Monday, October 10, 2011

Are There Pro-Oxidant, Cytotoxic and DNA-Damaging Anti-Oxidants in Your Supplements? Not all Forms of Vitamin C and Vitamin A are Equally "Harmless"!

Image 1: The common believe that you could not satisfy your micro-nutrient requirements without the use of a daily multi-vitamin flushes >8 billion US$ into the coffers of the industry.
I assume just one of the consumers who spent about >8 billion dollars in total for multi-vitamins each year (Balluz. 2000), aren't you? Now, tell me: Did it ever occur to you that some of those cheap vitamins, which are meant to provide you with all the vital nutrients you are supposed to be missing, because you are eating pizza, pasta and burgers instead of real food, could actually be killing your cells and damaging your DNA? In case you haven't I suggest you read on and take a look at the results of a recently published study by Therese Bergström, Jan Bergmann and Lennart Möller from the Department of Biosciences and Nutrition in Huddinge, Sweden (Bergström. 2011).

Before we get to the details, I want you to to go and take a look at the bottle of the multivitamin (or multiple vitamin products) in your supplement stash... ok, look at the label - what does it say? I suppose somewhere on the top you will find both Vitamin A and vitamin C listed, maybe even with the adjunct "antioxidants". If you bought one of the higher quality products it will probably also say "from..." followed by the specific type of vitamin A or C used in your product - usually this is hardly legible, so you better have your magnifying glass at hand ;-) If you bought your supplement in Europe, you could find one of the following ingredients there:
Figure 1: Compounds permitted in supplements by the European Parliament’s directives 2002/46/EC (EU. 2002)
Strange, isn't? While most people have been indoctrinated to believe that beta-carotene was the "better", "healthier" and "safer" alternative to "real vitamin A", few know that there are three different forms of preformed, i.e. "real" vitamin A, and even fewer people are aware of the broad range of vitamins C that can be used in dietary supplements according to the European Parliament and Council’s directive 2002/46/EC.
Image 2: Partly apoptotic HL-60 cells
under the microscope (img.
Why leukemia cells? It is certainly a valid question to ask, why the researchers used leukemia cells in this trial, although their intention was not to investigate desirable cytotoxic effects of high dose anti-oxidants on cancer cells, but to find out whether or not high doses of commonly used dietary supplements could exhibit undesirable cytotoxic, pro-oxidant and DNA-damaging side-effects. The reason is pretty simple a a typical example of how counter-intuitive science can be: Those cells are simply and easily available and highly versatile and standardized(!) model for studying the molecular events of myeloid differentiation and the effects of physiologic, pharmacologic, and virologic elements on this process. Asking "why HL-60" is thus similar to asking "why rats" - convenience and conventionalism!
A similar ignorance exists towards the possible pro-oxidant and cytotoxic effects of those "vital" nutrients, everyone believes he would be deficient in, if he did not pop one of those high dosed vitamin preperations on a daily basis - better safe than sorry, no? According to the results of the study at hand, that largely depends on a) how high-dosed your vitamin supplement really is, and b) on which form of the antioxidants the manufacturer of your supplement has used (and you bet that you get what you pay for ;-)
Figure 2: Cytotoxicity [in % of non-viable cells] of vitamin A compounds on the viability of HL-60 cells after 24-h exposure at a concentration of 20µM; each bar represents the average of a minimum of four independent experiments; * p<0.001 (data adapted from Bergström. 2011).
As far as its cytotoxity is concerned retinal certainly stands out (cf. figure 2). Of the five tested vitamin A compounds, retinal was the only one with literally sure-fire cytotoxic effects (94%; p<0.001!) on HL-60 cells after 24h of incubation at 10 times the normal plasma concentration of retinol (2µM; cf. Zemplini. 2007). Unfortunately, that does not mean that the other vitamins A are "safe": Both, plain retinal and retinol at physiological concentrations of 2µM, as well as the cheap and widely used retinyl acetate at slightly super-physiological doses of 6µM did increase dG oxidation (the occurrence of 8-oxo-dG, the oxidized form of deoxyguanosine is an indicator of DNA damage) in a acetate buffer (0.03M Zn2+). Interestingly, the oxidative damage due to both retinal and retinol, but not the one caused by retinyl acetate, were significantly reduced, if, instead of the acetate buffer, a phosphate buffer was used.
Figure 3: Cytotoxicity [in % of non-viable cells] of vitamin C compounds on the viability of HL-60 cells after 24-h exposure at a concentration of 500µM (for AA6P solubility was so low that a lower dose had to be used); each bar represents the average of a minimum of four independent experiments; ** p<0.01, * p<0.05 (data adapted from Bergström. 2011).
As the data in figure 3 goes to show, at 10x the normal plasma concentration (50µM; cf. Duarte. 2005), which is basically what is supposed, but actually won't be achieved* (see red box below) by the mega-doses of vitamin C you see in recommended treatments for all sorts of ailments on various more or less reliable health-related websites, plain ascorbic acid (28%) and sodium ascorbate (28%) exhibit statistically significant cytotoxity. In case of the "buffered vitamin C" calcium ascorbate, even only 32 out of 100 promyelocytic leukemia cells (HL-60) survived the profoundly cytotoxic anti-oxidant bath.
*Note: In view of the ability of your body to clear "superfluous" vitamin C from the blood and regulate serum vitamin C levels so, that they will constantly remain in the < 200µM range, it is very unlikely that oral supplementation with whatever form of vitamin C will be actually suffice to induce cytotoxic damage to your cells (thx. to majkinetor for the heads up). On the other hand, this does also mean that you are unlikely to achieve those exorbitant levels which have been associated with the often touted active (not preventive) anti-cancer effects of vitamin C. Keeping an eye on your daily intake to maintain adequate levels is thus probably a very good idea, The use of high dose supplements (>500-1,000mg) for the average human being (including athletes) however is probably unnecessary.

My multi has one of the "toxic" vitamins in it! I will throw it away, right?

Table 1: Overview of the tested compounds and their potentially deleterious side-effects in the petri dish (Bergström. 2011)
All this may now easily sound like you have to flush your beloved vitamins down the toilette - at least, if you do not happen leukemia or any other form of cancer and wanted to use your A's and C's as a natural chemotherapy. In view of what you probably have read about "hormesis" and the general idea of balance and  moderation it would however be much wiser to...
  • first, evaluate how much of each of the vitamins you are already getting from your diet - chances are this is much more than you have been made to believe (at least if you stick to a whole foods diet)
  • second, select those supplements you really need and do not apply the "more helps more principle", the opposite is usually the case
In the unlikely case that you are following a whole-foods diet and still find that you are deficient in any of those vitamins you could not get by simply adding another servings of vegetables or fruit to your diet, pick the next best low dose supplement to meet your requirements.