True or False: Mycotoxins in Coffee Are a Serious Threat to Our Health and the Only Way to Avoid Them is Abstinence

As we are about to see coffee is by far not the worst aflotoxin offender in the human diet. Still, that does not mean that the coffee related exposure to this form of mold that can befall all sorts of grains, nuts and seeds is harmless.

You will probably remember that I casually touched on the possibility of being exposed to aflotoxins and more importantly ochratoxins as a result of the consumption of mold-infected coffee in previous coffee articles. When the issue of the « coffee ➲ aflotoxin / ochratoxin exposure ➲ serious health » triage resurfaced in a brief facebook conversation, recently, I realized that I was not 100% sure if these mycotoxins that are produced by Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, and Aspergillus carbonarius do or don't pose a serious health risk.

Well, you know how much I hate unanswered questions, so I kept digging until I'd found what I consider to be a half-way satisfying answer to this life-or-death question ;-)

You can learn more about coffee at the SuppVersity

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Coffee + Cacao for Breast Cancer Prevention

"Decaf" Won't Help With Weight, ... Ahh, Fat Loss

"The amount of aflotoxin in the average cup of coffee is a serious threat to our health!"

In 90% of the cases the above statement is FALSE! Despite the fact that it is difficult to tell how much mycotoxins you've been flushing down with your morning coffee today, the exact amount of the more common aflotoxins and their similarly kidney-toxic, pro-carcinogenic and coffee-loving cousins, the ochratoxin, in the average cup of (roasted) coffee is probably way too low to be worried about.

Figure 1: Mycotoxin exposure from coffee; calculated based on data from Europe (Vd Stegen. 1997)

Based on the studies I have reviewed for this article, it appears almost certain to say that the relatively low amount of coffee beans per cup (4-8g) reduces your average mycotoxin exposure from 1-4 cups of coffee per day to levels that are almost certainly within one of the various (multi-)national reference ranges (Van Egmond. 2007):

• Europe: 5µg/kg
• Africa: 10µg/kg

• North America: 20µg/kg

• Asia: 15µg/kg
• Latin America: 20µg/kg

Using a selection of commercially available regular and soluble coffee brands, van der Stegen et al. have actually calculated the average daily mycotoxin intake of Europeans with an average coffee consumption of 1-4  to be in the nanogram range - 19ng and 10ng, specifically, for regular and instant coffee drinkers, respectively.

Please remember: We don't know if / what happens or doesn't happen if you stay within or break the arbitrary intake limits. If you take a look at the available evidence from rodent studies, the results Epstein et al. presented in their 1969 paper (see Table 1) later in this article, would suggest that a sensible intake limit would have to be below the 1µg range if we assume a similar toxicity for the average mycotoxin mixture as for the aflotoxin B Epstein et al. used. This assumption is obviously not realistic, but it should remind you of the arbitrariness of the "intake limits".

Just to make sure, we understand each other, here: That's 1000x less than you'd get from only 20g of some of the Brazilian peanuts Freitas and Brigido analyzed in a 1998 study for their mean and maximal mycotoxin content. With 1099µg/kg the worst offenders in this study would easily have you breach the already lax American (North & Latin America) max. tolerable intake limit of 20µg/kg per day - and that despite the fact that this limit is more than 1000x higher than the amount of mycotoxins you'll have in your average cup of coffee.

Green vs. roasted coffee: Do we have to chose between anti-oxidants and mycotoxins?

It goes without saying that the mere fact that the dangers of being exposed to high amounts of mycotoxins from adequately processed and stored coffee appears negligible, does not warrant ignoring the problem completely. The effects of processing and storage are and will thus always be an important issue.

Given the fact that humid and cool (but not cold) is what mold needs to thrive, you will probably already have suspected that the hot and arid environment of a coffee roastery is not exactly the favorite growth environment for the Aspergillus family.

Figure 2: Ochratoxin content (µg/kg) in green, roasted & soluble coffee, left; total antioxidant activity in TROLOX essay of green (=unroasted), lightly, medium and dark roast coffee (Blanc. 1998; del Castillo. 2002)

A lower mycotoxin content is yet not the only beneficial effect of roasting. Contrary to what 99% of the people will tell you, when you ask them, the roasting process increases not decreases the total antioxidant capacity of coffee (TAC).

What does the latest review say about regular coffee consumption and cancer? "The epidemiological evidence consis-tently indicates that coffee protects against liver cancer, and also point toward protective effects for risk of colorectal cancers (with relative risks of 0.50 (95% CI: 0.42–0.59) and 0.83 (95% CI: 0.75–0.92), respectively, in the most recent meta-analyses)" (Bøhn. 2013). The evidence for protective effects against breast and prostate cancer on the other hand is inconclusive - irrespective of the established chemo-preventive effects of coffee phytochemicals Bøhn et al. list in their soon to be published paper in Molecular Nutrition & Food Research early in 2014.

Due to the formation of a whole host of new antioxidant molecules during the roasting process, light or mildly roasted coffee beans have a higher total antioxidant capacity than green ones - irrespective of the reduced chlorogenic acid content, for which the green beans are currently (over-)hyped. If you look at the data in Figure 2 you will see that even the heavily roasted, tar-black coffee beans still have a minimally higher anti-oxidant activity than the "natural" green coffee beans (del Castillo. 2002) - an observation that has been made both in the Petri dish, as well as ex vivo rodent studies (Daglia. 2002).

You've been drinking tons green coffee, lately?

Don't worry! Your past green coffee consumption probably isn't a real problem either. It does after all look as if those beans were yet another example for the infamous "nature kows best" principle. I mean, can it really be "coincidence" that the beans come with "anti-mycotoxin agents" in form of cafestol and kahweol. These coffee-specific diterpenes have been shown to ameliorate the aflotoxin B induced genotoxicity (Cavin. 1998) and the subsequent pro-carcinogenic effects (Cavin. 2001) and can be expected to exert protective effects against ochratoxin toxicity, as well.

In view of the fact that similar evidence exists for chlorogenic acids (CGA), dodecyl chlorogenates (DCGA) and a high(er) coffee consumption, in general (Suárez‐Quiroz. 2013; Ferk. 2013), it is actually not surprising that studies like Shank et al. (1972)  or Bulatoa-Jaym et al. (1982) found links between aflotoxin contaminated corn, grains, potato, peanuts & co., but could not identify an increased risk in liver cancer for coffee aficionados. Consequently, it's (imho) relatively unlikely that your health has already taken a beating - irrespective of the amount of the number of cups of green coffee you've been consuming over the past weeks.

Coffee is not the worst mycotoxin offender in the human diet: In a case-control dietary study of primary liver cancer in humans Bulatoa-Jaym et al. found that 51.2% of their subjects daily aflotoxin exposure came from cassava, 20.3% from corn, 6.8% frompeanuts and 5.8% from sweet potato (Bulatao-Jaim. 1982). On the other hand, many scientists argue that the ochratoxin content of coffee is the real danger, anyway (Bayman. 2006)

I would still like to remind you that even the sum of the previous remarks must not be misunderstood as an incentive to willy nilly forget all previously harbored concerns about "moldy coffee beans"... and I say this in spite of the existing epidemiological evidence that a high coffee consumption decreases the risk of developing and dying from liver cancer (Kurozawa. 2005; El–Serag. 2007). The liver is after all not the only organ that may be affected by the pro-carcinogenic toxins. The kidneys are at least as susceptible to the toxic assault from the mycotoxin filtrate that passes through them.

The liver is not the only organ that's taking a beating

It may thus be hypothetical, but not impossible that a 2x / 2.6x increased risk to develop renal cell carcinoma Mimi et al. report in a 1986 paper on the associations between coffee consumption and kidney cancer may at least be partially related to the higher mycotoxin exposure in those 61 study participants who consumed 1-4 cups of coffee per day (Mimi. 1986).

That mycotoxins can promote the development of kindey cancer had been demonstrated 17 years before the publication of Mimi's paper by Epstein, Bartus & Farber (1969) whose Wistar rats developed renal epithelial neoplasms after being exposed to food-borne aflatoxin B1.

Table 1: Incidence of renal epithelial and malignant hepatic tumors in male Wistar rats ingesting aflatoxin B1 for 147 days; the indces a, b, c provide irrelevant (in this context) extra information (Epstein. 1969)

In that, it's quite remarkable that even the lowest aflotoxin dosage the researchers used in their study (0.25µg/kg chow; HED  ~1.2ng/kg body weight, ; see Table 1) lead to significant rates of cancerous growth in both kidney (28%, if we count both developing and full-blown renal neoplasms) and liver  within the 21 week study period.

There is just one no-go: Storing unroasted beans for years in your humid basement

In view of the large regional difference in aflotoxin infection rates, the different susceptibility of the various coffee cultivars and the influences of weather, storage conditions, blending, processing, and all the other factors that increase or decrease the amount of mold and mycotoxins on coffee (see Figure 3), I would still be hesitant to exclude the possibility that stocking up on highly aflotoxin contaminated unroasted coffee you possibly even stock in a very humid basement of yours to consume the coffee over the course of months if not years could have negative effects on the health of your organs, in general, and the function of your kidney and liver, in particular.

Figure 3: Percent infection of coffee cherries and beans byAspergillusspecies potentially capable of producing ochratoxin A in four Brazilian coffee growing regions from the 1999 and 2000 harvests (Taniwaki. 2003)

I mean, look at the data in Figure 3. It's probably no coincidence that the otherwise virtually uninfected beans of coffee from the 1999 and 2000 harvests in the Cerrado Miniero exhibits a 4% infection rate after being stored intermediately before it is either roasted, shredded or both or simply forwarded "raw" to the mailbox of an "unroasted coffee enthusiast". Similar effects can be expected when the huge coffee manufacturers mix harvests from various regions. If only one is infected, all it takes to have Aspergillus flavus literally "all over the place" is enough time in one of the huge storage silos or the hold of one of the container ships that transport coffee from the "New" back into the "Old World".

Figure 4: The amino acid make up of coffee changes upon roasting (data from Cirilo. 2003).

Life kills, anyway! Let's be honest. In the end, living is a pretty deadly undertaking, anyway. Against that background the uncertainty with respect to the tolerable intake of mycotoxins should not bother you so much to ignore the existing evidence of the beneficial effects of regular coffee consumption (see "Coffee - The Good, the Bad & The Interesting" | read more).

One thing you may keep in mind, though, is that this evidence is based on data from average coffee drinkers, people who drink coffee that's made of roasted beans. Beans that are virtually mycotoxin-free (see Figure 2, left) and have a higher, not lower antioxidant capacity than green coffee beans.

What roasted beans lack, though, are chlorogenic acid and trace amounts of amino acids (see Figure 4), including serotonin. If you are looking for one of these molecules specifically, you are yet probably better of with a hopefully aflotoxin and mycotoxin free green coffee extract and a bottle of pills with the serotonin precursor 5-HTP, anyways.

References:

• Bayman, P., & Baker, J. L. (2006). Ochratoxins: a global perspective. Mycopathologia, 162(3), 215-223. 
• Blanc, M., Pittet, A., Muñoz-Box, R., & Viani, R. (1998). Behavior of ochratoxin A during green coffee roasting and soluble coffee manufacture. Journal of agricultural and food chemistry, 46(2), 673-675.
• Bøhn et al. (2013) Coffee and cancer risk, epidemiological evidence, and molecular mechanisms. Molecular Nutrition & Food Research [early view article]
• Bulatoa-Jaym J, et al. (1982). A Case-Control Dietary Study of Primary Liver Cancer Risk from Aflatoxin Exposure*. International journal of epidemiology, 11(2), 112-119.
• Cavin, C., Holzhäuser, D., Constable, A., Huggett, A. C., & Schilter, B. (1998). The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism. Carcinogenesis, 19(8), 1369-1375.
• Cavin, C., Mace, K., Offord, E. A., & Schilter, B. (2001). Protective effects of coffee diterpenes against aflatoxin B< sub> 1</sub>-induced genotoxicity: mechanisms in rat and human cells. Food and Chemical toxicology, 39(6), 549-556.
• del Castillo, M. D., Ames, J. M., & Gordon, M. H. (2002). Effect of roasting on the antioxidant activity of coffee brews. Journal of Agricultural and Food Chemistry, 50(13), 3698-3703. 
• Cirilo, M. P., Coelho, A. F. S., Araújo, C. M., Gonçalves, F. R., Nogueira, F. D., & Glória, M. B. A. (2003). Profile and levels of bioactive amines in green and roasted coffee. Food Chemistry, 82(3), 397-402.
• Daglia, M., Papetti, A., Gregotti, C., Bertè, F., & Gazzani, G. (2000). In vitro antioxidant and ex vivo protective activities of green and roasted coffee. Journal of Agricultural and Food Chemistry, 48(5), 1449-1454.
• Epstein, S. M., Bartus, B., & Farber, E. (1969). Renal epithelial neoplasms induced in male Wistar rats by oral aflatoxin B1. Cancer Research, 29(5), 1045-1050.
• El–Serag, H. B., & Rudolph, K. L. (2007). Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology, 132(7), 2557-2576. 
• Ferk, F., Huber, W. W., Grasl‐Kraupp, B., Speer, K., Buchmann, S., Bohacek, R., ... & Knasmüller, S. (2013). Protective effects of coffee against induction of DNA damage and pre‐neoplastic foci by aflatoxin B1. Molecular nutrition & food research. 
• Freitas, V. P., & Brigido, B. M. (1998). Occurrence of aflatoxins B1, B2, G1, and G2 in peanuts and their products marketed in the region of Campinas, Brazil in 1995 and 1996. Food Additives & Contaminants, 15(7), 807-811.
• Kurozawa, Y., Ogimoto, I., Shibata, A., Nose, T., Yoshimura, T., Suzuki, H., ... & Tamakoshi, A. (2005). Coffee and risk of death from hepatocellular carcinoma in a large cohort study in Japan. British journal of cancer, 93(5), 607-610. 
• Mimi, C. Y., Mack, T. M., Hanisch, R., Cicioni, C., & Henderson, B. E. (1986). Cigarette smoking, obesity, diuretic use, and coffee consumption as risk factors for renal cell carcinoma. Journal of the National Cancer Institute, 77(2), 351-356.
• Shank, R. C., Wogan, G. N., & Gibson, J. B. (1972). Dietary aflatoxins and human liver cancer. I. Toxigenic moulds in foods and foodstuffs of tropical South-East Asia. Food and Cosmetics Toxicology, 10(1), 51-60.
• Taniwaki, M. H., Pitt, J. I., Teixeira, A. A., & Iamanaka, B. T. (2003). The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. International Journal of Food Microbiology, 82(2), 173-179. 
• Van Egmond, H. P., Schothorst, R. C., & Jonker, M. A. (2007). Regulations relating to mycotoxins in food. Analytical and bioanalytical chemistry, 389(1), 147-157. 
• Vd Stegen, G., Jörissen, U., Pittet, A., Saccon, M., Steiner, W., Vincenzi, M., ... & Schlatter, C. (1997). Screening of European coffee final products for occurrence of ochratoxin A (OTA). Food Additives & Contaminants, 14(3), 211-216. 
• Yamato, T., Yamasaki, S., Misumi, Y., Kino, M., Obata, T., & Aomine, M. (2002). Modulation of the stress response by coffee: an in vivo microdialysis study of hippocampal serotonin and dopamine levels in rat. Neuroscience letters, 332(2), 87-90.

Green Tea Extracts, Athletes and a Preliminary Answer to the Question: "Are Anti-Oxidants For Athletes Not?" No True Benefits or Negative Effects of 1g GTE in Sprinters

The supplement that was used in the study at hand was a commercially available product from Olimp Labs, a Polish producer of bodybuilding and fitness supplements.

Before I even go into more detail, I would like to point out that the study today's SuppVersity article will talk about is not able to answer the question whether anti-oxidants are for athletes once and for all. Why? Well, the subjects in the recently conducted experiment by Ewa Jówko, Barbara Długołecka, Beata Makaruk and Igor Cieslinski were sprinters from a University Sports Club, and they received a green tea supplement - so who can guarantee that a bodybuilder taking vitamin C would not have a totally different reaction to a totally different anti-oxidant?

No one can and that's why I'd like to ask you to go back to some of the previous articles on that matter and remind yourself that there is evidence that the provision of significant amounts of supplemental antioxidant can blunt the beneficial adaptive response to exercise (learn more and even more).

Learn more about hormesis and potential neg. effects of antioxidants at the SuppVersity

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NAC Impairs Anabolic Effects of Exercise

Vitamin C + E Hamper Gains in the Elderly

C+E Useless or Detrimental for Healthy People

Vitamin C and Glucose Management?

Antiox. & Health Benefits Don't Correlate

Apropos "significant amounts"! If we take a look at the amount of green tea extract, the 16 male sprinters (21.6 y; 76.9kg; 11.8% body fat) received, a question arises: Are 2x250 mg of standardized GTE (245 mg polyphenols, including 200 mg catechins, among them 137 mg epigallocatechin-3-galate) a "significant amount" of green tea extract (GTE) if they are consumed twice daily?

I guess there may be supplement junkies out there who consume way more than this 1g of green tea extract everyday. Based on the dosages in studies that report beneficial effects of GTE, 1g is yet already on the high(er) side of the dosing continuum and thus unquestionably a "significant amount" of green tea extract, which was administered in a randomized controlled crossover study that was conducted during preparatory phase of yearly training cycle (after transition period) of the sprinters all of whom had more than 4 years of training experience.

What do the latest reviews say about exercise + antioxidant supplementation? In spite of the fact that you will find reviews with different undertones, the vast majority of reviewers concludes that convincing evidence of the long-assumed benefits of anti-oxidant supplementation does not exist.Whether this warrants conclusions as the one Mari Carmen Gomez-Cabrera, Michael Ristow and Jose Viña formulate in their 2012 paper(s) and "the vast majority of experimental evidence clearly advises against this supplementation" (Gomez-Cabrera. 2014), however is still a matter of open debate (Holloszy. 2012).

The two 4-week treatment periods (during which half of the subjects received GTE and the other half PL, and vice versa) were separated by a 4-week washout period. The duration of the washout period was selected based on the results of one previous study (Brown. 2011), in which 6-week supplementation with higher amounts of catechins (800 mg/day) was used on obese subjects. In view of the fact that the plasma catechin concentration in this study returned to its baseline level after at least 2 weeks of washout period, we can safely assume that a 6-week washout in highly active non-obese individuals should be enough to get rid of all the effects of only 250mg of catechins.

Both GTE and PL were administered in the form of dark gelatin capsules (Olimp Labs, De˛bica, Poland), identical in appearance (i.e., size, shape, and color); the same dosage regimen was used (two capsules twice a day). One GTP capsule contained 250 mg of standardized GTE (245 mg polyphenols, including 200 mg catechins, among them 137 mg epigallocatechin-3-galate) and additional substances (maltodextrin, microcrystalline cellulose, and magnesium stearate). Therefore, each participant was administered 980 mg polyphenols daily. PL capsules contained microcrystalline cellulose, magnesium stearate, and maltodextrin instead of GTP."

Compliance was measured by counting the capsules the subjects returned. Participants who returned no more than 15 % of their capsule dose were classified as "compliant". At the end of each of the two 4-week treatment periods, the sprinters performed a repeated cycle sprint test (RST) on a cycle ergometer (Ergomedic 839E, Monark, Sweden).

Based on the food logs, the scientists decided that there were no significant nutritional differences between the two phases of the study (Jówko. 2014)

Dietary standardization: The participants were asked to not modify their diet for the duration of the study, except for refraining from consuming any products containing green tea and limiting the intake of caffeine-containing drinks to one cup per day. Moreover, they were asked to maintain a similar
diet for both treatment periods. During both the first and the second treatment periods (during 7 days preceding each RST), the participants filled out a 3-day dietary record (covering 2 week days and 1 day of the week end).

The test consisted of four consecutive 15-s bouts (4 x 15 s), each of them with base set according to the Wingate procedure and separated by 1-min rest intervals. The subjects were asked to cycle for 15 s, as fast as possible, against a constant load (75 g/kg body weight).

The performance tests were performed in the morning following 12-hovernight fast, at air temperature between 19 and 21°C and with 40–60 % relative humidity. The subjects were instructed to not perform hard physical training for 48 h and avoid drinking tea and caffeinated beverages within
24 h prior to each of the RSTs.

Figure 1: Changes in blood indices of acid–base balance & lactate concentration induced by the repeated sprint test (49 x15 s) in sprinters (n=16) after 4-week supplementation with placebo (PL) or green tea extract (GTE; Jówko. 2014)

As you can see in Figure 1, there were no treatment (only time) effects as far as the acute changes in blood indices of acid–base balance and plasma lactate concentration are concerned. Against that background it's not surprising that there were no changes in the performance results of the repeated sprint test, either. Peak power, mean power, total work output, and fatigue index during the Wingate protocol were identical.

Table 1: Changes in blood parameters of oxidative stress and muscle damage induced by the repeated sprint test (49 x15 s) in sprinters (n=16) after 4-week supplementation with placebo (PL) or green tea extract (GTE; Jówko. 2014)

An observation that certainly raises the question, whether the treatment effects that were observed for the total antioxidant capacity and Superoxide Dismutase (SOD) levels (see Table 2) are even physiologically significant. Personally, I'd say no, because higher TAC and SOD levels have no health or performance value on their own.

Previous studies suggest that NAC impairs the adaptive response to exercise | learn more

Bottom line: In spite of the fact that the study at hand does not provide evidence that the commonly assumed beneficial ergogenic effects of green tea supplements exists, the results are still good news for green tea supplement users. They do after all suggest that the provision of significant amounts of anti-oxidant catechins does not appear to hamper the adaptive response to exercise.

In that, it's important to mention that the study at hand acquits only green tea, yet not vitamin C, NAC & co which act via different mechanisms of the charge of having potentially detrimental effects on the adaptive response of athletes, average joes and/or obese type II diabetics... and just to remind you: Theoretically the response of all three of them could be totally different | comment on Facebook!

Reference:

• Brown, A. L., et al. "Health effects of green tea catechins in overweight and obese men: a randomised controlled cross-over trial." British Journal of Nutrition 106.12 (2011): 1880-1889.
• Gomez-Cabrera, Mari Carmen, Michael Ristow, and Jose Viña. "Antioxidant supplements in exercise: worse than useless?." American Journal of Physiology-Endocrinology and Metabolism 302.4 (2012): E476-E477. 
• Holloszy, J. O., et al. "Response to letter to the editor by Gomez-Cabrera et al." American Journal of Physiology Endocrinology and Metabolism 302 (2012): E478-E479.
• Jówko, Ewa, et al. "The effect of green tea extract supplementation on exercise-induced oxidative stress parameters in male sprinters." European Journal of Nutrition (2014): 1-9.

Study Puts "?" Behind Beneficial Health Effects of Veggies! Is There No Correlation Between Antioxidant Content & Beneficial Health Effects of Cucumber, Lotus & Rape!?

Don't obsess about "optimal" antioxidant contents, just eat your veggies!

Over the past couple of weeks, ... no actually over the past years I have repeatedly written about the concept of (mito-)hormesis and its consequences for the well-established, but not necessarily accurate free radical theory of aging (and for some people everything else). ROS, i.e. reactive oxygen species, have been established as an important signalling molecule that is - among other things - heavily involved in the insulin sensitizing effects of exercise. "Inflammation" makes muscles grow and burns body fat and the "what doesn't kill me makes me strong" principle appears to reign everywhere you look.

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That being said, the latest study from the Institute of Health and Environmental Medicine in Tianjin, China, opens another "anti-antioxidant" Box of Pandora. One that puts a huge questionmark behind the implications of hundreds of thousands of scientific studies, when it says in it's title, already: "No correlation is found for vegetables between antioxidant capacity and potential benefits in improving antioxidant function in aged rats"

"Skin of Grape Tomatoes Contains Max. Amount of Antioxidants" - You can find this and dozens of other daily updated SuppVersity Science News on www.facebook.com/SuppVersity

This is a title that may in fact change the way we look at study results like those of a recent study by Valdez-Morales, et al. (2014) investigating the "best" = highest antioxidant tomato, the results of which you are about to find among the ~20/day SuppVersity Facebook News @ www.facebook.com/SuppVersity - don't forget to like it, or you'll miss out on the latest science news!

If the results of the study can be confirmed by an independent team for vegetables other than lotus root, rape or cucumber and if there is an identical mismatch between the in-vivo anti-oxidant capacity and the potential benefits in improving antioxidant function in (aged) humans.

This would be big and highly consequential news for nutrition experts, scientists and average Joes and Janes like you and me. Why? Well,...

• any ranking of "superfoods" that was based even partly on in vitro data derived with the good old ferric reducing antioxidant power (FRAP) assay would be invalid, ...
• every scientist who has been following up on "promising" data from FRAP assays would have been wasting his time, ...
• and you may have been eating all the wrong foods for years...

... hell no, as long as you ate your veggies over the past years, I wouldn't worry if you may have made a "suboptimal" selection (which would be different based on whatever new criteria you select).

Figure 1: FRAP value, vitamin C and vitamin E content and total amount phenolics in the powdered vegetables that were added to the rodent diets in the study at hand (Ji. 2014)

Honestly, I'd hope that you didn't select your foods only based on the orthorexic principle of maximal antioxidant content, anyways. 

Never forget the three principles of veggie eating: Variety, seasonality, colorfulness

Against that background I'd recommend you keep eating your lotus roots, if you like them, although, they have a significantly lower beneficial effect on SuperOxide Dismutase (SOD, a group of antioxidant enzymes) than rape and cucumber.

Figure 2: Serum markers of anti-oxidant status / oxidative damage after 6 weeks on the three experimental diets (Ji. 2014)

Moreover, if you look closely at the data in Figure 1+2, you will realize that lotus may suck at SOD and its ability to reduce hemolysis (the destruction of red blood cells), but will have the most profound beneficial effects on the levels of malondealdehyde (MDA), a marker of lipid oxidation, and the amount of plasma carbonyls, which have - just as in cellular regulation, aging, and disease (Levine. 2002). Just like their similarly radical cousins, carbonyls will thus play a dual role so that in the end, their reduction may not be beneficial in each and every case.

Figure 3: Blood mononuclear cell DNA damage expressed as total injury rate (%) and total tails low (% of all) in male Wistar rats on control and experimental diets (Ji. 2014)

The Take Away: Whatever the role of carbonyls, MDA & co may be and no matter what you believe which of the three tested vegetables may be the "best" one, if there is one definite message you can take home from today's SuppVersity article, it's not to overly rely on the abstract data from chemical tests the reliability of which appears to be inversely proportional to their accuracy.

Trust your instincts and go for a broad variety of vegetables. Eat seasonal! Eat colorful! And most importantly eat plenty. Optimal or not, none of the vegetables in the study at hand would harm you - all of them would help you defy diabesity and slow the aging process as best mother nature allows.

Reference: 

• Ji, Linlin, et al. "No correlation is found for vegetables between antioxidant capacity and potential benefits in improving antioxidant function in aged rats." Journal of Clinical Biochemistry and Nutrition 54.3 (2014): 198-203.
• Levine, Rodney L. "Carbonyl modified proteins in cellular regulation, aging, and disease^{2, 3}." Free Radical Biology and Medicine 32.9 (2002): 790-796.
• Valdez-Morales, Maribel, et al. "Phenolic content, and antioxidant and antimutagenic activities in tomato peel and seeds, and tomato by-products." Journal of Agricultural and Food Chemistry (2014). Accepted Manuscript.

6mg Melatonin 30min Before HIT Will Increase Fatty Acid Oxidation, Boost Your Antioxidant Capacity, Reduce MDA Levels and Modulate Your Immune Response

Image 1: Other than you may have expected, this is exactly not what happened when the soccer players took 6mg of melatonin before their workouts.

The blogpost on melatonin's anti-Alzheimer's + anti-obesity effects from last week caused quite a stir, both in the comment area, here at the SuppVersity, as well as on facebook. Even friends in the gym came up with questions. Therefore, I suppose that you won't mind, if I re-address the topic today. This time, however, with data from a human study, of which I bet that it will catch your interest... after all, the study, which has been published ahead of print two days before Christmas could hold the key to winning the FIFA World Cup 2014 ;-)

Melatonin makes you sleepy? I don't think so!

I guess, even after last week's news, most of you will still think of melatonin as the "sleep hormone". At least those of you who follow my recommendation to make sure that they get their daily dose of SuppVersity news should yet already be familiar with the notion that melatonin may also be a very effective ergogenic aid / adaptogen to be taken not at night, but right before an event! Contrary to the Ococha study, which, due to its awkward "loading protocol" and the ultra-endurance setting, had little relevance for the average trainee, the recently published study by M.D. Maldanado and his (or her) colleagues (Maldando. 2011) from the Department of Medical Biochemistry and Molecular Biology at the University of Seville Medical School and the Andalusian Centre of Sports Medicine in Spain is approaching an area of physical activity that is much more dear to my heart: Football! Or as you, my American friends would say, "soccer" ;-)

Note: The scientists make a very useful comment with regards to the timing of exogenous melatonin, I guess you will be interested in: "Melatonin itself has a very short half-life in the blood (range of 20–40 min, depending on condition), so that elevated levels cannot persist after many hours. According with concentration-time curve of our laboratory and other researchers, 30-45 min is [sic!] the necessary time so that oral melatonin is absorbed in the gastrointestinal tract (GIT) and can be detected in blood before its metabolism and elimination. On the other hand, considering that melatonin is not toxic and has no undesirable effects, the 6 mg administered assured us their absorption by the mucous and is within the ranges recom- mended for use in humans (3–20 mg)." I hope that will spare me at least a few of the question some of you are probably already harboring ;-)

To study the effects of pre-workout melatonin supplementation on markers of oxidative damage, the Spanish scientists recruited sixteen 18 to 20 year old professional soccer players, and randomized them to receive either placebo or 6mg of melatonin 30 minutes prior to an intense (HR >135bpm; speed 25km/h) 60-min training session on stationary bikes.

Figure 1: Plasma total antioxidant activity (TAS) and lipid oxidation (MDA) before, during and right after 60min of high intensity (heart rate >135bpm) exercise on stationary bike (data adapted from Maldando. 2011)

As you can see in figure 1 the 6mg of melatonin the subjects received immediately before the arduous steady state high intensity "cardio" exercise, did not just ameliorate the -22% (60min mark) decrease in total antioxidant capacity (TAS) in the soccer players, it did in fact raise the TAS levels to +15% over baseline, while the subjects were pedaling on their bikes. Consequently, the increase in lipid oxidation (MDA) levels was profoundly reduced, yet not completely blunted (let alone reversed).

Figure 2: Plasma triglyceride before, during and after 60min HIT training (data adapted from Maldando. 2011)

For those of you who, besides being athletic, also like to look athletic, it may also be of interest that Maldando et al. ascribe the statistically signifant decrease in plasma triglyceride levels in the melatonin group to what they call an increased "catchment and lipids consumption by cells" - and for the native speakers out there - this is the Spanish way of saying that melatonin increases fatty acid oxidation during high intensity steady state exercise and could thusly be highly beneficial for anyone who wants to shed some additional pounds (in addition to what you diet will do for you) on the treadmill, bike or similar "cardio equipment" - and before you ask, I guess this will work for HIIT, as well ;-)

Melatonin increases immune response to 60-min HIT exercise

Its antioxidant and fat-burning activity aside, the 6mg of melatonin eight of the sixteen soccer players consumed before they hopped onto their stationary bikes exerted a statistically significant modulatory effect on plasma IgA levels (ca. +40% after 60min), about which Maldano et al. state that it

[...] indicates the start of a humoral immune response, for which it takes between 5 and 7 days [...so that melatonin] could act to promote the adaptation between plasma and mucosal IgA during the exercise.

Overall, I am becoming more and more convinced that my statement from last week that "melatonin is probably one of the most underrated supplements you can still buy without a script" is actually pretty accurate and that melatonin, as Maldono et al. put it, "could be a plausible therapeutic option for professional athletes that should make major exercises throughout their working lives" ;-)

Exercise Intensity, Oxidative Damage, Glycogen Depletion and Supercompensation. Plus: Optimal 0-12h Post Workout Glycogen Repletion Protocol For Performance Athletes

Do they train at the right intensity and what is the right intensity? What's right, anyway? Lot's of questions, tons of words, a couple of answers and some interesting revelations in today's 2nd article of the SuppVersity Exercise Science Week.

This is day 2 of the SuppVersity Exercise Science Week -- another day, another news. After you've learned about the various mechanisms by which exercise will induce structural changes to your beer belly, lover handles and other problem and non-problem areas, in yesterday's first article of the SuppVersity Exercise Science Week, today's post does actually pick up on the notion of the superiority of high intensity exercise and takes a look at how low vs. high(er) intensity endurance exercise effects the antioxidant defense system of the body. This will lead us to an issue that was once considered to be a downside of high intensity workouts: their notoriousness to deplete muscle glycogen, of which we now know that it is actually one of their fundamental strengths. When we are done with that, it's about time for the sweet dessert. The latter is going to have three courses and will help you achieve maximal muscle glycogen supercompensation after a workout.

Where does the idea that you better work out at low intensities come from?

I have made it a (enervating?) habit to include a small reminder of the "dark side" the same beneficial exercise stress that elicits muscle gains, fat loss, and improvements in conditioning and overall health can have, whenever you don't allow for adequate recovery and nutrient supply, in almost every of the articles pointing to the superiority of high intensity training vs. training in the comfort zone (click here to read up on a couple of these articles).

Figure 1: A comprehensive study by Carey revealed that the increase in ratio of fat-calories to total energy ependiture, when you train in the "fat burning zone" is 3% for men, 5% for women. The total amount of fat is yet higher above the "zone" and, most importantly, the current research suggests that the glycolytic effect, which is inversely related to the relative fat oxidation, is what triggers most of the beneficial metabolic effects.

The question, how pronounced the differences actually are, on the other hand, is not just rarely addressed here at the SuppVersity, it's also something scientists are still trying to elucidate. Usually you will see creatine kinase, an accepted marker of skeletal muscle damage being accessed before and after a workout, but as I have pointed out in previous articles, my personal experience tells me that an intense strength workout is - despite its ability to increase CK levels in training noops by up to 10,000% (x100, no typo - eg. Sewright. 2008) less prone to send you down into the abyss of the Athlete's Triad, than working out for hours (worst on a daily basis) in the purported fat burning zone, i.e. the target heart rate where you'll satisfy the greatest part of your metabolic demands from body fat and of which Carey has been able to show in "relatively fit" male and female runners that it is at least 30% below the anaerobic threshold (AT: 155Hb/min; Fat Burning Zone: 105Hb/min; cf. Carey. 2009).

Aside from that, Carey's results also support the observation Wilson et al. formulate in their recent review of concurrent training, namely that "most dramatic loss in fat mass occurr[s] from moderately high to very high intensities" (Wilson. 2012). In this context, the scientists' definition of "moderately high" is already way beyond the alleged zone of maximal fat loss. "Dramatic" is by the way also an excellent attribute for the 4.5x higher fat loss effect Wilson et al. computed for the highest vs. medium exercise intensities  (91-100% vs. 61-80% HRMax) based on the data they collected for their review.

"Better fat loss, w/ high intensity, aha... but isn't that at the cost of increased oxidation?"

In view of the fact that will be coming back to the issue of "optimal fat loss" later this week, anyway, I guess it's best we get back to the topic at hand and take a look at the toll endurance workouts at different exercise intensities actually take on your antioxidant defense system. As mentioned before, it is still far from being certain which markers you would actually have to measure to get a clear picture of how much stress and damage a given exercise regimen is inflicting. Compared to the creatine kinase levels, the measurement of markers of the activity and status of the anti-oxidant defense system, which was the main outcome variable in a study by Takahasi et al. does yet appear to be more relevant - if not with respect to exercise performance than certainly with respect to overall and metabolic health.

Figure 2: Changes in myeloperoxidase, heart rate, rate of perceived exertion and trolox equivalent antioxidant capacity (TAEC) in eight healthy and untrained males aged 22.6 ± 1.4 years (mean ± SD), with 67.7 ± 4.1 kg body mass, 175.2 ± 3.7 cm height, and 15.1 ± 2.2% body fat after 20min of exercise at 70%, 100% or 130% of the anaerobic threshold.

On three separate occasions, the Japanese researchers studied the effect of different exercise intensities. The latter ranged from 70% over 100% to 130% of the anaerobic threshold and would thus represent exercising in the "fat burning zone" at moderately high intensities and high intensities.

The first thing the scientists registered was that the pre to post increase in oxidative stress at the low and medium intensities did not even reach statistical significance. The "pro-oxidative" effects of the high intensity trial, on the other hand, were statistically significant. Yet, if you look at the actual data in figure 2, I'd guess that you will - just like me - ask yourselves what all the hoopla was about: The absolute differences are mediocre, at best and their physical not statistical significance is highly questionable; and that's not just because the trolox equivalent antioxidant capacity (TEAC) actually increased from pre to post exercise (from allegedly lower pre levels in the 130% trial than before the other exercise tests.

Training at higher intensities is demanding, yeah... but not overtly demanding!

Now, all these statistical significances were calculated on a pre vs. post basis. Intensity-specific differences on the other hand were not observed. We do therefore have to be cautious not to misinterpret the scientists very own and actually non-judgmental conclusion ...

"We found that plasma concentrations of d-ROMs increased as a result of 20 min of exercise above AT. Exercise above AT also increased enzymatic and nonenzymatic antioxidant capacity. On the other hand, there was no effect after 20 min of exercise at 70–100% AT, suggesting that exercise under the AT level does not produce oxidative stress damage." (Takahashi. 2013)

... as an advice to stick to "exercise under the AT [anaerobic threshold]". There are already way too many people wasting their time on the cross-trainers of this word - don't join them, but don't overexert yourself either.

The "Iranian HIIT Solution" has already proven that a minimalist HIIT regimen in the form of 3x200m sprint sessions per week can make all the difference esp. for someone who has never participated in regular activity before (read more).

A single bout of intense exercise leads to significant improvements in glucose and lipid metabolism in obese individuals, that's the latest result of another very recent study that was conducted at the University of Glasgow (Whyte. 2013). The protocol consisted of nothing more than " four maximal 30-s sprints, with 4.5min recovery between each (HIIT), or a single maximal extended sprint (HIT) matched with HIIT for work done". With 20% higher mean power during the sprints the temporary intensity was higher, in view of the fact that the overall exercise duration was longer and there was no time for in-between sprint glycogen replenishment. Thu it's actually not surprising that the acute increase in insulin sensitivity did reach statistical significance only after the extended sprint session. The overall metabolic benefits (non-significant improvements in glucose and lipid metabolism) on the day after, of which we can assume that they were not brought about by the immediate reduction of muscle glycogen, were identical for both conditions, while the the total and relative increase in fasting fatty oxidation was more pronounced after the HIIT protocol (total: 63% and 38%; relative, based on RER: 11% and 8% ).

Figure 3: Oxidative stress and glycogen depletion are important triggers of the beneficial effects of exercise on glucose metabolism ( (based on Kawanaka. 2012).

If we go a step further and think about whether or not oxidative stress is actually something you would want to avoid at all costs, the figure from Kentaro Kawanaka's recently published alongside review of the regulation of glucose transport in skeletal muscle during and after exercise (see figure 3) can help us make up our minds. If you take a look at my mark-ups it's plain to see that ROS production and the increase in AMP (quasi "used ATP") and decreases in ATP and phosphocreatine (PCr) are major signals for the activation of a hitherto incompletely understood signaling cascade that results in increased glucose uptake by the muscle. That's the same glucose uptake, by the way that makes the most significant difference between the "normal" and, insulin-intolerant individual and makes an ideal stepping stone to full-blown diabesity (=obesity + diabetes type II).

"So, what exactly is the effect size of these improvements? Are the worth the sweating?"

To illustrate the quantity of these effects, Kawanaka uses data from a 2009 study by Koshinaka et al. who subjected rats to an acute bout of 3x20s "high-intensity sprint interal swimming" and measured muscle glycogen levels and glucose transport at different timepoints in the 16h window after the workout.

Figure 4: Insulin and non-insulin stimulated glucose transport in rat epitrochlearis muscle at rest and 4 hours after cessation of HIIT exercise (left); muscle glycogen repletion and supercompensation after a workout (from Kawanaka. 2012 based on Koshinaka. 2009)

If we take into account that 3h(!) of continuous swimming elicited the exact same improvement in glycogen uptake as those 3x20s all out "sprints", I probably don't have to say it "appears" as if the synergistic combination of brief HI(I)T training and an appropriate diet will be more productive than the endless hours on an elliptical way too many (often unfortunately female) trainees are still performing in the desperate hope to finally shed the fat from whatever problem areas they have or believe they'd have.

Glycogen supercompensation: This is how it's done

There is yet more to the Koshinika study than another confirmation of the usefulness of HI(I)T exercise for fat loss, fitness and fabulous health. The data Koshinaka et al. collected does also tell us something about post workout glycogen repletion. Most importantly (at least in my humble opinion) that the first, immediate post-exercise phase is characterized by a rapid non-insulin dependent increase in glucose uptake. The latter is actually just as high (>5µmol/g/20min; respective data is not shown in figure 4) as the maximally measured glucose uptake in phase II, in the course of which the presence of insulin has a dose-dependent beneficial effect on the total amount of glucose that's going to be shuttled into the muscle (see figure 4, left). With phase III being characterized by saturated (in fact more than saturated) glycogen stores, these observations would suggest that an "optimal" glycogen replenishment protocol would look somewhat like this:

When you increase your calorie intake on a bulk, you better go really high carb + low fat, if lean gains are what you're looking for. This is at least what a 2011 study by Mendes-Netto suggests (read more)

1. phase I: immediately post > fast absorbing carbohydrate source -- what's important during the immediate post-workout phase is exclusively the availability of glucose, insulin the presence of extra high insulin levels is more or less unnecessary
2. phase II: post workout phase (<8h) > high GI carbohydrate source -- once the glycogen levels have reached a certain level the supercompensation process requires the presence of additional insulin, therefore your post-workout meal should not be carb-free or extremely low GI
3. phase III: recovery phase (>8h) > low GI carbohydrate source -- the glycogen stores have already reached higher than baseline levels, the presence of high levels of insulin in this phase would be counterproductive as it would actually drive glucose uptake by the adipose, not the muscle tissue

Whether this maximum glycogen repletion protocol does in fact make sense for everyone is yet another question, though. For someone who trains twice a day, like Arnold, it certainly does. The same goes for endurance athletes looking for maximal performance. If Lance Armstrong, for example, would ever be allowed to compete again, he would best go for a fast absorbing carbohydrate source like Vitargo right after the race, a huge bowl of pasta and some sugary grape juice as his first meal after the race and some slow digesting carbs like a couple of bowls of oats later that day to ensure optimal glycogen levels on the next day of the Tour -- what neither Lance nor you should not forget, though, is to add some protein to the equation, even if building muscle is not your goal, the protein will speed up the replenishment of muscle glycogen (Zawadski. 1992)

"But how important is muscle glycogen, anyway?"

For the average trainee it does yet remain questionable whether or not this protocol will actually yield noticeable benefits. While it is important to replete the glycogen stores, the advantages of doing this as fast as possible are actually not really relevant for someone who trains 3-4 times per week in order to promote health, well-being and a leaner, more muscular (but not freakish) physique. Especially with respect to the latter, the majority of the more recent studies clearly suggests that muscle protein synthesis is, in the short run, not impaired by low levels of muscle glycogen (click here to learn more).

What you should never forget, though, is that your body will interpret chronically low muscle and liver glycogen levels as a clear-cut indicator that you're starving. The results are a reduced metabolic rate and the shut down of "auxilliary" and costly bodily functions such as the reproductive machinery, etc. - and we don't want that to happen, right?


References:

• Kawanaka K. Regulation of glucose transport in skeletal muscle during and after exercise. 2012. J Phys Fitness Sports Med, 1(4): 563-572.
• Koshinaka K, Kawasaki E, Hokari F, Kawanaka K. Effect of acute high intensity intermittent swimming on postexercise insulin responsiveness in epitrochlearis of fed rats. Metabolism. 2009; 58: 246-253.
• Takahashi M, Suzuki K, Matoba H, Sakamoto S, Obara S. Effects of different intensities of endurance exercise on oxidative stress and antioxidant capacity. J Phys Fitness Sports Med. 2013 1(1): 183-189.
• Sewright KA, Hubal MJ, Kearns A, Holbrook MT, Clarkson PM. Sex differences in response to maximal eccentric exercise. Med Sci Sports Exerc. 2008 Feb;40(2):242-51.
• Whyte LJ, Ferguson C, Wilson J, Scott RA, Gill JM. Effects of single bout of very high-intensity exercise on metabolic health biomarkers in overweight/obese sedentary men. Metabolism. 2013 Feb;62(2):212-9.
• Wilson JM, Marin PJ, Rhea MR, Wilson SM, Loenneke JP, Anderson JC. Concurrent training: a meta-analysis examining interference of aerobic and resistance exercises. J Strength Cond Res. 2012 Aug;26(8):2293-307. 
• Zawadzki KM, Yaspelkis BB 3rd, Ivy JL. Carbohydrate-protein complex increases the rate of muscle glycogen storage after exercise. J Appl Physiol. 1992 May;72(5):1854-9.