Distribution of phosphate in body fluid compartments

This chapter is relevant to Section I3(ii) of the 2017 CICM Primary Syllabus, which expects the exam candidates to "describe the function, distribution, regulation and physiological importance of sodium, chloride potassium, magnesium, calcium and phosphate ions". The phosphate anion has been largely ignored by the CICM examiners. Question 20.1 in the first Fellowship Exam paper of 2017 potentially counts as a "phosphate question" because it had a hyperphosphataemic patient, but the college did not require the candidates to perform anything in the way of a detailed analysis ("interpret the biochemical results, giving underlying reasons" they asked). The Part I questions have repeatedly probed calcium metabolism, to which much of the phosphate material is linked, but phosphate itself has only come up once, as  Question 20 from the first paper of 2020. 

The exam candidate with near-infinite time resource may continue into the peer-reviewed publications on the topic of phosphate regulation and metabolism. One could do no better than Takeda et al (2004), which at the moment appears to be available as a free full text PDF. Lederer (2014) or Penido & Alon (2012) are also excellent.

Chemical properties

The phosphate anion (PO₄^-) is basically phosphoric acid (H₃PO₄). It has three pKas (2.2, 7.2 and 12.4), and can exist as (PO₄^-), (PO₄^2-) and (PO₄^3-), though at body fluid pH it is usually a mixture of (PO₄^-) and (PO₄^2-). It has excellent water solubility and does act as a weak acid, to the point where its accumulation in the body fluids can lead to a metabolic acidosis, though some might point out that the main reason for this will be the failure to properly use it in urinary buffering. 

Absorption of exogenous phosphate

Phosphate is abundant in the Western diet, and it is unlikely that there will ever be an episode of dietary phosphate depletion outside of a generalised starvation state.  On average, it seems there is a daily oral intake of about 40mmol of phosphate, and there is an additional daily generation of about 5mmol in the process of metabolism of phospholipids and phosphate-contaning proteins. A great deal of dietary variation in phosphate intake should be expected, as the modern gamer may consume truly bone-demineralising quantities of phosphate in the course of a normal day of glorping down litres of energy drinks. In case one reads this and feels seen, here is an excellent table of phosphate content in common beverages from Lindley et al, 2014:

 In the intestine, phosphate is absorbed by two main processes: one, a highly regulated sodium-dependent active co-transport, and the other a completely unregulated paracellular transport which is what probably accounts for the toxicity seen in inadvertent phosphate overdose.

Sabbagh et al (2011) points out that fortunately at least the active sodium-based transport mechanism is under close regulatory control; i.e. for physiologically normal doses (i.e not far exceeding normal dietary supplementation) the intestinal transporter can help by simply shutting down transport, and allowing the extra phosphate to simply remain in the bowel, generating diarrhoea. 

Distribution of phosphate and total body phosphate content

How much phosphate is in you, right now? All textbooks seem to have a slightly different value for intracellular and total body phosphate content. Guyton and Hall (11th ed. ) give the intracellular concentration as 11 mOsm/L (p. 294), saying that "approximately 85 per cent of the body’s phosphate is stored in bones, 14 to 15 per cent is in the cells, and less than 1 per cent is in the extracellular fluid".  Koam and Walsley's A Primer on Chemical Pathology gives the figure of total body phosphate as 25 moles, and says that "80% is complexed with calcium in bones, 10% incorporated into organic compounds and 10% combined with carbohydrates, proteins and lipids" (p.103). Ganong's Review of Medical Physiology (23rd ed.) gives the total body phosphorus mass as "500 to 800 g (16.1–25.8 mol), 85–90% of which is in the skeleton" (p.365). Neither of these textbooks gives anything in the way of references. Having said that, the 85-14-1% breakdown seems to be repeated with such seductive consistency that the author was compelled to reproduce these numbers here.

• Total body phosphate: 1% of body mass, 320 mmol/Kg, or 22.5 mol in a 70kg person (700g).
• Of this, the vast majority (85%, 19.1 moles or 595g) is bound in the hydroxyapatite matrix of bone.
• Of this bony phosphate, 300 mg o is exchanged every day as part of normal bone turnover. The other 15% is dissolved, and exists largely as the phosphate ion, PO₄^3-.
• The overwhelming majority of this 15% (say, 14.99%) is intracellular, which makes about 3.4 moles.
• Extracelllular phosphate is about 0.7mmol/L, or 12mmol in the total extracellular fluid. 
   

So where do these numbers come from, originally? They may sound authoritative, but in fact the basis for them is not completely scientific. Generally speaking, whenever a textbook mentions some value for body tissue or fluid composition, they are probably quoting this source, even when they don't realise it. Iyengar, Kollmer & Bowen published their "Normale Konzentrationen verschiedener Elemente tin Organen und Körperflüssigkeiten" in 1978. The book immediately came under fire for containing essentially unfiltered experimental data without much of an attempt to perform a synthesis of the findings. E.I Hamilton, a reviewer,  wrote, "the data presented in this compilation covers several decades of reporting but it is only really during the last five or so years that serious attention has been paid to quality control of analytical data; hence, as there appears to be no selection of preferred values, how accurate are the data?" This, as far as one can tell, is the source for the typically quoted textbook values for phosphate. 

There is indeed significant variation in the peer-reviewed literature with regards to body fluid and total body electrolyte content, and phosphate is severely affected by this issue, though perhaps not as much as something like cadmium or molybdenum, which would vary by factors of magnitude depending on how close your sample subjects live to the molybdenum-smelting plant.  Henry Lukaski (1987) offers an excellent discussion of the various means of body content measurement, and the ways in which it changes the results.  In short, depending of what you measure and how, you might come to very different conclusions. Because many of the studies come from a dark age of biochemistry, analytic quality controls and methodology are often unsatisfactory.

A favourite method of measuring total-body elemental content is to remove all volatile components and measure just the "dry" mass of the organism. For example, one of the more famous estimates for mammalian phosphate content is the historical 1926 study by Sherman and Quinn, who presented tabulated information regarding the Average Phosphorus Content of Normal Male Rats .  It turned out to be about 0.75% of total dry body mass, close to the 1% quoted above. The authors described their methods:

   "For the determination of phosphorus, the body of the rat was placed in a silica dish, charred slowly over a free flame, and burned at a dull red heat in an electric muffle to a white or light gray ash. The ash was dissolved with nitric or hydrochloric acid, the solution filtered and made up to volume."

Of course the use of incinerated or pureed organisms is relatively useless for the determination of body composition, because the most important matter is the distribution of the substance among the body compartments, as opposed to the total content of homogenised ash. Even relatively non-invasive techniques are fairly destructive. For instance, Cohn and Dombrovski (1971) developed a method that requires the investigator to  "deliver a moderated beam of fast neutrons to the subject", shot out of a cyclotron or a lump of plutonium-238.  The 3.5-8 MeV neutrons, captured by body atoms, create unstable isotopes which then rapidly revert to a more stable condition by emitting a gamma ray of characteristic energy, which you then measure with  "a detector array in a highly shielded facility". This permits a measurement of in vivo body elemental content without the need to incinerate your subject, but it does certainly make them quite radioactive, and so cannot be viewed as a totally benign investigation.  The article is ancient, and the only evidence of it now is this abstract in the Office of Scientific and Technical Information of the US Department of Energy. It is therefore impossible to say what became of their volunteers, or what the exact numbers were which they had generated.

Distribution of phosphate in the body fluid compartments

Intracellular phosphate content

Phosphate is the most abundant intracellular anion. Exactly how abundant is a matter of some scholarly debate. The clinical literature is awash with articles where the matter is either totally ignored or swept under the carpet, as by Penido et al (2012), where the authors dismissively claim that the intracellular concentration is "100-fold greater than that in the plasma" giving no reference or justification for the statement. 

This is because those references are hard to find. Bevington, Kep & Russell, in their chapter for Phosphate and Mineral Homeostasis (1985, pp 469-478) lamented the lack of attention to phosphate, jealous of the unfair spotlight which constantly seems to be shining on calcium. Without digressing into this, it is possible to say that intracellular phosphate is available in several species.  Of these, the species immediately available to use in reactions such as making ATP is P_i, "inorganic phosphate" or orthophosphate as it is known in some publications. This species is the "free" dissolved phosphate anion, which is a mixture of H₂PO₄^- and HPO₄^2-. It can be measured non-destructively by ³¹P-NMR nuclear magnetic resonance spectroscopy (Bevington et al, 1986). The role of phosphate as an intracellular buffer makes it difficult to generalise intracellular concentration data from disembodied strips of muscle to living organisms, and even within the same organism to different states of health or different tissues. As such, the author will offer a list of different ranges from different organisms and tissues, so that the reader can form their own impression. 

• 14 mmol/L (Bowen et al, 1982; Ehrlich ascites tumour cells from cancerous mice)
• 10 mmol/L (Knochel et al, 1980, defrosted leg muscles of a dog)
• 0.78 mmol/L (Noorwali et al, 1982;  human erythrocytes)
• 2.01 mmol/L (Noorwali et al, 1982;  human leukocytes)
• 4.12 mmol/L (Noorwali et al, 1982;  human platelets)

In summary, it's all a bit variable. If one were for whatever reason compelled to average these out to some sort of Representative Human Cell, one might settle for 10 mmol/L as a completely arbitrary value.

Apart from the immediately useful inorganic phosphate, intracellular phosphate is also bound to a variety of other molecular species. Of these, the most numerous is probably phosphocreatine, described as a high-powered intracellular energy reserve. Obviously cells differ in their need for battery storage, and so the levels of phosphocreatine vary from 10 to 30 mmol/L (Saks & Strumia, 1993). ATP, ADP and various other something P species of energy molecule are also responsible for intracellular phosphate stores, but because these are in a constant state of flux the exact concentration in any given cell at any given time is a complete guess. if one needed a range to use in some sort of childish cylinder diagram, one could fall back on sources such as Ataullakhanov et al (1997), which quotes the range of concentration for the relatively idle human erythrocyte as being between 1.0 and 5.0mmol/L.

Some of the intracellular phosphate is also bound to glucose as glucose-6-phosphate, which will again be highly variable between tissues and nutritional states (eg. before vs. after doughnut). According to a popular textbook by Stipanuk & Caudill, this concentration is usually 0.05-1.0 mmol/L; however it is probably lower at rest. Rothman et al (1992) used ³¹P-NMR to demonstrate that even under the effects of insulin the human skeletal muscle cells have a glucose-6-phosphate no higher than 0.22 mmol/L. 

Extracellular phosphate content

The extracellular concentration of phosphate is about 1.0mmol/L. In total, no more than 12mmol is available in the extracellular fluid at any given time.  This is the only phosphate source to the cells. Most of it is present as a univalent species (i.e. with a negative charge of 1). Even though the phosphate ion, when fully ionized, is a trivalent PO₄^3-  at physiological pH the divalent PO₄^2-  species is the more prevalent (about 80%), and the univalent PO₄^-  species is the other 20%. 

 Extracellular phosphate behaves in a manner similar to calcium. An authoritative paper by Baker and Worthley on calcium magnesium and phosphate metabolism reports that about 15% of it is bound to protein, another 53% free and ionised, and another 47% compelled with sodium, calcium and magnesium. The attentive reader will point out that this adds up to 115%, which seems to be the result of treating protein-bound phosphate as a separate percentage for some reason. Fortunately, these numbers mean spectacularly little, as any precise percentages of circulating phosphate described in a textbook or diagram are always going to be an idealised artists's impression.

The variability of phosphate distribution between the protein-bound and complexed states is dependent on a variety of factors, including pH, albumin level, sodium  calcium and magnesium concentrations, and the concentration of other molecules which compete for the same ions (mainly lactate and citrate). If one really needs a representative set of values, one may be directed to an ancient paper by Walser (1961), which presents the empirical data on ion fractions from 22 healthy volunteers. The mean protein-bound fraction of phosphate was 13%, but the range extended from 2% to 25%.  The author, after some considerable internal conflict, surrendered to his shameful urge and presented Walser's original data as a shiny cylinder diagram:

As you can see, Walser's data describe complexes between phosphate and other ions, where the term "complex" was used rather frivolously. For example, of Walser's complexes, the vast majority appeared to be formed between phosphate and sodium (around 0.3-0.4 mmol/L), with calcium and magnesium apparently contributing minimally. However, as chemically savvy readers might point out, sodium and phosphate have excellent solubility, and should not have any lasting interest in one another, certainly not to the point of forming some kind of chemical union. More likely, these were transient ion-ion interactions, which would not meet any modern definition of "complex" (since 1990, IUPAC would prefer us to use the term "coordination entity" to describe this loose noncovalent association of molecules). In this, the sodium-phosphate interactions are probably more like the interactions between sodium and dermal glycosaminoglycans, which researchers (eg. Hanson et al, 2021) are careful to never describe as "complexes".

This means the "complex" between sodium and phosphate is probably not a clinically relevant entity, as it shouldn't prevent phosphate from taking part in its various physiologically important molecular interactions. Unfortunately, not only is this sort of discourse missing from textbooks, but also there does not appear to be any authoritative published material on the subject. This section was enlightened mainly by crowdsourced peer review by people like Paul Tamas, whose expert contributions helped disperse some of the fog.

Movement of phosphate between the intracellular and extracellular compartments

Phosphate equilibrates easily between the extracellular fluid compartments. There really isn’t anything stopping it. It is strongly anionic, so there is slightly more in the interstitial fluid (as it is repelled by the anionic charge of plasma proteins). Phosphate is sucked into the cell by a sodium-phosphate co-transporter, which is more active in the presence of insulin. This process is powered by the awesomely powerful sodium concentration gradient. There are three types (inventively nominated as Type1, 2 and 3); the Type 3 co-transporter seems to be the ubiquitous one, which all of the human cells express.

Elimination of phosphate

Of the total daily phosphate loss, about 30mmol is excreted renally, and 15mmol is lost via stool. Whatever phosphate is not bound to protein gets filtered freely by the glomerulus, and then most of is reabsorbed in the proximal (70%) and distal (10-20%) tubules. The resorption occurs with the help of a sodium-phosphate cotransporter (one of the various sodium-organic anion cotransporters),  which is driven by the sodium gradient.

Renal reabsorption of phosphate is presented in more detail locally, as well as in a paper by Gmaj & Murer (1986). In summary, the renal excretion of phosphate is one of the regulatory mechanisms for acid-base homeostasis, and a disruption of those excretory processes can result in accumulating phosphate contributing to the metabolic acidosis of renal failure. It is mainly reabsorbed in the proximal tubule by a mechanism that is highly pH-dependent, i.e. with lower systemic pH the reabsorption of inorganic phosphate decreases because more of it ends up being present in H₂PO₄^- form, and the affinity of those sodium-phosphate cotransporters decreases.

With chronic metabolic acidosis, the reabsorption of phosphate here decreases because of decreased apical membrane channel expression, apparently under the control of PTH. This is a handy segue into:

Regulation of phosphate turnover

The following diagram is offered as a means of avoiding a deep dive into the endocrinology of parathyroid hormone and the like. If that's what you want, you will find it in Penido & Alon (2012).

In summary, the transient pool of extracellular inorganic phosphate originates from multiple sources. Much of it is a byproduct of the metabolism of bone hydroxyapatite, and some is being absorbed from the gut and recirculated from the kidneys. The size of this pool is therefore regulated by:

• Regulation of bone metabolism
• Regulation of gut absorption
• Regulation of renal reabsorption

Bone phosphate metabolism is regulated by parathyroid hormone. PTH

• Calcitriol increases the absorption of phosphate from bone
• PTH increases the absorption of phosphate from bone, and stimulates the production of calcitriol

Intestinal phosphate absorption  is regulated by:

• Dietary sodium: it is a co-factor necessary for active phosphate transport in the gut
• Phosphate concentration in the gut (a high concentration is necessary for passive transport)
• Calcitriol increases the absorption of phosphate (and conversely, a low phosphate stimulates the synthesis of calcitriol).
• PTH does not seem to have direct control over phosphate reabsorption in the gut, but it increases the synthesis of calcitriol, which in turn increases phosphate reabsorption. So, indirectly, PTH increase gut absorption of phosphate.

Renal phosphate reabsorption is regulated by:

• PTH, which decreases the proximal tubular reabsorption. In the absence of PTH, reabsorption is greater. When PTH release is stimulated by hypocalcemia, renal expression of apical sodium-potassium cotransporters decreases.
• Calcitriol increases the reabsorption of phosphate in the proximal tubule
• Acid-base balance regulates phosphate homeostasis, as phosphate is a urinary buffer; more phosphate is eliminated under conditions of acidosis

Notably, though calcitriol uniformly does everything possible to maximise extracellular phosphate, you will notice that PTH does not. PTH causes increased bone turnover and increased GI absorption of phosphate, but this effects are more than compensated for by its effects on renal phosphate clearance, such that in hyperparathyroidism, there is usually hypophosphataemia. 

Now; you may notice that, though a large proportion of phosphate is intracellular, it does not get involved overmuch in the overall accounting of body phosphate turnover. Intracellular phosphate tends to stay intracellular, as it is doing important work there, and should not be disturbed. 

The physiological role of phosphate

Phosphate is damn near everywhere. It generally seems to have a central role in life as we know it.

There is no way to cover these functions other than in the form of a brief summary:

• Structural role
  • Bone mineral
  • Phospholipid of cell membrane
  • Incorporated into DNA and RNA molecules
• Regulatory role
  • Secondary messenger (IP3)
  • Protein activity is turned on and off by by phosphorylation and dephosphorylation
• Metabolic role
  • Co-factor in oxygen transport (as 2,3-DPG)
  • Trapping glucose in cells (as glucose-6-phosphate)
  • Synthesis of ATP (it's the P in ATP)
  • Acid-base regulation (urinary and intracellular buffering)