Pigs Would Pick MSG - Glutamate Seals the Gut, Decreases Liver & Muscle Fat & Increases Plasma Amino Acids in Swine

Piglets would buy MSG food ;-)

Mono-sodium glutamate (MSG) and the "Chinese restaurant syndrome", obesity and overeating are often thrown together into a single psedo-scientific crock pot with the result being a brew that's 50% hear-say, 40% fear and 10% science. The study we are going to look at today is unquestionably part of the latter ingredient and its results do stand in line with my previously stated concern "that MSG is one of those substances that is usually found in foods with a whole host of other nutrient-poor ingredients, anti-nutrients and proven obesogenic, pro-inflammatory and otherwise unhealthy substances and food additives" ("MSG, NFALD, Leaky Gut & Brain ...") and could thus rather be corollary to, than causative of the toll the fast, convenient and nutrient deficient foods in the Western diet are taking on our health.

Published ahead of print in the online version of the journal Amino Acids you will find a study by a group of researchers from the Texas A&M University. The study was, according to the authors intended to "fill [the] important gap of knowledge about glutamate nutrition and metabolism in animals" (Rezaei. 2013). Luckily their study subjects were pigs, allegedly young pigs, but still omnivores like us and one of the best models of the human digestive tract we have:

"Both humans and pigs are highly dependent on dietary quality since symbiotic microorganisms within the gut play a relatively minor role in modifying the nutrients that are ingested. Intestinal  transit times and digestive efficiencies are comparable. Postabsorptive metabolism is also similar in many respects, although the wide differences in length of gestation and the numbers of young born introduce a potentially significant divergence in nutrient needs for reproduction. [...] Nevertheless, when minimum nutrient requirements of swine and established recommended daily allow­ ances of humans are expressed per kilogram of dietary dry matter (assuming an intake of 500 to 800 g of dry matter per day by teenagers and adults), these values are highly related. It is only reasonable that one not draw unsupport­able inferences from one species to another, but with the possible exception of nonhuman primates, it is apparent that the omnivorous pig is one of the best models for study of nutrition issues in the omnivorous human." (Miller. 1987)

Against that background it is quite intriguing that Rezaei. et al. did not find any of the suspected negative side effects of MSG up to a dosage of 4% in the diet of their piglets.

Figure 1: Weight development and feed intake and effciacy in pigs on diet containing different amounts of supplemental MSG (data based on Rezaei. 2013)

In fact, instead of eating more, the pigs that received the MSG-supplemented diets consumed slightly, but significantly less food than their peers. Despite these appetite suppressing effects of the diet, the piglets in the high MSG arm of the study still gained the most body weight and thusly had the 'optimal' (for lovers of Chines restaurant probably rather 'most detrimental') gain:feed ratio.

The amino acid modifying effects of MSG

When we are seaching for the underlying reasons of these changes, it may be worth taking a look at the amino acid composition of the plasma of the piglets after 21 days on diets supplemented with different amounts of MSG at 1 and 4 h after feeding. During this prostprandial phase, the scientists observed

• 

  More about MSG in human health

  significant increases  in aspartate, glutamate, glutamine, histidine, citrulline, arginine, taurine, alanine, methionine, valine, phenylalanine, isoleucine, leucine, proline, cysteine, ornithine, and lysine in plasma at both time points, i.e. one and four hours after feedin,
• highly significant increases in asparagine, serine, threonine, tryptophan, and tyrosine 1h after feeding and
• significant increases in alanine, citrulline, glutamate, methionine, ornithine, phenylalanine, proline, and tryptophan in the first hour of the postprandial window

If we also take into account previous rodent studies which have shown that MSG reduces the deposition of fatty acids in white adipose tissue (Kondoh. 2008), it cannot be ruled out though that these increases in weight gain were related to increases in lean- not fat tissue (remember: muscle is heavier than fat); after all we are dealing with growing young pigs, in which you would expect an increase in essential and non essential amino acid availability to help with skeletal muscle metabolism (Mahan. 1998).

Figure 2: Total lipid content in percent of control in response to MSG feeding at different doses (left) and the modulatory effects of sodium intake (NaCl) on the effects of MSG (right; data based on Rezaei. 2013)

As the data in figure 2 goes to show this hypothesis appears to stand in line with the decreased fatty acid deposition in liver and skeletal muscle, which will at the same time prevent negative side effects of intra-hepatic and -skeletal lipid accumulation on liver and muscle glucose uptake.

Does salt modify the effects of MSG? And what's the role of the gut in all this?

Against that background it is actually a pitty that we don't have data on the fatty acid content of liver and muscle tissue in response to the different levels of dietary salt in the diets (figure 2, right). I mean, at first sight it appears that more salt could 'ameliorate' the detrimental effects of MSG feeding on the body weight of the rodents, but if the latter was not detrimental, but beneficial, this would certainly entail the question if it's not MSG per se, but rather it's co-appearance with too much, or due to it's ability to boost all taste perception to little sodium in the previously mentioned fast, convenient and nutrient deficient foods, way too many people have gotten addicted to.

You see, just as so many times before things are way more complex than they may seem at first sight and if the interactions of body weight, lean mass, intrahepatic and intramuscular lipids and dietary salt with MSG was not already enough, the data in figure 3 brings another (side?) effect into play the importance of which must not be underestimated - the effect of MSG on the intestinal morphology of the pigs:

Figure 3: Jejunal morphology and jejunal concentrations of DNA, RNA, protein, ATP, and glutathione in 28-day-old pigs weaned at 21 days of age (Rezaei. 2013)

I don't know if you remember the side effect of the chronic ingestion of zinc on the intestinal structure of rodents that caused quite a stir in the zinc-loving bodybuilding community back in June!? In essence, the effects of mono-sodium glutamate on the microvilli, which are responsible for the absorption of nutrients look very similar to the ones that were observed by Taneja et al.in response to Zinc supplementation (SuppVersity: June 13, 2013). As previously mentioned this is per se not a bad thing and could in fact come very hand to people with chronic inflammatory conditions suffering from a "leaky gut" or people who want to protect their gut from the side effects of the chronic use of NSAIDs, where MSG has only recently been implicated as a viable tool to prevent and heal mucosal damage (Amagas. 2013).

Figure 4: Postprandial glucose levels (left) and intestinal morphology (right) of mice on diets with different concentrations of mono-sodium glutamate (Rezaei. 2013)

As figure 4 goes to show this could actually work with MSG without the zinc-induced increases in insulin and blood glucose (see figure 2 in previous article). Whether these effects are directly related to the ingestion of MSG or its glutamin-sparing effects n the gut cannot be said for sure, though:

"Grant alert" Despite the fact that I am pretty sure that the actuall data in this study is accurately reported, I still want to point out that the scientists received "a grant from the International Glutamate Technical Committee". It's explicitly listed in the "acknowledgments" and probably not much of an issue outside of the discussion in which you will obviously miss references to potential negative side effects (which have not been observed in the study, though).

"Thus, dietary supplementation with glutamate may enhance the availability of dietary glutamine in plasma. As a versatile amino acid, glutamate participates in both synthetic and oxidative pathways in the small intestine, resulting in the production of proteins, ornithine, citrulline, proline, arginine, alanine, aspartate, glutathione, CO2, and ATP. Therefore, dietary supplementation with glutamate increased the plasma concentrations of these amino acids  and jejunal concentrations of glutathione in weaned pigs. Compelling evidence shows that dietary glutamate is a major energy substrate for the small intestine, which is an organ with a particularly high met- abolic rate. In support of this notion, we found that dietary MSG supplementation increased jejunal concentrations of ATP in weaned pigs. Additionally, glutamate is an excitatory neurotransmitter, thereby regulating the motility of the gastrointestinal tract. Thus, when a weaning diet is deficient in glutamate, gut atrophy occurs and the efficiency of utilization of dietary protein for growth and other physiological functions is greatly decreased." (Rezaei. 2013)

As evidence from previous studies by Kondoh et al. suggests, the effects of glutamate do not end at the intestinal brush border. Its centrally mediated downstream effects after interacting with l-Glutamate receptors in the intestines are however still not fully understood and could either be beneficial (as the work by Kondoh et al. would suggest; Kondoh. 2008 & 2009), be without physiological consequences or - as the mainstream myth suggests - "be the devil"; with the latter being much more likely in people with genetic or already established metabolic problems which result in a deficiency of glutamate dehydrogenase (Stanley. 2009).

Bottom line: The last mentioned problems certain individuals who have inherited or acquired problems with the enzymatic conversion of glutamate are yet not the only reason why I strongly caution against taking the results of the study at hand as a free ticket for limitless MSG consumption. If it's not the MSG that's going to make you fat, I can assure you that those 'foods' in which it is used will be getting the job done pretty quickly and will thus compensate for any possibly existent improvements in intestinal and whole body amino acid metabolism.

Parmigiano Reggiano aside from seaweed the #1 "real food" offender in terms of MSG and still good for your bones (Pampaloni. 2011) - one of many examples of the fallacy of black-and-white thinking. To heal your gut, glutamine would yet still be your better choice, I suppose ;-)

That said, there are still unresolved issues related to the negative effects of MSG on the immune system and the thymus. The dosages that are required to observe toxic effects may be hilarious if you take into account how much of it you find in an individual food item, and even if you started supplementing with MSG, or lived on fast- and convenient food, only, you will probably be hard pressed to get up to the 50g+ human equivalent of mono-sodium glutamate which was sufficient to significantly decrease thymus cell viability in rats (Pavlovic. 2009). In case you feel you are endangered and belong to the people who rather wear a helmet than stop hammering their head against a wall, you could try to counter that with an additional 6-7g of vitamin C (for the rodents that worked)... but let's be honest, wouldn't your life be much easier, if you simply stuck to whole foods and don't worry about the occasional piece of aged Parmesan cheese with 1680 mg glutamate per 100g. It could not just be good for your gut, but has been shown to be good for your bones (Pampaloni. 2011), probably not because, but at least despite the high MSG content.

References:

• Amagase K, Ochi A, Kojo A, Mizunoe A, Taue M, Kinoshita N, Nakamura E, Takeuchi K. New therapeutic strategy for amino acid medicine: prophylactic and healing promoting effect of monosodium glutamate against NSAID-induced enteropathy. J Pharmacol Sci. 2013;118(2):131-7.
• Kondoh T, Torii K (2008) MSG intake suppresses weight gain, fat deposition, and plasma leptin levels in male Sprague-Dawley rats. Physiol Behav 95:135–144.
• Kondoh T, Mallick HN, Torii K. Activation of the gut-brain axis by dietary glutamate and physiologic significance in energy homeostasis. Am J Clin Nutr. 2009 Sep;90(3):832S-837S.
• Mahan DC, Shields RG Jr. Essential and nonessential amino acid composition of pigs from birth to 145 kilograms of body weight, and comparison to other studies. J Anim Sci. 1998 Feb;76(2):513-21.
• Miller ER, Ullrey DE. The pig as a model for human nutrition. Annu Rev Nutr. 1987;7:361-82. 
• Pampaloni B, Bartolini E, Brandi ML. Parmigiano Reggiano cheese and bone health. Clin Cases Miner Bone Metab. 2011 Sep;8(3):33-6.
• Pavlovic V, Pavlovic D, Kocic G, Sokolovic D, Sarac M, Jovic Z. Ascorbic acid modulates monosodium glutamate induced cytotoxicity in rat thymus. Bratisl Lek Listy. 2009;110(4):205-9.
• Stanley CA. Regulation of glutamate metabolism and insulin secretion by glutamate dehydrogenase in hypoglycemic children. Am J Clin Nutr. 2009 Sep;90(3):862S-866S.
• Rezaei R, Knabe DA, Tekwe CD, Dahanayaka S, Ficken MD, Fielder SE, Eide SJ, Lovering SL, Wu G. Dietary supplementation with monosodium glutamate is safe and improves growth performance in postweaning pigs. Amino Acids. 2013 Nov 2.

Calcium, Magnesium, Potassium & Co in Food, Water & Supps - Getting Enough is Easy, Knowing How Much Is Not!

Image 1: "Minerals? Yeah that's the stuff you need to avoid cramping" While this is certainly true, the mineral loss during "normal" workouts is largely overblown, the most important and actually only necessary ingredients in respective drinks, even for Ironman Triathletes, are salt, water and sugar and what's worse this prejudice conceals the importance of electrolytes for our general health.

While it is Thursday, it is plain to see that this is not Adelfo Cerame's weekly SuppVersity post. There have been a couple of issues with the promised workout videos and neither I nor Adelfo wanted to postpone them yet another week so that we decided to rather publish videos + Adelfo's weekly update tomorrow instead of a reduced snippet today. To make sure you have more than enough food for thought to bridge the time, I applied a coupe of tweaks to a longer snipped from the next installment of On Short News that dealt with the protective effects of high(er) intakes of calcium, magnesium and potassium on the incidence of vascular dementia (=dementia in response to low blood flow to / oxygenation of the brain) and Alzheimer's dementia (=dementia due to the build up of plaque in the brain). As you may already have seen, the result got somewhat epic, so let's not waste anymore time and get straight into the original data before it's too late and we have already become demented ;-)

Don't forget your minerals or they'll soon be just one of the many things you tend to forget

While the studies and reviews on the effects of minerals, especially calcium (and as of late also magnesium), on cardiovascular health is about as abundant as the assessments of their individual and joint benefits and / or pitfalls, their role in the etiology of another, quieter, but not less prevalent pandemic is still insufficiently studied. Against that background, the results Ozawa et al. present in a recently published paper in the Journal of the American Geriatric Society could well provide some novel insights on whether or not forgetting to keep an eye on your mineral intake now will make you forget more than just a couple of minerals in the more or less distant future - and that even if none of the 1081 community dwelling elderly (>60y) Japanese the scientists followed up for 17 years is even remotely related to you ;-)

Figure 1: Hazard ratios for all-cause, vascular and Alzheimer's dementia for patients in the lowest to highest quartiles of potassium (≤1,856 / 1,857–2,149 / 2,150–2,559 / ≥2,560), calcium (≤431 / 432–531 / 532–638 / ≥638) and magnesium (≤147 / 148–169 / 170–195 / ≥196) intake in mg/day (top) and difference in intake of selected foods in the highest vs. lowest quantile of overall mineral intake (bottom; all calculated based on Ozawa. 2013)

Aside from the general association of higher potassium, calcium, and magnesium intake with lower incidence of dementia, which was - given the overall low median intake - more or less to be expected, there are a couple of other very noteworthy things I want you to take note of (see figure 1; data adjusted for age; sex; low education; history of stroke; hypertension; diabetes mellitus; total cholesterol; body mass index; smoking; alcohol intake; regular exercise; and energy, vitamin C, cholesterol, saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid intake):

• a higher mineral intake was had a more pronounced beneficial impact on vascular compared to Alzheimer's dementia (-77% vs. -46% max. reduction)
• for potassium and calcium the general rule of thumb is "the more, the better" (the deviation from that rule in the individual analysis for Alzheimer's is statistically nonsignificant), but I don't this is mediated by the overall low intake of both and thus only valid within the given range of ~700-900mg of calcium and ~2600-3000mg of potassium - intakes you can by the way easily get from your diets alone
• aside from the usual suspects, i.e. (green) vegetables, fruits and fish, dairy is among the most important source of minerals and high dairy eaters tend to be high mineral consumers, while low / no dairy eaters tend to be in the lowest quartiles of overall mineral intake

as well as a couple of things you cannot read off the graphs, e.g.:

• women had  significantly higher mineral intakes than men, i.e. 68.3% of the persons in Q4 for overall mineral intake were women
• age had no effect whatsoever on the overall intake of potassium, calcium and magnesium
• a low(er) education (<6 years of schooling) was a good predictor of low total mineral intakes, just as it is by the way in view of an overall worse diet quality (this is however less pronounced than conventional wisdom would suggest)
  
• intriguingly, people with diabetes had on average higher mineral intakes than people without diabetes, almost certainly a non-causative relationship that is probably mediated by supplements and nutritional counseling the diabetics received
• contrary to the US, there was no association between high salt and low Ca, Mg, or K intakes, this is also surprising because the "average" middle aged Japanese consumes way more than 5g of salt (Nagata. 2004) and thus 2x more than the US "tolerable upper intake level" of 2300 mg (Cogswell. 2013)

Apropos US, in view of a couple of other studies that have only recently provided support for the widely held, but in fact rarely scrutinized believe that, the lack of adequate amounts of potassium and magnesium, in particular, is associated not only with the age-related cognitive decline and even dementia, but also with such profane things as "simple" obesity, e.g. ...

• Donfrancesco et al. report higher potassium and magnesium intakes were associated with lower BMIs in 1168 men and 1112 women aged 35-79 yrs from 12 Italian regions (Donfrancesco. 2013)
• almost identical results in a study by Shay et al. that found associations with lower BMIs for potassium and magnesium 1794 men and women (ages 40-59 y) from 8 US population samples (Shay. 2013)

... it would unquestionably make sense to eventually stop bashing on sodium and start promoting the consumption of magnesium- and potassium-rich foods, instead.

Figure 2: Percentage of the population mineral intakes below the EAR for individuals aged ≥2y (data from NHANES 2003–2006; n = 16,110; Fulgoni. 2011).

Did you know that according to latest data from the CDC (Cogswell. 2013) less than 2% of US adults meet the dietary recommendations for potassium (≥4700 mg K/d) and that the lack of potassium was even more pronounced in the elderly (0.5% of the >72y-old US citizens meet the dietary requirements) and obese (0.7% meet the recommendations. With two out of five Americans also failing to meet even the required amount of magnesium in the diet, it appears more than questionable why the good-meaning (I don't doubt they are but too often they are mislead of have the good of the wrong people in mind) policy makers don't put magnesium and potassium into the water supply instead of toxic junk such as chlorine and fluoride...

I mean, you will probably remember from "On Short News on July 28, 2013" that each milligram of magnesium per liter drinking water could decrease the heart disease risk of people with an unbalanced mineral intake by 5%! But, alas, who am I to make such bold suggestions?

Now, while the importance of watching your dietary magnesium and potassium are pretty obvious and probably nothing you have not heard before, there is still one question left to be answered - a question that will point us away from RDAs and EARs and back to foods, which never contain only one of the aforementioned minerals in isolation. So here is the question: What do we make of calcium? In the Ozawa study it appeared to be clearly useful, but that was with intakes of >638mg/day in the highest quintile of the study population! The average European citizen, on the other hand, consumes roughly 1g = 1,000mg, i.e. 36% more than the Japanese and still we (us Europeans) are about as sick, if not sicker than the average Japanese? How come?  

Potassium, check; magnesium, check; calcium, ... wait a minute! What about phosphorus? 

Aside from the mere possibility that we could already be consuming way too much calcium (which is not supported by science as long as those 1,000mg come from your diet and not from supplements; cf. "Higher Calcium Intake Greater Fatty Acid Oxidation"), the most straight forward explanation would be an imbalanced intake of phosphorus. For the average European the latter is at about 1,675mg/day (mostly from dairy, cereals and meats - 27.9, 23.4, 17.4.% of daily intake, respectively) and thus clearly twice the amount our (the European) version of the well-meaning policy makers are telling us each and every one of us should be consuming on a daily basis.

Figure 4: Relative potassium intake in European countries according to source; note: with 4,110mg/day the average potassium intake in the Euopean Union is much higher than in the US, highest intakes were observed in Spain, lowest in Germany (Welch. 2009)

Did you know that the average magnesium intake in Europe (409mg/day) is much higher than in the US? And guess what, the usual suspects, i.e. dairy and cereals aside, non-alcoholic beverages are the #2 source (19% of total mg intake) of dietary magnesium in Europe! I would, an observation Welch et al. attribute just like the almost "optimal" (wrt to the US recommendations) average potassium intake of 4,110mg/day to the high quality tap and bottled mineral water, and other non-alcoholic beverages (and certainly not to reverse osmosis or the consumption of mineral-free distilled water, which is something you can use to satisfy the water requirements of your radiator or  iron, but not those of your body ;-).

Figure 3: Hazard risk analysis based on the Cholesterol and Recurrent Events (CARE) study (n = 4127; Tonelli. 2005)

In fact, we have broached on another of these imbalances in the context of the effects that were observed with higher magnesium : calcium ratios in drinking water (cf. red box above + "On Short News on July 28, 2013"), before. With phosphor we have yet another "antagonistic partner" of calcium, of which Ritz et al. have only recently argued that its increasing use as a food additive (check out the label of whatever processed food you buy, chances you find a XZY-phosphate on it are >50%) poses a serious health risk. To support their argument, the researchers cite data from a 2005 study by Tonelli et al. that indicates that even serum phosphor levels that are well within the normal range (2.0-4.0mg/dl) were associated with significantly increased CVD risks (cf. figure 3; suggested read: "Does Low Vitamin D Protect Us From Dietary Phoshporus Overload?").

These are only two selected examples of the available evidence that suggests that we are still totally underestimating the effects of "electrolytes", in general, and their ratios, in particular, on our neurological and metabolic health - and, even worse, doctors, policy makers, experts and gurus keep making mostly unwarranted recommendations to increase our intake of one and decrease the intake of another mineral, when in fact the lack of synergists (e.g. normal amounts of dietary magnesium to complement calcium) and absence or abundance of antagonists (e.g. potassium and magnesium for salt and calcium, magnesium and potassium for phosphorus) are the actual problems we are dealing with.

Figure 5: Don't forget that there are personal, regional and historical difference in total and relative mineral intakes and never supplement, high amounts of isolated minerals simply because Mr or Mrs average would benefit, without checking how "average" you actually are in terms of your solid, fluid and supplemental mineral intake (data for image based on Crawford. 1971; data on US water hardness according to the Water Research Center)

Implications: I guess based on all the information on the allegedly complicated interactions between the different minerals, you will by now have realized that statements like "everybody will benefit from taking 300mg of supplemental magnesium" let alone "everybody must take at least 300mg of supplemental magnesium" are about as useful as the constant advice to cut your salt, cut your fat and cut your calories people are confronted with on a daily basis. The chances that person X may benefit are probably high, but they are certainly much lower than the chances that you will survive the sting of a bee - and even that will still kill 53 US citizens per year.

Individualization, evaluation are therefore obligatory steps which must necessarily come before supplementation, which would - as some of the data in the figure 4 did already suggest - rarely be necessary, if the average inhabitant of the Western hemisphere did not top his sugary, salty and phosphate-laden fast-food diet with beverages that are either devoid of any minerals or will simply exasperate the existing imbalances.

Too many people (and I believe this is particularly true for the US) seem to have forgotten that we have not always been forced to filter all the minerals out of our water just to make the chlorinate, fluorinated, and "estrogenated" sludge that streams out of the faucet suitable for human consumption. Think of that and the data in figure 5, the next time the as of late often second-guessed recommendation that you got to have "at least X cups of water per day" resurfaces and of how little use each of them is, when it does not contain any of the electrolytes your body needs to handle the water appropriately.

References:

• Cogswell ME, Zhang Z, Carriquiry AL, Gunn JP, Kuklina EV, Saydah SH, Yang Q, Moshfegh AJ. Sodium and potassium intakes among US adults: NHANES 2003-2008. Am J Clin Nutr. 2013 Aug 1. 
• Crawford MD, Gardner MJ, Morris JN. Cardiovascular Disease and the Mineral Content of Drinking Water. Br. Med, Bull. 1971; 27,1: 21-24.
• Donfrancesco C, Ippolito R, Lo Noce C, Palmieri L, Iacone R, Russo O, Vanuzzo D, Galletti F, Galeone D, Giampaoli S, Strazzullo P. Excess dietary sodium and inadequate potassium intake in Italy: Results of the MINISAL study. Nutr Metab Cardiovasc Dis. 2013 Jul 24.
• Fulgoni VL 3rd, Keast DR, Bailey RL, Dwyer J. Foods, fortificants, and supplements: Where do Americans get their nutrients? J Nutr. 2011 Oct;141(10):1847-54.
• Ozawa M, Ninomiya T, Ohara T, Hirakawa Y, Doi Y, Hata J, Uchida K, Shirota T, Kitazono T, Kiyohara Y. Self-Reported Dietary Intake of Potassium, Calcium, and Magnesium and Risk of Dementia in the Japanese: The Hisayama Study. J Am Geriatr Soc. 2013 Aug 2. 
• Ritz E, Hahn K, Ketteler M, Kuhlmann MK, Mann J. Phosphate additives in food--a health risk. Dtsch Arztebl Int. 2013 Jan;109(4):49-55. Epub 2013 Jan 27. 
• Shay CM, Van Horn L, Stamler J, Dyer AR, Brown IJ, Chan Q, Miura K, Zhao L, Okuda N, Daviglus ML, Elliott P; for the INTERMAP Research Group. Food and nutrient intakes and their associations with lower BMI in middle-aged US adults:  the International Study of Macro-/Micronutrients and Blood Pressure (INTERMAP). Am J Clin Nutr. 2013 Aug 1. 
• Tonelli M, Sacks F, Pfeffer M, Gao Z, Curhan G; Cholesterol And Recurrent  Events Trial Investigators. Relation between serum phosphate level and cardiovascular event rate in people with coronary disease. Circulation. 2005 Oct 25;112(17):2627-33. 
• Water Research Center. Hard Water  Hardness Calcium Magnesium - Water Corrosion Mineral Scale. < http://www.water-research.net/hardness.htm > retrieved Aug 16, 2013.
• Welch AA, Fransen H, Jenab M, Boutron-Ruault MC, Tumino R, Agnoli C, Ericson U, Johansson I, Ferrari P, Engeset D, Lund E, Lentjes M, Key T, Touvier M, Niravong M, Larrañaga N, Rodríguez L, Ocké MC, Peeters PH, Tjønneland A, Bjerregaard L, Vasilopoulou E, Dilis V, Linseisen J, Nöthlings U, Riboli E, Slimani N, Bingham S. Variation in intakes of calcium, phosphorus, magnesium, iron and potassium in 10 countries in the European Prospective Investigation into Cancer and Nutrition study. Eur J Clin Nutr. 2009 Nov;63 Suppl 4:S101-21.