels. All six participants tested below calibration levels at baseline and in final testing.

6 Table 2 Heavy metal and elemental analysis at baseline and SUBJECT As Cd Pb Cu Ni Sn Hg Al <.020* <.014* <.30* * * * < 0.18* <1.8* * Reference ranges established by the testing laboratory Decrease Increase - statistically insignificant Dark shading indicates above reference range at baseline

7 Discussion The study demonstrated that whole dried Dunaliella salina cells contain bioavailable carotenoid antioxidants and significantly elevated blood serum carotenoid levels in all participants. It is interesting to note that at baseline all participants had carotenoid levels at the lower end of the laboratory reference range, indicating that the wider Australian population may have low carotenoid levels and that supplemental sources may be of value in the diet. Of further interest is the number of subjects who had significant levels of heavy metals in hair specimens above the reference range at baseline. This suggests that heavy metal contamination may possibly be a feature of the general population. Results showed significant reductions in the hair levels of heavy metals and toxic elements across most participants. These results strongly suggest that whole dried Dunaliella salina may be extremely useful in detoxification and chelation and warrant further investigation. While we hypothesize that the high level of carotenoid antioxidants, nutrient minerals other properties found within the algae are primarily responsible for the detoxification results, the exact mechanism is unproven. Conclusion Whole dried Dunaliella salina cells are an effective source of bioavailable carotenoids and 14 weeks of supplementation with Dunaliella salina can produce favourable shifts in heavy metals and elemental minerals. The results of this preliminary observational study now need to be confirmed in a more rigorous randomized controlled trial. For more information contact: lab@interclinical.com.au References Braun, L., Cohen, M. (2005) Herbs and Natural Supplements : An evidence based guide. Sydney, Elsevier Mathews-Roth, M. M. "Carotenoid functions in photoprotection and cancer prevention" Journal Of Environmental Pathology, Toxicology And Oncology: Official Organ Of The International Moreira, A., P. Moreira, et al. (2004). "Increased dietary beta-carotene intake associated with better asthma quality of life." Alergologia e Inmunologia Clinica 19(3): Neuman, I., H. Nahum, et al. (1999). "Prevention of exercise-induced asthma by a natural isomer mixture of beta-carotene." Annals Of Allergy, Asthma & Immunology: Official Publication Of The American College Of Allergy, Asthma, & Immunology 82(6): Ben-Amotz, A., S. Yatziv, et al. (1998). "Effect of natural beta-carotene supplementation in children exposed to radiation from the Chernobyl accident." Radiation And Environmental Biophysics 37(3): Levy, Y., H. Zaltsberg, et al. (2000). "Dietary supplementation of a natural isomer mixture of

8 beta-carotene inhibits oxidation of LDL derived from patients with diabetes mellitus." Annals Of Nutrition & Metabolism 44(2): Chao, J. C.-J., C.-H. Huang, et al. (2002). "Effects of Β-carotene, vitamin C and E on antioxidant status in hyperlipidemic smokers." Journal of Nutritional Biochemistry 13(7): Heinrich, U., C. Gartner, et al. (2003). "Supplementation with beta-carotene or a similar amount of mixed carotenoids protects humans from UV-induced erythema." The Journal Of Nutrition 133(1): Lavy, A., Y. Naveh, et al. (2003). "Dietary Dunaliella bardawil, a & Beta;-carotene-rich alga, protects against acetic acid-induced small bowel inflammation in rats." Inflammatory Bowel Diseases 9(6): Takenaka, H., H. Takahashi, et al. (1993). "Protective effect of Dunaliella bardawil on waterimmersion-induced stress in rats." Planta Medica 59(5): Ben-Amotz, A., B. Rachmilevich, et al. (1996). "Natural beta-carotene and whole body irradiation in rats." Radiation And Environmental Biophysics 35(4): Bitterman, N., Y. Melamed, et al. (1994). "Beta-carotene and CNS oxygen toxicity in rats." Journal Of Applied Physiology (Bethesda, Md.: 1985) 76(3):