Wednesday, May 31, 2017

The Great Cholesterol Deception

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Millions of Australians are prescribed cholesterol-lowering drugs – statins like Pravachol®, Zocor® and Lipitor® – each year at a cost of more than $1 billion dollars with very little, if any, benefit. In the US, some 40 million people currently take statins at a cost of more than $3.00 per pill, more than $1,000 per year, totalling more than $40 billion a year.

While there are many exaggerated claims and a lot of hype about the benefits of statins, there are also many studies showing no benefits at all. The pro-statin hype is based on the misuse and abuse of statistics.

Various independent studies in prestigious, peer-reviewed journals have shown that statin use in primary prevention – that is, to save lives – has minimal or no value in reducing mortality and certainly nothing that is considered anywhere near clinically significant to warrant their widespread use. It does not matter how one manipulates the statistics, the results just aren’t there.

In data gathered in 2009 from six trials, a review of the efficacy in lowering the risk of death with statins found virtually no difference between the treatment group and the control group.[1] There are many more of these studies.

In an independent meta-analysis (when a number of studies are put together to achieve more statistical power) of randomised controlled trials in patients without CVD, statin therapy decreased the incidence of major coronary and cerebrovascular events and revascularisations but not coronary heart disease or overall mortality.[2]

Taking statins for a number of years will not reduce mortality: “Primary prevention with statins provides only small and clinically hardly relevant improvement of cardiovascular morbidity/mortality.”[3] “Hardly relevant” means there is virtually no clinical benefit; as the authors of these particular studies are independent, they gain nothing by stating this.

Another review found that “current clinical evidence does not demonstrate that titrating lipid therapy (trying to lower cholesterol with statins) to achieve proposed low LDL cholesterol levels is beneficial or safe.”[4] In other words, lowering lipids has no real benefit and has the potential for adverse effects.

Following up on this, in a major independent review of studies funded by the Ministry of Health of British Columbia (Canada) on statins and primary prevention, researchers reported that “statins have not been shown to provide an overall health benefit in primary prevention trials.”[5] This is a government report carried out by an independent university yet its findings are still ignored.

The problem really comes down to vested interests and the abuse of statistics. To overcome the limitations of small studies, vested parties combine many studies into a meta-analysis. The researchers themselves select the studies used in the meta-analysis. A fundamental problem is that researchers with direct links to drug companies have the authority to select the most positive studies and ignore the rest – including independent studies not funded by pharmaceutical companies. Despite this, they have still not been able to show any clinically significant findings.

As readers of the scientific journals, we should not be confused between statistical significance and clinical significance. For an outcome to be “statistically significant” means that the outcome was likely a result of the treatment – whether the result was 100% effective or less than 0.1% effective. That is, if you treat 1,000 people to save one life (0.1%) it may be statistically significant but it is not clinically significant. “Clinical significance” means 20% to 30% or more. The drug companies’ most positive studies on statins for prevention of CVD report statistical significance, mostly 1% or less, and none have found any clinical significance.

Busy medical professionals don’t have time to review the statistics; few of them may be aware of the different ways the statistics are manipulated. So if the experienced professionals don’t understand the results of these studies, how do we expect the media or public to understand?

More Deception

The studies on statins also report “relative risk,” not “absolute risk” or “real risk.” The relative risk reduction is highly misleading [6,7,8,9,10] if not deceptive. An example of relative risk is: if you have four people in a study who die in the placebo group (no drug) compared to three people who die in the drug treatment group – that is, four were expected to die but with the drug only three did – then there is a 25% relative risk reduction. However, to get this effect of saving one life you would have to treat 1,000 people and the real risk reduction is 0.1%. Relative risk is like adding 1+1 to get 11 or 2+5 to get 25 or more. How can the pharmaceutical companies and the researchers working for them get away with this? This is probably because (at least in my experience) most people are afraid of statistics.

In studies by the Medical Research Council dating back to the late 1980s, researchers found that of 1,000 men ranging in age from 35 to 64 who received treatment for mild hypertension over five years, there were six fewer strokes and two fewer cardiovascular events than would be expected.[11,12] The real risk reduction over five years was 0.9%.

Ten years later, a study of Pravachol® was released in the media, with much fanfare, as having a 22% drop (relative risk, not real risk) in mortality. However, when one looks at the numbers and statistics behind the calculations, treating 1,000 middle-aged men who had hypercholesterolemia (high cholesterol) and no evidence of a previous myocardial infarction with pravastatin for five years resulted in seven fewer deaths from cardiovascular causes, and two fewer deaths from other causes than would be expected in the absence of treatment.13 The real risk reduction, however, was a mere 0.9%, less than 1% or nine lives out of 1,000 when treated for five years. The research was sponsored by Bristol-Myers Squibb Pharmaceutical (West of Scotland Coronary Prevention Study).

Conservatively, put another way, researchers treated 1,000 people for five years at a total cost of over $5 million to save seven people from CVD. One might wish to compare this to the cost and efficacy of adopting healthy lifestyle choices.

In the Heart Protection Study in the United Kingdom, more than 20,000 participants aged 40 to 80 years with high risk of cardiovascular disease but average-to-low levels of total cholesterol and LDL cholesterol were treated with 40mg daily of simvastatin (marketed under several trade names including Zocor). Of 20,500+ study participants, 577 on statins died from a heart attack, 701 not treated died from a heart attack. That is a 25% relative risk reduction over five years.[14] Sounds good, doesn’t it? The real percentage improvement is actually 1.7%. Over the five-year study, they saved 25 people per year in a high-risk population with previous cerebrovascular disease, peripheral artery disease, renal impairment or diabetes. These are seriously ill people and the researchers still achieved a benefit of only 1.7%. Researchers neglected to mention that around 30,000 people were not allowed in or dropped from the study and not counted in the percentage of people with side effects. There were 10,269 people on statins and 10,267 people on a placebo.[15]

A study of 90,056 participants combining 14 randomised trials looked at the best outcome for people who had pre-existing conditions: 47% had pre-existing chronic heart disease, 21% had a history of diabetes and 55% a history of hypertension. The death rate was 8.5% among the statin group compared to 9.7% in the control group. This difference represents 1.2%.[16]

The well-known JUPITER study compared a placebo group to a statin-taking group. The study found that there were 68 heart attacks in the placebo group and 31 heart attacks in the drug treatment group – a 58% relative risk reduction. There were 64 strokes in the placebo group, compared to 33 strokes in the treatment group, a relative risk reduction of 48%.[17]Sounds good, doesn’t it? However, the drug treatment group had 8,901 participants in it. In real terms, the heart attack risk went from a very low 0.76% to 0.35% and the risk of stroke went from 0.72% to 0.37%.

Effectively, if you treat 300 people with expensive and dangerous drugs you might save one life. Under the best possible scenario, the real risk reduction was well under one half of one percent. The real risk reduction of consuming a handful of raw mixed nuts is much higher. It is interesting to note that one of the risk factors used to select the participants in the study was C-Reactive Protein (CRP) an indicator of inflammation, the real cause of CVD.

In an independent assessment of the same statistics in 2010 titled “Cholesterol Lowering, Cardiovascular Diseases, and the Rosuvastatin-JUPITER Controversy. A Critical Reappraisal” by Michel de Lorgeril and her 8 colleagues found that “the JUPITER Study” was severely flawed.[18] This recent analysis did a careful and independent review of both results and methods used in the JUPITER Study and reported that the “trial was flawed.”
In an unprecedented attack on the study they (scientists other than myself usually don’t say boo even when it is serious) stated that, “The possibility that bias entered the trial is particularly concerning because of the strong commercial interest in the study.” In other words, the big pharmaceutical money influenced the study. And concluded, “The results of the trial do not support the use of statin treatment for primary prevention of cardiovascular diseases and raise troubling questions concerning the role of commercial sponsors.”

This is a scathing attack in scientific terms of the earlier drug company sponsored study. Scientist do not go out of their way to create waves but these ones have not just found different results but also criticised the earlier studies link with pharmaceutical industry. It highlights not only that the studies don’t show any significant results but these studies and the education of our doctors is strongly influenced by the drug companies.[19]

More recently, a study reported in the BMJ was a meta-analysis of 10 randomised clinical trials of about 70,000 people followed for an average of four years.[20] In these trials, people with risk factors for cardiovascular disease but no history of existing disease were randomised to receive statins or no treatment. The relative risk reduction was 12% for total mortality, 30% for coronary event and 19% for a cerebrovascular event (stroke). However, the real risk reduction was 0.6%, 1.3% and 0.4% respectively. The actual number needed to treat to save one life was 167. Despite this outcome the authors of the study concluded, “In patients without established cardiovascular disease but with cardiovascular risk factors, statin use was associated with significantly (statistical not clinical) improved survival and large (statistical) reductions in the risk of major cardiovascular events.” (emphasis added.).

In fact, the authors had significant associations with the drug companies and failed to mention it was statistically significant but not clinically significant. Again, busy medical professionals tend to read only the abstracts; claims like this are pretty convincing, though very misleading.

More telling however, is the latest findings in June 2010 where two major independent studies, one the re-analysis of the Jupiter Study reported above and the other “A Meta-analysis of 11 Randomised Controlled Trials Involving 65,229 Participants” (don’t worry about the title) by Ray Kausik and 6 other independent researchers. The study, wait for it, found the use of statins in high-risk individuals was not associated with a statistically significant reduction in mortality. That is, they don’t save lives. Their data combined from 11 studies with 65,229 participants followed for approximately 244,000 person-years, a very big study, reported that this “meta-analysis did not find evidence for the benefit of statin therapy on all-cause mortality in a high-risk primary prevention set-up.” In other words they don’t save lives even in a high risk group. Even if you have all the elevated risk factors these drugs don’t work.
How many more studies to we need to do to show these drugs don’t work?

Professor Peter Dingle’s new book on the truth about cholesterol and cholesterol lowering medication, The Great Cholesterol Deception, is available. 


1 Bartolucci, A.A., S. Bae, et al. (2009). A Bayesian meta-analysis approach to address the effectiveness of statins in preventing death after an initial myocardial infarction. 18th World IMACS/MODSIM Congress. Cairns, Australia. 2009. Cairns, Australia.

2 Thavendiranathan, P., A. Bagai, et al. (2006). “Primary prevention of cardiovascular diseases with statin therapy: A meta-analysis of randomized controlled trials.” Archives of Internal Medicine 166: 2307-2313.

3 Vrecer, M., S. Turk, et al. (2003). “Use of statins in primary and secondary prevention of coronary heart disease and ischemic stroke. Meta-analysis of randomized trials.” International Journal of Clinical Pharmacology and Therapeutics 41(12): 567-577. M.Turk, S.Drinovec, J.Mrhar, A.International Journal of Clinical Pharmacology and Therapeutics. International Journal of Clinical Pharmacology and Therapeutics 567-57741122003

4 Hayward, R.A., T.P. Hofer, et al. (2006). “Narrative review: Lack of evidence for recommended low-density lipoprotein treatment targets: A solvable problem.” Annals of Internal Medicine 145(7): 520-530.

5 University of British Columbia (2003). “Do statins have a role in primary prevention? A review by the Therapeutics Initiative of the Department of Pharmacology & Therapeutics of the University of British Columbia.” Therapeutics Letter (48).

6 Fidan, D., B. Unal, et al. (2007). “Economic analysis of treatments reducing coronary heart disease mortality in England and Wales, 2000–2010.” QJM 100: 277-289.

7 Franco, O.H., A. Peeters, et al. (2005). “Cost effectiveness of statins in coronary heart disease.” Journal of Epidemiology and Community Health 59: 927-933. O.H.

8 Franco, O.H., E.W. Steyerberg, et al. (2006). “Effectiveness calculation in economic analysis: the case of statins for cardiovascular disease prevention.” Journal of Epidemiology & Community Health 60: 839-845.

9 Capewell, S. (2008). “Will screening individuals at high risk of cardiovascular events deliver large benefits? No.” British Medical Journal 337: a1395. S. British Medical Journal Capewell200816161617

10 Nuovo, J., J. Melnikow, et al. (2002). “Reporting number needed to treat and absolute risk reduction in randomized controlled trials.” Journal of American Medical Association 287: 2813-2814.

11 Medical Research Council Working Party (1985). “MRC trial of treatment of mild hypertension: principal results.” British Medical Journal 291: 97-104.

12 Miall, W.E. and G. Greenberg (1987). Mild Hypertension: Is There Pressure to Treat? An account of the MRC trial. New York, Cambridge University Press.

13 Shepherd, J., S.M. Cobbe, et al. (1996). “Prevention of coronary heart disease with Pravastatin in men with hypercholesterolemia.” New England Journal of Medicine 333: 1301-1307. P.W.McKillop, J.H.Packard, C.J.New England Journal of Medicine. New England Journal of Medicine 1301-13073331996

14 Heart Protection Study Collaborative Group (2002). “MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: A randomised placebo-controlled trial.” Lancet 360: 7-22.

15 Ibid.

16 Cholesterol Treatment Trialists’ Collaborators, C. Baigent, et al. (2005). “Efficacy and safety of cholesterol lowering treatment: Prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.” Lancet 366: 1267-1278. L.Buck, G.Pollicino, C.Kirby, A.Sourjina, T.Peto, R.Collins, R.Simes, R.Lancet, Lancet 1267-12783662005

17 Ridker, P.M., E. Danielson, et al. (2008). “Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein.” New England Journal of Medicine 359(21): 2195-2207. J.G.Nordestgaard, B.G.Shepherd, J.Willerson, J.T.Glynn, R.J.JUPITER Study Group, New England Journal of Medicine 2195-2207359212008

18 Ray, K.K., S.R.K. Seshasai, et al. (2010). “Statins and all-cause mortality in high-risk primary prevention: A meta-analysis of 11 randomized controlled trials involving 65 229 participants.” Archives of Internal Medicine 170(12): 1024-1031.

19 de Lorgeril, M., P. Salen, et al. (2010). “Cholesterol lowering, cardiovascular diseases, and the Rosuvastatin-JUPITER controversy: A critical reappraisal.” Archives of Internal Medicine 170(12): 1032-1036.

20 Brugts, J.J., T. Yetgin, et al. (2009). “The benefits of statins in people without established cardiovascular disease but with cardiovascular risk factors: meta-analysis of randomised controlled trials.” British Medical Journal 338: b2376.

DR. PETER DINGLE, Ph.D,  has a Bachelor of Education in Science, Bachelor of Environmental Science with first class honours, and a Ph.D. Dr. Dingle is an Associate Professor in Health and the Environment at Murdoch University and one of Australia’s best motivational health speakers. He has spent the past twenty years as a researcher, educator, communicator and author. He has more than 100 scientific papers. Dr. Dingle currently has 6 books in print with the latest ones My Dog Eats Better Than Your Kids and Is Your Home Making You Sick. He is a regular in state and national print media and may be heard weekly on radio and TV, reporting on health and the environment. He was the presenter on the award winning SBS program “Is Your House Killing You”, he is seen regularly on “Can we help” on ABC and is currently filming his second TV series. His website is

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Intracellular Water

There is a dynamic coupling between intracellular water and active metabolic processes

 V Intracellular solutions contain more K+ ions
V Membranes help create a tendency towards low density water in cells
V The effect of intracellular protein on water structuring
V The importance of protein carboxylate groups
V The importance of protein mobility
V Cooperative conversion of the water structuring
V Actin, tubulins and the intermediate filaments

"the molecular structure of water is the essence of all life"  
Dr. Albert Szent-Gyorgy, Nobel Prize winner.          

Intracellular water is the water inside cells that bathes all the necessary biological molecules including the proteins and nucleic acids. a Cells contain differing amounts of water dependent more on their purpose (and hence expressed proteins) rather than their source organism (for example, the % water contents of red blood cells are about 64% whether from man or dog whereas frog heart contains 80% but a frog egg only 49% water) [762]. Intracellular water is in more ordered state than pure water, as demonstrated by its significantly shorter (NMR) spin-lattice and spin-spin relaxation times [2321]. Very little of this water can be considered as solely a medium [2753] but is a metabolic reactant, product, catalyst, chaperone, messenger and controller (see for example, [1194]). Intracellular water is responsible for the conformation and function of all biomolecules through direct interaction with their hydration shells [2845]. However and importantly the water's structuring may also be controlled by some cellular constituents. Cellular macromolecules ‘‘dance’’ to the tune of intracellular water and, to a more restricted extent, vice versa. The water is essential for biomolecular recognition [1787] and orchestrates the cell machinery [1785]. The complexity and organization within the cytoplasm is expressed in a comprehensive review of intracellular water [1191] and an interesting current review has considered the versatility and adaptability of intracellular water in engaging in a wide range of cellular biochemistry [1359]. A key feature of intracellular water is its ability to convert between high and low density states [1364] (as exemplified by the ESreversible arrowCS equilibrium). Such changes have been linked to the state of autothixotropy [1898].

An increase in cell size, following cell membrane depolarization, occurs together with a reduction in water's diffusivity, indicating the very different characteristics that intracellular water appears to possess when compared to extracellular water. b Experimentally, this area presents a difficult problem as the properties and functioning of the cell in vivo cannot be easily determined from the sum of its parts in vitro; indeed the commonly practiced in vitro experiments on the homogenized and dilute contents of dead cells may be entirely misleading. For examples, CO2 production in metabolizing cells may give rise to complex changes in water movement and osmotic pressure changes as neutral CO2 diffuses readily but ions formed from it on hydration do not [1137] and actively metabolizing cells may be producing a significant amount of their intracellular water [1139]. Study of the live cell is fraught with difficulty as most procedures may alter the in vivo conditions, although dynamic phase microscopy shows promise [1332]. The structure and hydrogen bonding of intracellular water is clearly central to any theory concerning intracellular cell dynamics with intracellular protein dynamics and internal protein fluctuations both slaved to the water dynamics [1856]. There are two theories concerning intracellular water that have greatly influenced current thinking concerning the complex roles of intracellular water. These are the 'polarized multilayer theory' of Gilbert Ling [634, 2807] and the 'gel sol transition' of Gerald Pollack [351, 635]. On this page, the combination of aspartic and glutamic acid ion-pairing with K+ ions, changes in the mobility of key proteins and the natural low density clustering within intracellular water are all shown to contribute towards intracellular metabolic transitions and information transfer. Both prior theories are accommodated within this hypothesis [1093].

Water in cancerous breast tissue is markedly different from that in the normal tissue, in having more water plus a greater concentration of free hydroxyl groups [2280].
[Back to Top to top of page]
Intracellular solutions contain more K+ ions
The different characteristics of the intracellular and extracellular environments manifest themselves particularly in terms of restricted diffusion and a high concentration of chaotropic inorganic ions and kosmotropic other solutes within the cells. Note that both chaotropic inorganic ions and kosmotropic other solutes encourage low density water structuring. The difference in concentration of the ions is particularly apparent between Na+ and K+ (see below); Na+ ions creating more broken hydrogen bonding and preferring a high aqueous density whereas K+ ions prefer a low density aqueous environment. A 1000-fold preference for K+ over Na+ has been found in a halophilic organism without any energy expenditure but with a highly reduced intracellular water mobility [817]. As explained in discussion of the Hofmeister effect and shown by the negative apparent ionic volumes (that is, addition of the ions reduces the volume of the water, see below), the interactions between water and Na+ are stronger than those between water molecules, which in turn are stronger than those between water and K+ ions; all being explained by the differences in surface charge density. The interaction strength is reflected in that the distance between the Na+ ions and water is shorter than between two water molecules which is shorter than between K+ ions and water. Ca2+ ions have even stronger destructive effects, on the hydrogen bonding, than Na+ ions. Clearly, K+ ions are preferred within the intracellular environment.

Comparison of key cellular ions
Ionic radius
Surface charge density
Molar ionic volume
Water preference
100 pm
-28.9 cm3
0.1 μM
2.5 mM
High density
102 pm
-6.7 cm3
10 mM
150 mM
High density
138 pm
+3.5 cm3
159 mM
4 mM
Low density

Although somewhat contentious, it has been reported that the cellular membrane ion-pumps cannot produce these large differences in ionic composition in the absence of other mechanisms, simply as the (ATP) energy required appears to exceed the energy that is available to the cell and the gradients are maintained in the absence of intact membranes and/or the absence of active energy (that is, ATP) production [634, 635]. Also, in contrast to that written in several undergraduate textbooks, many studies show that cells do not need an intact membrane to function [635]. Instead, the intracellular water tends towards a low density structuring due to the kosmotropic character of the majority of the solutes, the confined space within the cell stretching the hydrogen-bonded water and the extensive surface effects of the membranes [1094]. The ions partition according to their preferred aqueous environment; in particular, the K+ ions partition into the cells. Ion pumps must thus be present for other (perhaps fail-safe) purposes, such as speeding up the partition process after metabolically linked changes in ionic concentration. [Back to Top to top of page]
Membranes help create a tendency towards low density water in cells

Membrane lipids contain hydrophilic kosmotropic groups such as the phosphatidylethanolamine, shown opposite, which encourages the lower density water structuring inside cells. This is particularly relevant as there is extensive membrane interfacial water within cells, e.g. liver cells contain ~100000 μm2 membrane surface area. The aqueous interface next to the membranes forms a functional unit linking the membrane to the water-soluble proteins of the cytosol, facilitating the rapid exchange of structural, dynamic and physiological information [1553]. [Back to Top to top of page]
The effect of intracellular protein on water structuring
The degree of density lowering inside cells is determined by the solutes, their concentration and the conformations and state of motion of the proteins; mobile proteins creating more disorder in the clustering compared with more static proteins. Many intracellular proteins are globular, so retaining rotational entropy notwithstanding the crowded environment. Water has conflicting effects in the mixed environments around proteins. Weak H-bonding between the protein and water molecules allows greater flexibility. Strong H-bonding endows the protein with greater stability and solubility. There is an ordered structure in first shell around the protein, with both hydrophobic clathrate-like and H-bonded water molecules each helping the other to optimize water's H-bonding network. Changes in the proteins' conformations may result in water influx into the cell or efflux from the cell [949] as well as density changes. Protein carboxylate groups are generally surrounded by strongly hydrogen-bonded water whereas the water surrounding basic chaotropic groups (arginine, histidine and lysine) tends towards a clathrate structuring. Clathrate formation over hydrophobic areas maximizes non-bonded interactions without loss of H-bonds whereas carboxylate groups usually only fit a collapsed water structure creating a reactive fluid zone. The diffusional movement of the proteins will cause changes in the water structuring outside the first hydration shell.
Rotation and translation of proteins

Translational diffusion involves breaking water-water links at a distance from surface whereas rotational diffusion involves breaking close water-water and protein-water links [640]. The surface area for translation and rotation is the same but the velocity differential is constant for all radial distances (r) for translation but varies with r2 with rotation.
At the breaking surface, half the H-bonds are ruptured, therefore creating a zone of higher density water (as explained elsewhere). Protein rotation thus creates a relatively close and surrounding high-density water zone with many broken hydrogen bonds, and as shown by some NMR studies. The volume of this interfacial region of perturbed water around the proteins is comparable to the volumes of the proteins. [Back to Top to top of page]
The carboxylate group showing the closeness of the oxygen atoms, 2.23 Angstrom
The importance of protein carboxylate groups
The side-chain carboxylate groups (that is, aspartic and glutamic acids) in proteins possess high dipole moments and contain two oxygen atoms that are nearer (~2.23 Å) than occurs between water molecules in bulk liquid water (~2.82 Å). These carboxylate oxygen atoms preferably hydrogen bond ~4 [1169] to ~6 [1781] water molecules which accounts for about 40% of the hydration free energy with the remainder mostly derived from outer aqueous shells [1781 a]. f This hydrogen bonding causes a localized high density water clustering due to the closeness of the water molecules as they gather around the carboxylate groups [1063]. Binding of water to the two oxygen atoms is anticooperative and instantaneously the oxygen atoms and C-O bonds are non-identical [1587].

Such hydrogen bonding induces a more negative charge on the carboxyl oxygen atoms, increasing the group's dipole and this leads to an increase in the carboxylate pKa, as is shown below.

Dipoles of the carboxylate groups of carboxylic acids plotted against their pKa The atomic charges and geometry of various related carboxylate were determined using ab initio molecular dynamics with the 6-31G** basis set. in Hyperchem. The dipoles of the carboxylate groups were calculate in Debye, units. A linear trend can be seen between the carboxylate group dipole and the pKa of the acids: a, trifluoroacetate; b, trichloroacetate; c, dichloroacetate; d, difluoroacetate; e, fluoroacetate; f, chloroacetate; g, thioglycollate; h, thiolactate; i, glycollate; j, lactate; k, 3-thiopropanoate; l, acetate; m, 2-methylpropanoate; n, propanoate; o, 2,2’-dimethylpropanoate. Most of the acidity constants were obtained from [1142].

The relationship between the pKa of carboxylate groups and the charge on the carboxylate oxygen atoms, as determined using ab initio modeling (6-31 G**)

If the charges on the carboxylate oxygen groups are reduced, by neighboring electron-withdrawing substituent groups or imposed negative electric field, the pKa is reduced. Note that the charge on the oxygen atoms lies between that on the ionic kosmotrope sulfate (–0.87; with hydrogen-bonds to water) and the ionic chaotrope perchlorate (–0.71; preferring to be surrounded by clathrate water). Thus, only a 5% reduction in the negative charge on the carboxylate oxygen atoms is necessary to switch from kosmotropic to chaotropic behavior. It is found that Na+ ions prefer binding to the weaker carboxylate groups (pKa > 4.5) whereas K+ ions prefer the stronger acids (pKa < 3.5) [634]. [Back to Top to top of page]
Part of the F-actin structure; 5 actin units (structure from Holmes KC and Eschenburg S (unpubl.))
The importance of protein mobility
Actin is a highly conserved and widespread eukaryotic protein (42-43 kDa) e responsible for many functions in cells. Non-muscle cells contain 5-10% (of all protein) actin whereas muscle cells contain about 20%. All actin molecules contain a conserved acidic N-terminus with several neighboring aspartic and/or glutamic acids (emphasized right) and a post-translation acetylated terminal amino group (for example, N-Ace-Asp-Glu-Asp-Glu in rabbit muscle α-actin). When actin forms a filament structure this highly charged grouping is placed on the outer exposed edge of the helix, where it may be additionally used as a binding site for other proteins, such as myosin. The positioning of the acidic N-terminal antennae are shown in red in the cartoon below. Tubulin and the intermediate filaments are further structural proteins that form immobile structures with ordered hydration within cells, possessing conserved acidic groupings that serve similar functions. e
Cartoon of the actin filament, showing the positioning of the acidic N-termini}
ATP conversion of G-actin to F-actin
Actin is converted between a freely rotating molecule (G-actin; about 4 - 6 nm diameter) and a static right-handed double helical protein filament (F-actin; up to several microns in length) by ATP; a process involving the conversion of an α-helix to a β-turn in one of the structural domains [636]. Each molecule of the freely rotating G-actin can influence a large volume of water within its effective radius of gyration, causing a reduction in the intracellular low density aqueous clustering. Filamentous actin (F-actin) is a much more ordered structure so creating more order in its surrounding water. Protein fibers trap water, which has decreased entropy. In order to attempt to keep the water activity constant, therefore, the water has to form bonds with a more negative enthalpy. This results in stronger bonds, causing greater structuring and lower density. Also, enclosure of water involves capillary action forming 'stretched' confined water that is much more highly structured (hence lower density) than the bulk water.

Comparison of isobutyrate with polyacrylate

As overlapping fields from nearby groups enhance counter ion association, F-actin's extensive negatively charged N-terminus will cause the partitioning of cations into its vicinity, in a similar manner to that well known from immobilized enzyme studies. This partitioning effect was originally shown by Kern [638] who established that the activity of Na+ with isobutyric acid is >0.9 whereas similarly with polyacrylate it is <0 .32="" cations="" font="" greater="" microenvironment="" multiply-charged="" of="" partitioning="" polymer.="" proving="" the="" thus="" within="">
Na+ and K+ ions behave differently when close to the carboxylate groups; K+ ions have a preference for forming ion pairs whereas Na+ ions form solvent separated pairings [637, 2035]. This is due to the Na+ ions holding on to their water strongly with the inner shell polarized water molecules being ideally situated for forming strong hydrogen bonds to the carboxylate oxygen atoms. The K+ ions prefer an inner shell clathrate water structuring that is reinforced its direct ion pairing to the carboxylate group. Also, Na+ ions cannot take part in clathrate structuring whereas K+ prefers this environment [641]. As the distance between the neighboring amino acid carboxylate groups (about 7.3 Å) is about the same as the diameter of clathrate water clusters (about 7.6 Å), extensive ion pairing of the adjacent carboxylate groups may be accommodated by neighboring clathrate clustering. K+ RCO2- pairs move from solvent separated to ion pair with reduction in the pKa. K+ ions prefer strong acids (that is, low pKa) whereas Na+ prefers weak acids (that is, higher pKa). Direct ion pair association discourages aqueous hydrogen bonding to the carboxylate oxygen atoms but encourages clathrate formation surrounding the ion-paired carboxylate group.

Note that the association of K+ with proteins' aspartate and glutamate groups is the central theme of Ling's fixed charge hypothesis [634] where evidence for the molecular mechanism for the association includes (1) the low intracellular electrical conductance, (2) the strongly reduced mobility of intracellular K+ ions, (3) the altered X-ray absorption fine edge structure, (4) the widely different activity coefficients of K+ ions in different cells, (5) K+ ion absorption shows one to one stoichiometric absorption to the carboxylate groups and (6) identification of K+ ion absorption sites as aspartate and glutamate side chains.
Equilibria involving the static protein

Under conditions when the carboxylate groups possess high pKa, both K+ and Na+ form solvent separated species when partitioned into the carboxylate environment. This gives a preference for Na+ and high-density water. However, rotation of the protein will tend to sweep such ions, and their associated water, away. If the protein stops rotating, Na+ ions tend to destroy any low density structuring around carboxylate groups of the protein. However generally the intracellular Na+ ion concentration is far lower than that of K+ ions (see above). At low pKa, K+ ion pairs to carboxylate groups, particularly when partitioned into their microenvironment due to the presence of a number of contiguous acidic amino acids.
Equilibria involving the rotating proteinWhen ion-paired the hydrogen bonding to the carboxylate oxygen atoms is prevented and a surrounding clathrate structuring is preferred. The clathrate clustering can signal their state to other neighboring carboxylate groups (see later).
When the static protein filaments converts to freely diffusing proteins, the ions are swept away due to the multiple contacts with surrounding water molecules and their inability to keep up with the rotating protein at increasing radial distances. This results in the conversion of any localized clathrate water structuring to high-density water involving hydrogen-bonded carboxylate groups. [Back to Top to top of page]
Cooperative conversion of the water structuring
Binding of K+ by the carboxylate groups lowers the ionic strength of the intracellular solution. As this ionic strength decreases, the pKa of phosphate groups increases resulting in the conversion of the more kosmotropic intracellular doubly-charged HPO42- ions to the singly-charged more chaotropic H2PO4- ions. d All intracellular phosphate entities (about 100 milli-equivalents L-1) will behave similarly, so increasing the tendency towards low-density water. Thus the cooperative effects of the change between static filament formation and freely-diffusional protein can be summarized as below.
Summary of cooperative changes creating low density water when a protein, such as actin, is polymerized

Further support for this process is given by F-actin becoming more static in the presence of about 100 mM K+ [642].
Cartoon showing the cooperative buildup of low density clustering around K+ on-paired carboxylate groups in proteins
Forward      Backward
Only the oxygen atoms of the water network are shown above.
Formation of K+-carboxylate ion pairs lead to the formation of a surrounding clathrate water structuring that further leads to icosahedral water structuring (so ensuring maximal hydrogen-bond formation) and informing neighboring carboxylate groups. This signaling cooperatively reinforces the tetrahedrality of the water structuring found between these groups. The mechanism is indicated on the right (press the Forward button). Note that the clathrate arrangement allows cluster mobility (like a ball-and-socket joint), enabling the hydrogen bonding to search out cooperative partners. The expected greater rigidity of such structures with lower temperatures has been proven [1466].

The diameter of the cylindrical water cluster appears to be less than 3% different from that invoked to explain the hydrophobic effect between flat hydrophobic surfaces and extending over some tens of nanometers [1316], indicating that the same clustering may be involved in both cases.

The cooperative nature of this signaling offers an explanation for the signal amplification seen in the conduction of impulses along the microtubules and actin [1434], e and perhaps linking to other parts of the cytoskeleton e [1307]. The importance of this cannot be overstated as microtubules are implicated in information processing and memory [1307]. The structuring of water may therefore be key to the conscious mind [1337].

Although the clustering (above right) involves a major drop in entropy, this is compensated by a more-negative enthalpy due to the stronger bonding of the fully-tetrahedral hydrogen-bonded structure. The phase transition between ordered and disordered water has been regarded as a mechanism to release energy for biological work rather than the membrane (pump) theory [1960]. It is consistent with Ling's association-induction polarized multilayer model [634], c as can be seen from the net dipoles emanating from the clathrate arrangement, but offers a more realistic explanation. The initial icosahedral size (3 nm diameter) also equals the water domain size proposed by Watterson [546]. Extensive (1.4 nm) long-lived (> 300 ps) aqueous dipole arrangements between biomolecules have been found in molecular dynamics studies [329]. Further support for this model is given by a number of studies concerning different types of intracellular water, such as "normal" bulk and "abnormal" osmotically inactive interfacial water [639]. Also, the water in the extreme halophile Haloarcula marismortui, which contains particularly large amounts of bound intracellular K+ ions, shows particularly slow translational diffusion [1253]. Idealized tetrahedral clustering caused by neighboring K+ carboxylate ion pairs, view 1 Idealized tetrahedral clustering caused by neighboring K+ carboxylate ion pairs, view 2

These orthogonal diagrams show the maximum limit of fully tetrahedral water clustering due to the cooperative clustering (see above) from two K+-carboxylate ion pairs. This fully tetrahedral structuring, which resembles a mixture of hexagonal and cubic ice structures but with 5-fold symmetry (such that it cannot be easily frozen), is idealized in the cartoons. An interactive structure is given (Jmol).

Extension of the clathrate network and its associated low density water enables K+ ion binding to all aspartic and glutamic acid groups, not just the key ones within the extensive acidic centers. Thus, the gel-sol transition of Pollack [635] is interpreted as the conversion to low density water clustering (the gel state) due to clathrate clustering around K+-carboxylate ion pairs. The changes in the functional state of the cell and its organisms has been shown to be reflected in the changes in the cell's refractivity [1997]. Certainly, the stiffness of the cytoplasm depends on the structure of the water as shown by changing the H2O water to D2O heavy water when the intracellular relaxation frequency reduces by between two and four orders of magnitude depending on the timescale [1856].

In the presence of raised levels of Na+ and/or Ca2+ ions, as occasionally occurs during some cell functions, these ions will replace some of the bound K+ ions. These newly formed solvent separated Na+ and/or Ca2+ ion pairings destroy the low-density clathrate structures and initiate a cooperative conversion of the associated water towards a denser structuring.
In conclusion the information transfer within the cell involves the following:
Intracellular water favors K+ ions over Na+ ions.
Freely rotating proteins create zones of higher density water, which tend towards a lower density clustering if the rotation is prevented.
Static charge-dense intracellular macromolecular structures prefer K+ ion pairs over freely soluble K+ ions.
Ion paired K+-carboxylate groupings prefers a local clathrate water structuring.
Clathrate water prefers local low density water structuring.
Low density water structuring can reinforce the low density character of neighboring site water structuring.
Na+ and Ca2+ ions can destroy the low density structuring in a cooperative manner. [Back to Top to top of page]

a  Philosophical, historical and scientific views of water in living systems have been reviewed [1011] [Back]

b   The cytoplasm of cells is described in [892]. Nuclear magnetic resonance signal widths are much broader inside cells, showing that intracellular water is far more structured than extracellular or pure water; for example, see [884]. For a review of the relationship between the intracellular water and the cytoskeleton see [880]). It is noteworthy that the amount of 'free' versus 'bound' water [969], K+ ions and the cytoskeleton are all intimately linked in the differences between normal and cancer cells [1333]. [Back]

c   The polarized multilayer theory relies on the hypothesis that static proteins in the cell change their secondary structure from compact to extended forms, so that the newly formed extensive lengths of polypeptide 'polarize' the water by means of their amide (N-H and C=O) backbone. Although there is support for the presence of polarized multilayers of water [634], there is no experimental support to this explanation for their presence. Indeed, most experimental data is contra-indicative, resulting in only minority support from scientists in this area. [Back]

d   The chaotropic/kosmotropic nature of these ions has been confirmed by FTIR [1072]. [Back]

Actin, tubulins and the intermediate filaments

Eukaryotic cells are dynamic structures, able to move, and divide. Such activities are achieved through the use of cytoskeletal filaments (actin, tubulins and intermediate filaments) and molecular motors (such as myosin) within the cell. The filaments lengthen, shorten and cross-link, regulated by other proteins. The actin filament is a helical ribbon formed of two parallel helical strands that each comprise a linear strands of small G-actin protein (42 kDa) molecules with uniform orientation along the ribbon axis. The ribbon has a width of ∼8 nm, a thickness of ∼5 nm, and a helical repeat of 36 nm. An analysis of the known actin structures from the UniProt Knowledgebase showed the 32 complete α-actin structures recorded from fish, birds, snakes and mammals all to have the conserved acidic N-terminal structure Acetyl- D/E- D/E- D/E- D/E- T/S/V- T- A- L- V- C- D- N- G- S- G- L- V- K- A- G- F- A- G- D- D- A- P- R- A- V- F- P- S- I- V- G- R- P- R- H- Q- G- V- M- V- G- M- G and the 70 complete β- and γ-actin structures recorded, from a similar range of species, to all have the conserved and closely related N-terminal structures of Acetyl- D/E- D/E- D/E- I/V/T- A/T- A/S- L- V- V/I/C- D/A- N- G- S- G- M/L- C- K- A- G- F- A/G- G/R- D- D/G- A- P- R- A- V/A- F- P- S- I- V- G- R- P- R- H- Q- G- V- M- V- G/R- M- G with even the variations limited to one or a very few molecules.

Tubulin showing the alpha/beta units and the acidic C-termini Eukaryotic tubulin is another structural protein that forms immobile structures with ordered hydration within cells [689]. It forms fat hollow and stiff microtubules (~25 nm diameter, containing low density water) making tracks through the intracellular space for the movement of organelles. These tubular structures are built up from pairs of similar subunits (α and β), each of about 450 amino acids [1140]. Tubulins have a GTP binding domain near their N-terminal and two separate β-sheets each surrounded by α-helices. The microtubules are formed from head to tail arrangement of α/ β dimers with the beta-subunit GTP at the open end. Only this GTP is hydrolyzed following polymerization. Outside the molecular core, two exterior antiparallel α-helices lead to highly acidic C-termini, which are thus situated on the outside of the microtubule and away from the tubulin surface [1306]. The α-tubulin structure is highly conserved with C-terminal consensus sequence - A- R- E- D- M- A- A- L- E- K- D- Y- E- E- V- G- V- D- S- V- followed by an extremely variable but highly acidic C- terminus exemplified by the human α- tubulin (type ‘ubiquitous’) structure - E- G- E- G- E- E- E- G- E- E- COOH. The β- tubulin structure is also highly conserved with C- terminal consensus sequence - A- E- S- N- M- N- D- L- V- S- E- Y- Q- Q- Y- Q- D- A- T- A- followed by an extremely variable highly acidic C- terminus exemplified by the human β- tubulin (type ‘4Q’) structure - E- E- E- E- D- E- E- Y- A- E- E- E- V- COOH. As with the actins, these examples indicate clearly that very strongly conserved structures exist for tubulins except for at an end (here the C-terminus) where glutamic acid and aspartic acid occur apparently interchangeably and in variable but always very acidic sub-structures. Improper functioning of tubulin has been linked with intracellular water clustering in the origin of cancer [1901].

Intermediate filaments are fibrous, flexible and elastic proteins formed mainly from coiled coils (‘ropes’, ~11 nm diameter) of multi-stranded acidic α-helices [1141]. There are a number of families of associated proteins such as the nuclear lamins, sarcomere desmins, neuron neurofilaments and epithelial cytokeratins. In addition, the vimentins generally support the organelles and maintain cellular integrity. All these proteins possess α-helical central ‘rod’ domains (~48 nm long) that contain about 310 amino acids plus more variable end domains. Whilst some acidic groups form salt links across to basic groups in other strands to build up and lengthen the resulting ‘elastic rope’ structure, there exists an excess of acidic groups on the surface of the filaments. Within the α-helices are acidic bulges (formed from single π-helical loops) containing clusters of three glutamic acids within four residues and creating flexible ‘linker’ regions. As different strands come together to form the fibrous ‘ropes’ these acidic clusters are grouped together with contributing acidic clusters from about 20 helices on the surface of the filaments. [Back] 
favored acetate hydration

f  Time-resolved infrared pump-prime spectroscopy of acetate in D2O suggests the bridge structure (right) as the most stable (single-hydrated) structure [1804], but other water molecules are also involved. [Back]


Introduction: Substances, such as water, ions, and molecules needed for cellular processes, can enter and leave cells by a passive process such as diffusion. Diffusion is random movement of molecules but has a net direction toward regions of lower concentration in order to reach an equillibrium.

Simple passive diffusion occurs when small molecules pass through the lipid bilayer of a cell membrane. Facilitated diffusion depends on carrier proteins imbedded in the membrane to allow specific substances to pass through, that might not be able to diffuse through the cell membrane.

Importance: The rate of diffusion is affected by properties of the cell, the diffusing molecule, and the surrounding solution. We can use simple equations and graphs to examine how particular molecules and their concentration affect the rate of diffusion. We can also compare simple and facilitated diffusion.

Question: How do rates of simple and facilitated diffusion differ in response to a concentration gradient?

Simple Diffusion



number of molecules inside cell (mol)


time (seconds)


permeability constant for a particular molecule (cm/sec)


surface area of the cell membrane (cm2)


concentration of diffusing molecule (mol/cm3)


width of cell membrane (cm)

Method: The rate of simple diffusion can be expressed by a modification of Fick's Law for small, nonpolar molecules. The rate of diffusion, dn/dt, is the change in the number of diffusing molecules inside the cell over time.

Since the net movement of diffusing molecules depends on the concentration gradient, the rate of diffusion is directly proportional to the concentration gradient (dC/dx) across the membrane. The concentration gradient, dC/dx, is the difference in molecule concentration inside and outside of the cell across a cell membrane of width dx. This is equivalent to (Cout - Cin)/Dx where Cout and Cin are the substrate concentrations inside and outside the cell, and Dx is the width of the cell membrane. When the concentration outside the cell (Cout) is larger than inside the cell (Cin), the concentration gradient (dC/dx) will be positive, and net movement will be into the cell (positive value of dn/dt).

We can describe the rate of diffusion as directly proportional to the concentration gradient by the following equation:

where A is the membrane area and P is the permeability constant. P is a constant relating the ease of entry of a molecule into the cell depending on the molecule's size and lipid solubility.

Notice that when A and P are constants, this equation simply describes a line where dn/dt is a function of dC/dx. If we graph the rate of diffusion as a function of the concentration gradient, we get a simple linear function.

Interpretation: Notice the rate of diffusion increases as the concentration gradient increases. If the concentration of molecules outside the cell is very high relative to the internal cell concentration, the rate of diffusion will also be high. If the internal and external concentrations are similar (low concentration gradient) the rate of diffusion will be low.

Facilitated Diffusion



number of molecules inside cell


time (seconds)


saturation constant (mol/cm3/sec)


constant determining speed of saturation (mol/cm4)


concentration of diffusing molecule (mol/cm3)


width of cell membrane (cm)

Method: Unlike simple diffusion, facilitated diffusion involves a limited number of carrier proteins. At low concentrations, molecules pass through the carrier proteins in a way similar to that of simple diffusion. At high solute concentrations, however, all the proteins are occupied with the diffusing molecules. Increasing the solute concentration further will not change the rate of diffusion. In other words, there is some maximum rate of diffusion (Vmax) when all the carrier pro teins are saturated. Therefore, we can not use a simple linear equation to describe the rate of diffusion. The rate of diffusion will increase with increasing solute concentration, but must asymptotically approach the saturation rate, Vmax. How quick ly the carrier proteins become saturated can be described by the variable K, the concentration gradient at which the rate of diffusion is 1/2 Vmax. K and Vmax depend on properties of the diffusing molecule, such as its permeability (P), as well as the surface area (A) of the cell, but for simplification we give the equation as:

We can graph this equation, dn/dt as a function of dC/dx, to see how the rate of diffusion changes with increasing solute concentration outside the cell.

Interpretation: By graphing this equation, we see that at low concentrations of solute, the rate of diffusion into a cell occurs almost linearly, like simple diffusion. Notice that at low solute concentrations, the slope is much steeper than that of simple diffusion. Facilitated diffusion can increase the rate of diffusion of particular molecules at low concentrations. However, the rate of facilitated diffusion levels off with increasing solute concentration. Additional increases in external solute concentration cannot increase the rate of diffusion once carrier proteins are saturated.

Conclusion: Passive diffusion of solute into a cell is linearly related to the concentration of solute outside the cell. Carrier proteins increase the rate of diffusion by allowing more solute to enter the cell. Facilitated diffusion, however, approaches a maximum rate as the carrier proteins become saturated with solute.

Additional Questions:

Simple Diffusion

1. We graphed dn/dt as a function of dC/dx. What is the slope of this line? What do increases or decreases in the slope mean biologically?

2. Now assume the concentration gradient is a constant. How does the rate of diffusion (dn/dt) change with the surface area (A) of the cell and the permeability (P) of the diffusing molecule? Graph dn/dt as a function of A or P and describe the function.

Facilitated diffusion

1. Look at the equation for facilitated diffusion and find the horizontal asymptote. What happens to dn/dt as dC/dx approaches infinity?

2. Try graphing this equation with different values for K. How does this change the concentration at which the carrier proteins are saturated?

3. Compare simple and facilitated diffusion of glucose into erythrocytes by graphing rate of diffusion (micromoles per hour) as a function of external glucose concentration (mmol/cm3). For facilitated diffusion, Vmax=500 micromoles per hour and K=1.5 mmol/cm3. For simple diffusion, A x P is 3 cm3/hour.

Sources: Darnell, J., H. Lodish, and D. Baltimore. 1986. Molecular Cell Biology. Scientific American Books, Inc., New York

LeFevre, P. G. 1961. Sugar transport in the red blood cell: Structure-activity relationships in substrates and antagonists. Pharmacological Review 13:39-70.

Water, Hydration and Health

This review attempts to provide some sense of our current knowledge of water including overall patterns of intake and some factors linked with intake, the complex mechanisms behind water homeostasis, the effects of variation in water intake on health and energy intake, weight, and human performance and functioning. Water represents a critical nutrient whose absence will be lethal within days. Water’s importance for prevention of nutrition-related noncommunicable diseases has emerged more recently because of the shift toward large proportions of fluids coming from caloric beverages. Nevertheless, there are major gaps in knowledge related to measurement of total fluid intake, hydration status at the population level, and few longer-term systematic interventions and no published random-controlled longer-term trials. We suggest some ways to examine water requirements as a means to encouraging more dialogue on this important topic.

Keywords: water, hydration, water intake, water measurement, recommended daily intake, water adequacy

Water is essential for life. From the time that primeval species ventured from the oceans to live on land, a major key to survival has been prevention of dehydration. The critical adaptations cross an array of species, including man. Without water, humans can survive only for days. Water comprises from 75% body weight in infants to 55% in elderly and is essential for cellular homeostasis and life.1 Nevertheless there are many unanswered questions about this most essential component of our body and our diet. This review attempts to provide some sense of our current knowledge of water including overall patterns of intake and some factors linked with intake, the complex mechanisms behind water homeostasis, the effects of variation in water intake on health and energy intake, weight, and human performance and functioning.

Recent statements on water requirements have been based on retrospective recall of water intake from food and beverages among healthy non-institutionalized individuals. We provide examples of water intake assessment in populations to clarify the need for experimental studies. Beyond these circumstances of dehydration, we do not truly understand how hydration affects health and well-being, even the impact of water intakes on chronic diseases. Recently, Jéquier and Constant addressed this question based on the human physiology.2 We need to know more about the extent that water intake might be important for disease prevention and health promotion.

As we note later, few countries have developed water requirements and those that do base them on weak population-level measures of water intake and urine osmolality.3, 4 The European Food Safety Authority (EFSA) has been recently asked to revise existing recommended intakes of essential substances with a physiological effect including water since this nutrient is essential for life and health.5

The US Dietary Recommendations for water are based on median water intakes with no use of measurements of dehydration status of the population to assist. One-time collection of blood samples for the analysis of serum osmolality has been used by NHANES. At the population level we have no accepted method of assessing hydration status and one measure some scholars use, hypertonicity, is not even linked with hydration in the same direction for all age groups.6 Urine indices are used often but reflect recent volume of fluid consumed rather than a state of hydration.7 Many scholars use urine osmolality to measure recent hydration status.8–12 Deuterium dilution techniques (isotopic dilution with D2O or deuterium oxide) allows measurement of total body water but not water balance status.13 Currently we feel there are no adequate biomarkers to measure hydration status at the population level.

When we speak of water we are essentially focusing first and foremost on all types of water, be they soft or hard, spring or well, carbonated or distilled water. Furthermore we get water not only directly as a beverage but from food and to a very small extent also from oxidation of macronutrients (metabolic water). The proportion of water that comes from beverages and food varies with the proportion of fruits and vegetables in the diet. We present the ranges of water in various foods (Table 1). In the United States it is estimated that about 22% of water comes from our food intake while it would be much higher in European countries, particularly a country like Greece with its higher intake of fruits and vegetables or South Korea.3, 14, 15 The only in-depth study of water use and water intrinsic to food in the US found a 20.7% contribution from food water;16, 17 however as we show later, this research was dependent on poor overall assessment of water intake.

Table 1
The Water Content Range for Selected Foods

Percentage Food Item
100% Water

90–99% Fat-free milk, cantaloupe, strawberries, watermelon, lettuce, cabbage, celery, spinach, pickles, squash (cooked)

80–89% Fruit juice, yogurt, apples, grapes, oranges, carrots, broccoli (cooked), pears, pineapple

70–79% Bananas, avocados, cottage cheese, ricotta cheese, potato (baked), corn (cooked), shrimp

60–69% Pasta, legumes, salmon, ice cream, chicken breast

50–59% Ground beef, hot dogs, feta cheese, tenderloin steak (cooked)

40–49% Pizza

30–39% Cheddar cheese, bagels, bread

20–29% Pepperoni sausage, cake, biscuits

10–19% Butter, margarine, raisins

1–9% Walnuts, peanuts (dry roasted), chocolate chip cookies, crackers, cereals, pretzels, taco shells, peanut butter

0% Oils, sugars

Source: The USDA National Nutrient Database for Standard Reference, Release 21 provided in Altman.

This review considers water requirements in the context of recent efforts to assess water intake in US populations. Relationship of water and calorie intake is explored both for insights into the possible displacement of calories from sweetened beverages by water and also to examine the possibility that water requirements would be better expressed in relation to calorie/energy requirements with the dependence of the latter on age, size, gender, and physical activity level. We review current understanding of the exquisitely complex and sensitive system which protects land animals against dehydration and comment on the complications of acute and chronic dehydration in man against which a better expression of water requirements might complement the physiological control of thirst. Indeed, the fine intrinsic regulation of hydration and water intake in individuals mitigates against prevalent underhydration in populations and effects on function and disease.

Regulation of fluid intake

To prevent dehydration reptiles, birds, vertebrates, and all land animals have evolved an exquisitely sensitive network of physiological controls to maintain body water and fluid intake by thirst. Humans may drink for various reasons, particularly for hedonic ones but most of drinking is due to water deficiency which triggers the so called regulatory or physiological thirst. The mechanism of thirst is quite well understood today and the reason non-regulatory drinking is often encountered is related to the large capacity of kidneys to rapidly eliminate excesses of water or reduce urine secretion to temporarily economize on water.1 But this excretory process can only postpone the necessity for drinking or for stopping drinking an excess of water. Non regulatory drinking is often confusing, particularly in wealthy societies facing highly palatable drinks or fluids that contain other substance that the drinker seeks. The most common of them are sweeteners or alcohol to which water is served as a vehicle. Drinking these beverages isn’t due to excessive thirst or hyperdipsia as it can be shown by offering pure water instead and finding out that the same drinker is in fact hypodipsic (Characterized by abnormally diminished thirst).1

Fluid balance of the two compartments

Maintaining a constant water and mineral balance requires the coordination of sensitive detectors at different sites in the body linked by neural pathways with integrative centers in the brain that process this information. These centers are also sensitive to humoral factors (neurohormones) produced for the adjustment of diuresis, natriuresis and blood pressure (angiotensin mineralocorticoids, vasopressin, atrial natriuretic factor). Instructions from the integrative centers to the “executive organs” (kidney, sweat glands and salivary glands) and to the part of the brain responsible for corrective actions such as drinking are conveyed by certain nerves in addition to the above mentioned substances.1

Most of the components of fluid balance are controlled by homeostatic mechanisms responding to the state of body water. These mechanisms are sensitive and precise, and are activated with deficits or excesses of water amounting to only a few hundred milliliters. A water deficit produces an increase in the ionic concentration of the extracellular compartment, which takes water from the intracellular compartment causing cells to shrink. This shrinkage is detected by two types of brain sensors, one controlling drinking and the other controlling the excretion of urine by sending a message to the kidneys mainly via the antidiuretic hormone vasopressin to produce a smaller volume of more concentrated urine.18 When the body contains an excess of water, the reverse processes occur: the lower ionic concentration of body fluids allows more water to reach the intracellular compartment. The cells imbibe, drinking is inhibited and the kidneys excrete more water.

The kidneys thus play a key role in regulating fluid balance. As discussed later, the kidneys function more efficiently in the presence of an abundant water supply. If the kidneys economize on water, producing a more concentrated urine, these is a greater cost in energy and more wear on their tissues. This is especially likely to occur when the kidneys are under stress, for example when the diet contains excessive amounts of salt or toxic substances that need to be eliminated. Consequently, drinking enough water helps protect this vital organ.

Regulatory drinking

Most drinking obeys signals of water deficit. Apart from urinary excretion, the other main fluid regulatory process is drinking, mediated through the sensation of thirst. There are two distinct mechanisms of physiological thirst: the intracellular and the extracellular mechanisms. When water alone is lost, ionic concentration increases. As a result, the intracellular space yields some of its water to the extracellular compartment. One again, the resulting shrinkage of cells is detected by brain receptors that send hormonal messages to induce drinking. This association with receptors that govern extracellular volume is therefore accompanied by an enhancement of salt appetite. Thus, people who have been sweating copiously prefer drinks that are relatively rich in Na+ salts rather than pure water. As previously mentioned, it is always important to supplement drinks with additional salt when excessive sweating is experienced.

The brain’s decision to start or stop drinking and to choose the appropriate drink is made before the ingested fluid can reach the intra- and extracellular compartments. The taste buds in the mouth send messages to the brain about the nature, and especially the salt of the ingested fluid, and neuronal responses are triggered as if the incoming water had already reached the bloodstream. These are the so-called anticipatory reflexes: they cannot be entirely “cephalic reflexes” because they arise from the gut as well as the mouth.1

The anterior hypothalamus and pre-optic area are equipped with osmo-receptors related to drinking. Neurons in these regions show enhanced firing when the inner milieu gets hyperosmotic. Their firing decreases when water is loaded in the carotid artery that irrigates the neurons. It is remarkable that the same decrease in firing in the same neurons takes place when the water load is applied on the tongue instead of being injected in the carotid artery. This anticipatory drop in firing is due to a mediation neural pathways departing from the mouth and by converging on to the neurons which simultaneously sense of the inner milieu (blood).

Non-regulatory drinking

Although everyone experiences thirst from time to time, it plays little day-to-day role in the control of water intake in healthy people living in temperate climates. We generally consume fluids not to quench our thirst, but as components of everyday foods (e.g. soup, milk), as beverages used as mild stimulants (tea, coffee) and for pure pleasure. As common example is alcohol consumption which can increase individual pleasure and stimulate social interaction. Drinks are also consumed for their energy content, as in soft drinks and milk, and are used in warm weather for cooling and in cold weather for warming. Such drinking seems also to be mediated through the taste buds, which communicate with the brain in a kind of “reward system” the mechanisms of which are just beginning to be understood. This bias in the way human beings rehydrate themselves may be advantageous because it allows water losses to be replaced before thirst-producing dehydration takes place. Unfortunately, this bias also carries some disadvantages. Drinking fluids other than water can contribute to an intake of caloric nutrients in excess of requirements, or in alcohol consumption that in some people may insidiously bring about dependence. For example, total fluid intake increased from 79 fluid ounces in 1989 to 100 fluid ounces in 2002 among US adults, all from caloric beverages.19

Effects of aging on fluid intake regulation

The thirst and fluid ingestion responses of older persons to a number of stimuli have been compared to those seen in younger persons.20 Following water deprivation older persons are less thirsty and drink less fluid compared to younger persons.21, 22 The decrease in fluid consumption is predominantly due to a decrease in thirst as the relationship between thirst and fluid intake is the same in young and old persons. Older persons drink insufficient water following fluid deprivation to replenish their body water deficit.23 When dehydrated older persons are offered a highly palatable selection of drinks, this also failed to result in an increased fluid intake.23 The effects of increased thirst in response to an osmotic load have yielded variable responses with one group reporting reduced osmotic thirst in older individuals24 and one failing to find a difference. In a third study, young individuals ingested almost twice as much fluid as old persons, despite the older subjects having a much higher serum osmolality.25

Overall these studies support small changes in the regulation of thirst and fluid intake with aging. Defects in both osmoreceptors and baroreceptors appear to exist as well as changes in the central regulatory mechanisms mediated by opioid receptors.26 Because of their low water reserves, it may be prudent for the elderly to learn to drink regularly when not thirsty and to moderately increase their salt intake when they sweat. Better education on these principles may help prevent sudden hypotension and stroke or abnormal fatigue can lead to a vicious circle and eventually hospitalization.


Hydration status is critical to the body’s process of temperature control. Body water loss through sweat is an important cooling mechanism in hot climates and in physical activity. Sweat production is dependent upon environmental temperature and humidity, activity levels, and type of clothing worn. Water losses via skin (both insensible perspiration and sweating) can range from 0.3 L/h in sedentary conditions to 2.0 L/h in high activity in the heat and intake requirements range from 2.5 to just over 3 L/d in adults under normal conditions, and can reach 6 L/d with high extremes of heat and activity.27, 28 Evaporation of sweat from the body results in cooling of the skin. However, if sweat loss is not compensated for with fluid intake, especially during vigorous physical activity, a hypohydrated state can occur with concomitant increases in core body temperature. Hypohydration from sweating results in a loss in electrolytes, as well as a reduction in plasma volume, and can lead to increased plasma osmolality. During this state of reduced plasma volume and increased plasma osmolality, sweat output becomes insufficient to offset increases in core temperature. When fluids are given to maintain euhydration, sweating remains an effective compensation for increased core temperatures. With repeated exposure to hot environments, the body adapts to heat stress, and cardiac output and stroke volume return to normal, sodium loss is conserved, and the risk for heat-stress related illness is reduced.29 Increasing water intake during this process of heat acclimatization will not shorten the time needed to adapt to the heat, but mild dehydration during this time may be of concern and is associated with elevations in cortisol, increased sweating, and electrolyte imbalances.29

Children and the elderly have differing responses to ambient temperature and different thermoregulatory concerns than healthy adults. Children in warm climates may be more susceptible to heat illness than adults due to greater surface area to body mass ratio, lower rate of sweating, and slower rate of acclimatization to the heat.30, 31 Children may respond to hypohydration during activity with a higher relative increase in core temperature than adults do,32 and sweat less, thus losing some of the benefits of evaporative cooling. However, it has been argued that children can dissipate a greater proportion of body heat via dry heat loss, and the concomitant lack of sweating provides a beneficial means of conserving water under heat stress.30 Elders, in response to cold stress, show impairments in thermoregulatory vasoconstriction and body water is shunted from plasma into the interstitial and intracellular compartments.33, 34 With respect to heat stress, water lost through sweating decreases water content of plasma, and the elderly are less able to compensate for increased blood viscosity.33 Not only do they have a physiological hypodipsia, but this can be exaggerated by central nervous system disease 35 and by dementia 36. In addition, illness and limitations in activities of daily living can further limit fluid intake. Coupled with reduced fluid intake, with advancing age there is a decrease in total body water. Older individuals have impaired renal fluid conservation mechanisms and, as noted above, have impaired responses to heat and cold stress 33, 34. All of these factors contribute to an increased risk of hypohydration and dehydration in the elderly.

In this section, the role of water in health is generally characterized in terms of deviations from an ideal hydrated state, generally in comparison to dehydration. The concept of dehydration encompasses both the process of losing body water and also the state of dehydration. Much of the research on water and physical or mental functioning compares a euhydrated state, usually achieved by provision of water sufficient to overcome water loss, to a dehydrated state, which is achieved via withholding of fluids over time and during periods of heat stress or high activity. In general, provision of water is beneficial in those with a water deficit, but little research supports the notion that additional water in adequately hydrated individuals confers any benefit.

Physical performance

The role of water and hydration in physical activity, particularly in athletes and in the military, has been of considerable interest and is well-described in the scientific literature.37–39 During challenging athletic events, it is not uncommon for athletes to lose 6–10% of body weight in sweat loss, thus leading to dehydration if fluids have not been replenished. However, decrements in physical performance in athletes have been observed under much lower levels of dehydration, as little as 2%.38 Under relatively mild levels of dehydration, individuals engaging in rigorous physical activity will experience decrements in performance related to reduced endurance, increased fatigue, altered thermoregulatory capability, reduced motivation, and increased perceived effort.40, 41 Rehydration can reverse these deficits, and also reduce oxidative stress induced by exercise and dehydration.42 Hypohydration appears to have a more significant impact on high-intensity and endurance activity such as tennis43 and long-distance running44 than on anaerobic activities45 such as weight lifting or on shorter-duration activities, such as rowing.46

During exercise, individuals may not hydrate adequately when allowed to drink according to thirst.32 After periods of physical exertion, voluntary fluid intake may be inadequate to offset fluid deficits.1 Thus, mild to moderate dehydration can therefore persist for some hours after the conclusion of physical activity. Research in athletes suggests that, principally at the beginning of the season, they are at particular risk for dehydration due to lack of acclimatization to weather conditions or suddenly increased activity levels.47, 48 A number of studies show that performance in temperate and hot climates is affected to a greater degree than performance in cold temperatures.41, 49, 50 Exercise in hot conditions with inadequate fluid replacement is associated with hyperthermia, reduced stroke volume and cardiac output, decreases in blood pressure, and reduced blood flow to muscle.51

During exercise, children may be at greater risk for voluntary dehydration. Children may not recognize the need to replace lost fluids, and both children as well as coaches need specific guidelines for fluid intake.52 Additionally, children may require longer acclimation to increases in environmental temperature than do adults.30, 31 Recommendations are for child athletes or children in hot climates to begin athletic activities in a well-hydrated state and to drink fluids over and above the thirst threshold.

Cognitive performance

Water, or its lack (dehydration), can influence cognition. Mild levels of dehydration can produce disruptions in mood and cognitive functioning. This may be of special concern in the very young, very old, those in hot climates, and those engaging in vigorous exercise. Mild dehydration produces alterations in a number of important aspects of cognitive function such as concentration, alertness and short-term memory in children (10–12 y),32 young adults (18–25y)53–56 and in the oldest adults, 50–82y.57 As with physical functioning, mild to moderate levels of dehydration can impair performance on tasks such as short-term memory, perceptual discrimination, arithmetic ability, visuomotor tracking, and psychomotor skills.53–56 However, mild dehydration does not appear to alter cognitive functioning in a consistent manner.53, 54, 56, 58 In some cases, cognitive performance was not significantly affected in ranges from 2–2.6% dehydration.56, 58 Comparing across studies, performance on similar cognitive tests was divergent under dehydration conditions.54, 56 In studies conducted by Cian and colleagues,53, 54 participants were dehydrated to approximately 2.8% either through heat exposure or treadmill exercise. In both studies, performance was impaired on tasks examining visual perception, short-term memory, and psychomotor ability. In a series of studies using exercise in conjunction with water restriction as a means of producing dehydration, D’Anci and colleagues56 observed only mild decrements in cognitive performance in healthy young men and women athletes. In these experiments, the only consistent effect of mild dehydration was significant elevations of subjective mood score, including fatigue, confusion, anger, and vigor. Finally, in a study using water deprivation alone over a 24-h period, no significant decreases in cognitive performance were seen with 2.6% dehydration 58. It is possible therefore, that heat-stress may play a critical role in the effects of dehydration on cognitive performance.

Reintroduction of fluids under conditions of mild dehydration can reasonably be expected to reverse dehydration-induced cognitive deficits. Few studies have examined how fluid reintroduction may alleviate dehydration’s negative effects on cognitive performance and mood. One study59 examined how water ingestion affected arousal and cognitive performance in young people following a period of 12-h water restriction. While cognitive performance was not affected by either water restriction or water consumption, water ingestion affected self-reported arousal. Participants reported increased alertness as a function of water intake. Rogers and coworkers60 observed a similar increase in alertness following water ingestion in both high- and low-thirst participants. Water ingestion, however, had opposite effects on cognitive performance as a function of thirst. High-thirst participants’ performance on a cognitively demanding task improved following water ingestion, but low-thirst participants’ performance declined. In summary, hydration status consistently affected self-reported alertness, but effects on cognition were less consistent.

Several recent studies have examined the utility of providing water to school children on attentiveness and cognitive functioning in children.61–63 In these experiments, children were not fluid restricted prior to cognitive testing, but were allowed to drink as usual. Children were then provided with a drink or no drink 20–45 minutes before the cognitive test sessions. In the absence of fluid restriction and without physiological measures of hydration status, the children in these studies should not be classified as dehydrated. Subjective measures of thirst were reduced in children given water,62 and voluntary water intake in children varied from 57 ml to 250 ml. In these studies, as in the studies in adults, the findings were divergent and relatively modest. In the research led by Edmonds and colleagues,61, 62 children in the groups given water showed improvements in visual attention. However, effects on visual memory were less consistent, with one study showing no effects of drinking water on a spot-the-difference task in 6–7 year old children 61 and the other showing a significant improvement in a similar task in 7–9 year old children62 In the research described by Benton and Burgess,63 memory performance was improved by provision of water but sustained attention was not altered with provision of water in the same children.

Taken together these studies indicate that low to moderate dehydration may alter cognitive performance. Rather than indicating that the effects of hydration or water ingestion on cognition are contradictory, many of the studies differ significantly in methodology and in measurement of cognitive behaviors. These variances in methodology underscore the importance of consistency when examining relatively subtle chances in overall cognitive performance. However, in those studies in which dehydration were induced, most combined heat and exercise, thus it is difficult to disentangle the effects of dehydration on cognitive performance in temperate conditions, from the effects of heat and exercise. Additionally, relatively little is known about the mechanism of mild dehydration’s effects on mental performance. It has been proposed that mild dehydration acts as a physiological stressor which competes with and draws attention from cognitive processes64. However, research on this hypothesis is limited and merits further exploration.

Dehydration and delirium

Dehydration is a risk factor for delirium and delirium presenting as dementia in the elderly and in the very ill.65–67 Recent work shows that dehydration is one of several predisposing factors in observed confusion in long-term care residents,67 although in this study daily water intake was used as a proxy measure for dehydration rather than other, more direct clinical assessments such as urine or plasma osmolality. Older people have been reported as having reduced thirst and hypodypsia relative to younger people. In addition, fluid intake and maintenance of water balance can be complicated by factors such as disease, dementia, incontinence, renal insufficiency, restricted mobility, and drug side effects. In response to primary dehydration, older people have less thirst sensation and reduced fluid intakes in comparison to younger people. However, in response to heat stress, while older people still display a reduced thirst threshold, they do ingest comparable amounts of fluid as younger people.20

Gastrointestinal function

Fluids in the diet are generally absorbed in the proximal small intestine, and absorption rate is determined by the rate of gastric emptying to the small intestine. Therefore, the total volume of fluid consumed will eventually be reflected in water balance, but the rate at which rehydration occurs is dependent upon factors which affect the rate of delivery of fluids to the intestinal mucosa. Gastric emptying rate is generally accelerated by the total volume consumed and slowed by higher energy density and osmolality.68 In addition to water consumed in food (1 L/d) and beverages (~2–3 L/d), digestive secretions account for a far greater portion of water that passes through and is absorbed by the gastrointestinal tract (~8 L/d).69 The majority of this water is absorbed by the small intestine, with a capacity of up to 15 L/d with the colon absorbing some 5 L/d.69

Constipation, characterized by slow gastrointestinal transit, small, hard stools, and difficulty in passing stool, has a number of causes including medication use, inadequate fiber intake, poor diet, and illness.70 Inadequate fluid consumption is touted as a common culprit in constipation, and increasing fluid intake is a frequently recommended treatment. Evidence suggests, however, that increasing fluids is only of usefulness in individuals in a hypohydrated state, and is of little utility in euhydrated individuals.70 In young children with chronic constipation, increasing daily water intake by 50% did not affect constipation scores.71 For Japanese women with low fiber intake, concomitant low water intake in the diet is associated with increased prevalence of constipation.72 In older individuals, low fluid intake is a predictor for increased levels of acute constipation73, 74 with those consuming the least amount of fluid having over twice the frequency of constipation episodes than those consuming the most fluid. In one trial, researchers compared the utility of carbonated mineral water in reducing functional dyspepsia and constipation scores to tap water in individuals with functional dyspepsia.75 When comparing carbonated mineral water to tap water, participants reported improvements in subjective gastric symptoms, but there were no significant improvements in gastric or intestinal function. The authors indicate that it is not possible to determine to what degree the mineral content of the two waters contributed to perceived symptom relief, as the mineral water contained greater levels of magnesium and calcium than the tap water. The available evidence suggests that increased fluid intake should only be indicated in individuals in a hypohydrated state.71, 69

Significant water loss can occur through the gastrointestinal tract, and this can be of great concern in the very young. In developing countries, diarrheal diseases are a leading cause of death in children resulting in approximately 1.5–2.5 million deaths per year.76 Diarrheal illness results not only in a reduction in body water, but also in potentially lethal electrolyte imbalances. Mortality in such cases can many times be prevented with appropriate oral rehydration therapy, by which simple dilute solutions of salt and sugar in water can replace fluid lost by diarrhea. Many consider application of oral rehydration therapy to be one of the signal public health developments of the last century.77

Kidney function

As noted above, the kidney is crucial in regulating water balance and blood pressure as well as removing waste from the body. Water metabolism by the kidney can be classified into regulated and obligate. Water regulation is hormonally mediated, with the goal of maintaining a tight range of plasma osmolality between 275 to 290 mOsm/kg. Increases in plasma osmolality, and activation of osmoreceptors (intracellular) and baroreceptors (extracellular) stimulate hypothalamic release of arginine vasopressin (AVP). AVP acts at the kidney to decrease urine volume and promote retention of water, and the urine becomes hypertonic. A hypertonic solution is a particular type of solution that has a greater concentration of solutes on the outside of a cell when compared with the inside of a cell. With decreased plasma osmolality, vasopressin release is inhibited, and the kidney increases hypotonic urinary output. In a hypotonic solution the total molar concentration of all dissolved solute particles is less than that of another solution or less than that of a cell. If concentrations of dissolved solutes are less outside the cell than inside, the concentration of water outside is correspondingly greater. 

In addition to regulating fluid balance, the kidneys require water for the filtration of waste from the blood stream and excretion via urine. Water excretion via the kidney removes solutes from the blood, and a minimum obligate urine volume is required to remove the solute load with a maximum output volume of 1 L/h.78 This obligate volume is not fixed, but is dependent upon the amount of metabolic solutes to be excreted and levels of AVP. Depending on the need for water conservation, basal urine osmolality ranges from 40 mOsm/kg up to a maximum of 1400 mOsm/kg.78 The ability to both concentrate and dilute urine decreases with age, with a lower value of 92 mOsm/kg and an upper range falling between 500–700 mOsm/kg for individuals over 70.79–81 Under typical conditions, in an average adult, urine volume of 1.5 to 2.0 L/d would be sufficient to clear a solute load of 900 to 1200 mOsm/d. During water conservation and the presence of AVP, this obligate volume can decrease to 0.75–1.0 L/d and during maximal diuresis can require up to 20 L/d to remove the same solute load.78, 80, 81 In cases of water loading, if the volume of water ingested cannot be compensated for with urine output, having overloaded the kidney’s maximal output rate, an individual can enter a hyponatremic state as described above.

Heart function and hemodynamic response

Blood volume, blood pressure, and heart rate are closely linked. Blood volume is normally tightly regulated by matching water intake and water output, as described in the section on kidney function. In healthy individuals, slight changes in heart rate and vasoconstriction act to balance the effect of normal fluctuations in blood volume on blood pressure.82 Decreases in blood volume can occur, through blood loss (or blood donation), or loss of body water through sweat, as seen with exercise. Blood volume is distributed differently relative to the position of the heart whether supine or upright, and moving from one position to the other can lead to increased heart rate, a fall in blood pressure and, in some cases, lead to syncope. This postural hypotension (or orthostatic hypotension) can be mediated by drinking 300–500 ml of water.83, 84 Water intake acutely reduces heart rate and increases blood pressure in both normotensive and hypertensive individuals.85 These effects of water intake on the pressor effect and heart rate occur within 15–20 minutes of drinking water and can last for up to 60 minutes. Water ingestion is also beneficial in preventing vasovagal reaction with syncope in blood donors at high risk for post-donation syncope.86 The effect of water drinking in these situations is thought to be due to effects on the sympathetic nervous system rather than to changes in blood volume.83, 84 Interestingly, in rare cases, individuals may experience bradycardia and syncope after swallowing cold liquids.87–89 While swallow syncope can be seen with other substances than water, swallow syncope further supports the notion that the result of water ingestion in the pressor effect has both a neural component as well as a cardiac component.


Water deprivation and dehydration can lead to the development of headache.90 Although this observation is largely unexplored in the medical literature, some observational studies indicate that water deprivation, in addition to impairing concentration and increasing irritability, can serve as a trigger for migraine and also prolong migraine.91, 92 In those with water deprivation-induced headache, ingestion of water provided relief from headache in most individuals within 30 min to 3 h.92 It is proposed that water deprivation-induced headache is the result of intracranial dehydration and total plasma volume. Although provision of water may be useful in relieving dehydration related headache, the utility of increasing water intake for the prevention of headache is less well documented.

The folk wisdom that drinking water can stave off headaches has been relatively unchallenged, and has more traction in the popular press than in the medical literature. Recently, one study examined increased water intake and headache symptoms in headache patients.93 In this randomized trial, patients with a history of different types of headache, including migraine and tension headache, were either assigned to a placebo condition (a non-drug tablet) or the increased water condition. In the water condition, participants were instructed to consume an additional volume of 1.5 L water/day on top of what they already consumed in foods and fluids. Water intake did not affect number of headache episodes, but was modestly associated with reduction in headache intensity and reduced duration of headache. The data from this study suggest that water is limited as prophylaxis in headache sufferers, and the ability of water to reduce or prevent headache in a broader population remains unknown.


One of the more pervasive myths regarding water intake is the improvement of the skin or complexion. By improvement, it is generally understood that individuals are seeking to have a more “moisturized” look to the surface skin, or to minimize acne or other skin conditions. Numerous lay sources such as beauty and health magazines as well as the Internet suggest that drinking 8–10 glasses of water a day will “flush toxins from the skin” and “give a glowing complexion” despite a general lack of evidence94, 95 to support these proposals. The skin, however, is important in maintaining body water levels and preventing water loss into the environment.

The skin contains approximately 30% water, which contributes to plumpness, elasticity, and resiliency. The overlapping cellular structure of the stratum corneum and lipid content of the skin serves as “waterproofing” for the body.96 Loss of water through sweat is not indiscriminate across the total surface of the skin, but is carried out by eccrine sweat glands, which are evenly distributed over most of the body surface.97 Skin dryness is usually associated with exposure to dry air, prolonged contact with hot water and scrubbing with soap (both strip oils from the skin), medical conditions and medications. While more serious levels of dehydration can be reflected in reduced skin turgor,98, 99 with tenting of the skin as a flag for dehydration, overt skin turgor in individuals with adequate hydration is not altered. Water intake, particularly in individuals with low initial water intake, can improve skin thickness and density as measured by sonogram,100 and offsets transepidermal water loss, and can improve skin hydration.101 Adequate skin hydration, however, is not sufficient to prevent wrinkles or other signs of aging, which are related to genetics, and sun and environmental damage. Of more utility to individuals already consuming adequate fluids, the use of topical emollients will improve skin barrier function and improve the look and feel of dry skin.102, 103

Hydration and chronic diseases

Many chronic diseases have multifactorial origins. In particular, differences in lifestyle and the impact of environment are known to be involved and constitute risk factors that are still being evaluated. Water is quantitatively the most important nutrient. In the past, scientific interest with regard to water metabolism was mainly directed toward the extremes of severe dehydration and water intoxication. There is evidence, however, that mild dehydration may also account for some morbidities.4, 104 There is currently no consensus on a “gold standard” for hydration markers, particularly for mild dehydration. As a consequence, the effects of mild dehydration on the development of several disorders and diseases have not been well documented.

There is strong evidence showing that good hydration reduces the risk of urolithiasis (See Table 2 for evidence categories). Less strong evidence links good hydration with reduced incidence of constipation, exercise asthma, hypertonic dehydration in the infant, and hyperglycemia in diabetic ketoacidosis. Good hydration is associated with a reduction in urinary tract infections, hypertension, fatal coronary heart disease, venous thromboembolism, and cerebral infarct but all these effects need to be confirmed by clinical trials. For other conditions such as bladder or colon cancer, evidence of a preventive effect of maintaining good hydration is not consistent (see Table 3).

Table 2

Table 2
Categories of evidence in evaluating the quality of reports

Category -Classification-Description
Ia Strong Evidence from a meta-analysis of randomized, controlled trials
Ib Strong Evidence from at least one randomized, controlled trial
IIa Less Strong Evidence from at least one controlled study without randomization
IIb Less Strong Evidence from at least one other type of quasi-experimental study
III Weaker Evidence from descriptive studies, such as comparative studies, correlation studies, and case control studies
IV Weaker Evidence from expert committee reports, opinions or clinical experience of respected authorities, or both
Inconsistent Evidence from small studies that are inconsistent in outcome
Speculative Posited relationships are based essentially on extrapolation from mechanism

Table 3

Table 3
Hydration Status and Chronic Diseases

Chronic Disease Evidence Level* Findings
Urolithiasis Strong Increased urine volume from increased fluid intake reduces stone recurrence. Favorable associations between increased hydration status and lower stone recurrence rate.

Bronchopulmonary Disorders Strong Exercise related asthma is linked with low fluid intake.128

Hypertonic Dehydration in Infants Less strong In infants with gastroenteritis, a high urine osmolality due to a high protein and sodium content of formula and weaning foods increases the risk of hypertonic dehydration.129

Diabetic Hyper glycemia and Ketoacidosis Less strong In diabetics, experimentally induced dehydration promotes development of hyperglycemia.130
Higher serum osmolality at time of hospital admission was the most important predictor of death in children with diabetic ketoacidosis.131
Morphological and Functional Changes in the Kidney Weaker In patients with polycystic kidney disease and chronic renal failure, sustained high urine volumes with urine osmolalities below plasma osmolality accelerate the decline of glomerular filtration rate.132
Hypertension Weaker In diabetic patients, lower urine flow and sodium excretion rates are associated with higher blood pressure during the day and a reduced fall in blood pressure at night.133 In a study of 1688 healthy men, a low urine production day-to-night ratio was not associated with hypertension.134 In one study, eight male hypertensive volunteers and eight controls were exercised in a hot environment with or without water ingestion. In hypertensive men, water ingestion increased exercise-related differences in their systolic and diastolic blood pressure.
Fatal Coronary Heart Disease Weaker High intake of water is associated with lower risk of fatal heart disease.135
Venous Thromboembolism Weaker High serum osmolality after stroke is associated with increased rate of thromboembolism.136
Cerebral Infarct (Stroke) Weaker Increased serum osmolality or hematocrit is associated with increased risk of stroke morbidity/mortality.137, 138 Stroke patients with initial mid-range hematocrit have better discharge outcomes.139
Dental Diseases Weaker Salivary output decreases with dehydration. Hypohydration may be linked with dental disease.
Urinary Tract Infection (UTI) Weaker Occurrence of UTI is associated with low fluid intake or low urine output.140, 141 No definitive evidence links susceptibility to UTI to fluid intake.
Bladder and Colon Cancer Inconsistent Generally show no association between fluid intake and cancer risk or tumor recurrence.142–144
Gallstone Speculative Water intake induces gallbladder emptying suggestive that a high daily water intake may prevent gallstone formation.145
Mitral Valve Prolapse Speculative Mitral valve prolapse developed after dehydration in one in 10 healthy men.146
Glaucoma Speculative Dehydration reduces intraocular pressure and elevated colloid osmotic pressure.147 Intraocular pressure increases minutes after water ingestion and remains elevated above baseline for up to 45 minutes post-ingestion.148

*Categories of evidence: Described in Table 2

III. Water consumption and requirements and relationships to total energy intake
Water consumption, water requirements and energy intake are linked in fairly complex ways. This is partially because physical activity and energy expenditures affect the need for water but also because a large shift in beverage consumption over the past century or more has led to consumption of a significant proportion of our energy intake from caloric beverages. Nonregulatory beverage intake, as noted earlier, has assumed a much greater role for individuals.19 In this section we first review current patterns of water intake, then refer to a full meta-analysis of the effects of added water on energy intake. This includes adding water to the diet and water replacement for a range of caloric and diet beverages, including sugar-sweetened beverages, juice, milk, and diet beverages. The third component is a discussion of water requirements and suggestions for considering the use of ml water/kcal energy intake as a metric.

A. Patterns and trends of water consumption

Measurement of total fluid water consumption in free-living individuals is fairly new in focus. As a result, the state of the science is poorly developed, data are most likely fairly incomplete, and adequate validation of the measurement techniques used are not available. We first present varying patterns and trends of water intake for the United States over the past three decades and review briefly the work on water intake in Europe.

We have really no information to allow us to assume that consumption of water alone or beverage containing water affects hydration differentially.3, 105 Some epidemiological data suggests water might have differential metabolic effects when consumed as water alone rather than water contained in caffeinated or flavored or sweetened beverages but these data are at best suggestive of an issue deserving of exploration.106, 107 We do show below from the research of Ershow that beverages not consisting of solely water do contain less than 100% water.

One study in the United States has attempted to examine all the sources of water in our diet.16, 17 These data are cited in Table 4 as the Ershow study and were based on National Food Consumption Survey food and fluid intake data from 1977–78. These data are presented in Table 4 for children 2–18 (Panel A) and for adults 19 and older (Panel B). Ershow and colleagues spent a great deal of time working out ways to convert USDA dietary data into water intake, including water absorbed during the cooking process, water in food, and all sources of drinking water.16, 17

Table 4
Table 4
Beverage Pattern Trends in the United States for Children aged 2–18 and Adults Aged 19 and older, Nationally Representative
These created a number of categories and used a range of factors measured in other studies to estimate the water categories. The water that is found in food, based on food composition table data, was 393ml for children. The water that was added as a result of cooking (e.g., rice) was 95 ml. That consumed as a beverage directly as water was 624 ml. The water found in other fluids as noted comprised the remainder of the ml with the most water coming from whole fat milk and juices (506 ml). There is a small discrepancy between the Ershow total fluid intake measures for these children and that of the normal USDA figure. That is because USDA does not remove milk fats and solids, fiber and other food constituents found in beverages, particularly, juice and milk.

A key point to be seen in these nationally representative US data is enormous variability in the amount of water consumed between survey waves (see Figure 1 which highlights that large variation in water intake measured in these surveys). Although adult and child water intake moved up and down at the same time, for reasons we cannot explain, the variation is greater among children than adults. This is partly that the questions asked have varied and there has not been detailed probing for water intake as the focus has been on obtaining measures of macro-and micronutrients. Dietary survey methods in the past have focused on obtaining foods and beverages containing nutrient and nonnutritive sweeteners but not on water. Related are the huge differences between the NHANES 1988–1994 and 1999 and later surveys and the USDA surveys. In addition even the NHANES1999–2002 and the 2003–6 surveys differ greatly. This represents a shift in mode of questioning to inclusion of water intake as part of a standard 24-hour recall rather than as standalone questions. Water was not even measured in 1965, and the way the questions have been asked and the limitation on probes for water intake are very clear from a review of the questionnaires plus these data. Essentially in the past people were asked how much water they consumed in a day and now they are asked these questions as part of a 24-hour recall survey. However, unlike other caloric and diet beverages, there are limited probes for water. These must be viewed as crude approximations of total water intake without any strong research to show if they are over- or underestimated. We know from our own research with several studies of water and two on-going random controlled trials that probes that include consideration of all beverages including water as a separate item provide more complete data.

Figure 1
Figure 1
Water Consumption Trends from USDA and NHANES Surveys (ml/day/capita), weighted to be nationally representative
Water consumption data for Europe are collected far more selectively than even the crude water intake questions from NHANES. The recent EFSA report provides measures of water consumption from a range of studies in Europe.4, 105, 108, 109 Essentially what these studies show is that total water intake is lower across Europe than the United States. As with the United States data, none are based on long-term carefully measured or even repeated 24-hour recall measures of water intake from food and beverages. In unpublished work Popkin and Jebb110 are examining water intake in UK adults in 1986–87 and 2001–2. Their intake increased by 226 ml/d over this time period but still is only 1787 ml/d in the latter period, far below the US figure for 2005–6 of 2793 or earlier figures for comparably aged adults.

There are a few studies in the US and Europe that utilize 24-hour urine and serum osmolality measures to determine total water turnover and this measure of hydration status. These studies suggest that US adults consume over 2100 ml of water per day while those from Europe consume less than a half liter or more.4, 111 Data on total urine collection would appear to be another useful measure for examining total water intake. Of course few studies aside from the Donald adolescent cohort in Germany have collected such data on population levels for large samples.109

B. The effects of water consumption on overall energy intake

There is an extensive literature that focuses on the impact of sugar-sweetened beverages on weight and risk of obesity, diabetes and heart disease; however the perspective of providing more water and its impact on health has not been examined. The water literature does not address portion sizes but rather focuses mainly on water ad libitum or in selected portions compared with other caloric beverages. Elsewhere we have prepared a detailed meta-analysis of the effects of water intake alone (adding additional water), replacing sugar-sweetened beverages, juice, milk and diet beverages.112

In general, the results of this review suggest that water, when replacing sugar-sweetened beverages, juice and milk is linked with reduced energy intake. This comes mainly from the clinical feeding studies but also from one very good random controlled school intervention and several other epidemiological and intervention studies. Aside from portion sizes, there are issues of timing of the beverage intake and meals (delay time from the beverage to the meal) and types of caloric sweeteners that remain to be considered. However when beverages are consumed in a normal free-living situations where 5–8 eating occasions are the norm, the delay time from the beverage to the meal may matter less.113–115

The literature on children is extremely limited as it relates to water intake. However, the excellent German school intervention with water would suggest the effects of water on overall energy intake of children might be comparable to that of adults.116 In this German study children were educated on the value of water and provided in school with special filtered drinking fountains and water bottles. The intervention school children increased by 1.1 glasses/day (P<0 .001="" 31="" and="" by="" of="" overweight="" p="0.40).</font" reduced="" risk="" their="">

C. Water requirements: Evaluation of the adequacy of water intake

Classically, water data are examined in terms of milliliters (or some other measure of water volume consumed per capita per day by age group). This measure does not link fluid and caloric intake. Disassociation of fluid and calorie intake difficult for clinicians dealing with an older person who has reduced caloric intake. This ml water measure assumes some mean body size (or surface area) and a mean level of physical activity – both determinants of not only energy expenditure, but also of water balance. Children are dependent on adults for access to water and studies suggest that a larger surface area to volume ratio makes them susceptible to changes in skin temperatures, linked with ambient temperature shifts.117 One option utilized by some scholars is to explore in ml/kcal of food and beverage intake as was done in the 1989 US RDAs.4, 118 This is an option interpretable for clinicians and does incorporate in some sense body size or surface area and activity. It has one disadvantage, namely that water as consumed with caloric beverages, affects both the numerator and denominator; however we do not know a measure that could be independent of this direct effect on body weight and/or total caloric intake.

Despite its critical importance in health and nutrition, as noted earlier, the array of available research that serves as a basis for determining requirements for water or fluid intake, or even rational recommendations for populations, is limited compared to most other nutrients. While this deficit may be partly explained by the highly sensitive set of neurophysiological adaptations and adjustments that occur over a large range of fluid intake to protect body hydration and osmolarity, this deficit remains a challenge for the nutrition and public health community. The latest official effort at recommending water intake for different subpopulations was a part of the Dietary Reference Intake process of the Institute of Medicine of the National Academy of Science’s Report on Dietary Reference Intakes on Water and Electrolytes as reported in 2005.3 As a graphic acknowledgement of the limited database upon which to express Estimated Average Requirements for water for different population groups, the Committee and the Institute of Medicine were forced to state “While it might appear useful to estimate an average requirement (an EAR) for water, an EAR based on data is not possible”. Given the extreme variability in water needs that are not solely based on differences in metabolism, but also on environmental conditions and activities, there is not a single level of water intake that would assure adequate hydration and optimum health for half of all apparently healthy persons in all environmental conditions. Thus, an Adequate Intake (AI) is established in place of an EAR for water.

The AI for different population groups was set as the median water intake in populations from the National Health and Nutrition Examination Survey, whose intake levels varied greatly based on the survey years (e.g., NHANES 1988–94 vs NHANES 1999–2002) and also were much higher than the USDA surveys (e.g., 1989–91, 1994–98, or 2005–6). If the AI for adults as expressed in Table 5 is taken as a recommended intake, we question the wisdom of the conversion of an AI into recommended water or fluid intake. The first problem is the almost certain inaccuracy of the fluid intake information from the national surveys, even though that problem may also exist for other nutrients. More importantly, from the standpoint of translating an AI into recommended fluid intake for individuals or populations, is the decision in setting the AI to add an additional roughly 20% of water intake, which is derived from some foods in addition to water and beverages. While this may be a legitimate effort to express total water intake as a basis for setting the AI, the recommendations that derive from this IOM report would better be directed at recommendations for water and other fluid intake on the assumption that the water content of foods would be a “passive” addition to total water intake. In this case, the observations of the DRI committee that water intake needs to meet needs imposed by not only metabolism and environmental conditions, but also body size, gender and physical activity. Those are the well studied factors which allow a rather precise measurement and determination of energy intake requirements. It is only logical that those same factors might underlie recommendations to meet water intake needs in the same populations and individuals, and therefore that consideration be given and data gathering be done by experimental and population research, to the possibility that water intake needs would best be expressed relative to the calorie requirements, as is done regularly in the clinical setting.

Table 5
Table 5
Water requirements expressed in relation to energy recommendations
It is important to note that only a few countries even include water on the list of nutrients.119 The European Food Safety Authority is developing a European wide standard.105 At present only the United States and Germany provide Adequate intake (AI) values but no other country does that.3, 120

Another way of considering an approach to the estimation of water requirements beyond the limited usefulness of the AI or estimated mean intake is to express water intake requirements in relation to energy requirements in ml/kcal. An argument for this approach includes the observation that energy requirements are strongly evidence-based in each age and gender group on extensive research which takes into account body size, and activity level which are crucial determinants of energy expenditure which must be met by dietary energy intake. Such measures of expenditure have used highly accurate methods such as doubly-labeled water and thus EERs (Estimated Energy Requirements) have been set based on solid data rather than the compromise inherent in the AIs for water. Those same determinants of energy expenditure and recommended intake are also applicable to water utilization and balance and this provides an argument for pegging water/fluid intake recommendations to the better studied energy recommendations. The extent to which water intake and requirements are determined by energy intake and expenditure is understudied but in the clinical setting it has long been practice to supply 1 ml per kcal administered by tube to patients unable to take in food or fluids. Factors such as fever or other drivers of increased metabolism affect both energy expenditure and fluid loss and are thus linked in clinical practice. This concept may well deserve consideration in the setting of population intake goals.

Finally, for decades there has been discussion of expressing nutrient requirements per 1000 kcal so that a single number would apply reasonable across the spectrum of age groups. This idea, which has never been adopted by the Institute of Medicine (IOM) and National Academy of Science, may lend itself to an improved expression of water/fluid intake requirements which must replace the AIs eventually. Table 5 presents the IOM water requirements and then develops a ratio of ml/kcal based on them. The European Food Safety Agency refers positively to the possibility of expressing water intake recommendations in ml/kcal as a function of energy requirements.105 Outliers in the adult male categories which reach ratios as high as 1.5 may well be based on American AI data which are above those in the more moderate and likely more accurate European recommendations.

Exploring the topic of utilizing ml/kcal as the way to examine water intake and water gaps, we take the full set of water intake AIs for each age-gender grouping and examine total intake in Table 6. They suggest a high level of fluid deficiency. Since a large proportion of fluids in the US are based on caloric beverages and this proportion has changed markedly over the past 30 years, fluid intake increases both the numerator and denominator of this ml/kcal relationship. Nevertheless even using 1 ml/kcal as the AI would leave a gap for all children and adolescents. We then utilize the NHANES physical activity data translated into METS/day to categorize all individuals by physical activity level and thus varying caloric requirements. Using these measures show a fairly large fluid gap, particularly for adult males as well as children.

Table 6
Table 6
Water intake and water intake gaps based on US Water Adequate Intake Recommendations (based on utilization of water and physical activity data from NHANES 2005–6.
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This review has pointed out a number of issues related to water, hydration and health. As undoubtedly the most important nutrient and the only one whose absence will be lethal within days, understanding of water measurement and requirements are very important. The effects of water on daily performance and short and long-term health are quite clear. There are few negative effects of water intake and the evidence of positive effects is quite clear from the literature.

Little work has been done to measure total fluid intake systematically and there is no understanding of measurement error and best methods of understanding fluid intake. The most definitive US and European documents on total water requirements as based on these extant intake data.3, 105 We feel that absence of validation of methods for water consumption intake levels and patterns represents a major gap. Little exploration of even varying methods of probing to collect better water recall data have been conducted.

Of course, the other half of the issue is the need for understanding total hydration status. We have no acceptable biomarkers of hydration status at the population level. Controversy exists about current knowledge of hydration status among older Americans.6, 121 This represents a topic understudied at the population level though certainly scholars are focused on attempting to create biomarkers for measurement of hydration status.

As we have noted, the importance of understanding the role of fluid intake on health has emerged as a more important topic partially because of the shift toward large proportions of fluids coming from caloric beverages. We summarized briefly a related systematic review of the clinical, epidemiological and intervention literature on the effects of added water on health. As a replacement for SSB’s, juice, or whole milk there are clear effects in that energy intake is reduced by about 10–13% of total energy intake. However on these topics, there are only a few longer-term systematic interventions and no published random-controlled longer-term trials. There is very minimal evidence on the effects of just adding water to the diet and of replacing water with diet beverages.

Limitations to this review are many. One certainly is the omission of discussions of the issue of potential differences in the metabolic functioning of different types of beverages122. There is little basis at this point for delving into this sparse literature. Another is omission of the potential effects of fructose (from all caloric sweeteners) when consumed in caloric beverages on abdominal fat and subsequently all the metabolic conditions directly linked with this (e.g., diabetes).123–126 We do not review in any detail all the array of biomarkers being considered to measure hydration status as there is just no sense in the field today that there is a measurement that covers more than a very short time period except for 24-hour total urine collection.

We suggest some ways to examine water requirements as a means to encouraging more dialogue on this important topic. Given the importance of water to our health and of caloric beverages to our total energy intake and potential risks of nutrition-related non communicable diseases, understanding both the requirements for water related to energy requirements, and the differential effects of water vs. other caloric beverages remain important outstanding issues.

In the end, this review has attempted to provide some sense of the importance of water to our health, its role in relationship to the rapid increases of obesity and other related diseases, and our gaps in understanding measurement and requirements. Water is essential to our survival and to our civilizations’ and we hope this critical role will sharpen our focus on water in human health.

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Funding was provided by the Nestlé Waters, Issy-les-Moulineaux, France, 5ROI AGI0436 from the National Institute on Aging Physical Frailty Program, and NIH R01- CA109831 and R01-CA121152. We also wish to thank Ms. Frances L. Dancy for administrative assistance, Mr. Tom Swasey for graphics support, Dr. Melissa Daniels for assistance, and Florence Constant (Nestle’s Water Research) for advice and references.

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Contributor Information
Barry M. Popkin, Department of Nutrition, University of North Carolina, Chapel Hill, NC.

Kristen E. D’Anci, Department of Psychology, Tufts University, Medford, MA.

Irwin H. Rosenberg, Nutrition and Neurocognition Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA.

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