Distribution and regulation of calcium in the body fluids

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". It is difficult to know where to put this material, as it could just as easily be classified as endocrinology. Calcium the electrolyte has appeared multiple times in the past papers, and the questions have generally been asking about its distribution and regulation in the body fluids, but a lot of the answers depended on whether or not the candidates knew what the regulatory hormones did and where they came from. 

Calcium-related SAQs were:

  • Distribution
    • > 99% of total body calcium is in bone and not easily accessible
    • Less than 1% is in the extracellular fluid (~20-30mmol total)
    • Minimal intracellular concentration (second messenger)
    • In the plasma:
      • 40-45% of calcium circulates bound to albumin
      • 10-15% is bound to other anions such as lactate and phosphate
      • 40-45 % is free and ionised
  • Regulation of calcium intake
    • Absorbed by combination of paracellular and transcellular transport in the ileum
    • Transcellular route carefully regulated by hormones
      • intestinal absorption increased by calcitriol and PTH, 
      • decreased by calcitonin
  • Regulation of bone resorption of calcium
    • Normal daily flux in and out of bone is 12-13 mmol
    • Bone is reabsorbed by osteoclasts; this liberates calcium
    • Osteoclast activity is increased by PTH and decreased by calcitonin
    • Calcitriol has mixed effects; in vitro increased osteoclast activity, and in vivo protective effects in the treatment of osteoporosis
  • Regulation of renal elimination of calcium
    • 70% is reabsorbed in the proximal tubule
    • 20% is reabsorbed in the thick ascending limb
    • 10% is reabsorbed in the distal convoluted tubule by a mechanism that is regulated by PTH calcitriol and calcitonin:
      • Calcitonin decreases the reabsorption of calcium and phosphate
      • PTH and calcitriol increase the reabsorption of calcium and phosphate 
      • Calcitriol increases the activity of calbindin, TRPV5 and TRP
  • Main hormones involved​​​​​​​
    • PTH is a polypeptide hormone secreted by the parathyroid glands
      • Stimulus for release: hypocalcemia, hypophosphatemia
      • Inhibitory factors: calcitriol, hypermagnesemia
      • Actions: increases plasma calcium by increasing its renal reabsorption and osteoclast activity
      • Also increases Vit D conversion into calcitriol
    • Calcitriol is the metabolic product of Vit D, a fat-soluble vitamin
      • Stimulus for release: hypocalcemia, hypophosphatemia, PTH 
      • Inhibitory factors: decreased sun exposure, decreased functional renal mass, elevated calcium and phosphate levels
      • Actions: increases plasma calcium by increasing its gut absorption and renal reabsorption
      • Inhibits the production of PTH; conflicting effects on osteoclast activity
    • Calcitonin is a polypeptide hormone secreted from parafollicular cells of the thyroid
      • Stimulus for release: hypercalcemia, gastrin 
      • Inhibitory factors: hypocalcemia, somatostatin
      • Actions: decreases plasma calcium by decreasing its renal reabsorption, as well as inhibiting osteoclast activity

If the time-rich reader wanted to lose a few hours, their tumble into the calcium rabbit-hole would likely begin with an article like Mundy & Guise's "Hormonal Control of Calcium Homeostasis" (1999). One would likely follow with Daniel Bickle's 2014 treatment of Vitamin D, a definitive resource that happens to be freely available online. 

Distribution of calcium in the body fluids

You have about 360mmol/kg of calcium in your body;  that makes about 25 moles of calcium. A 70kg male has about 25 moles of calcium, or about 1000g.  Of this, over 99% is locked up in bone. Some absolute minimum amount is present in cells, and about 20-30 mmol of calcium is in the extracellular fluid. The proportion of circulating calcium is slightly higher than what might be expected from the usual distribution of extracellular fluid, because it is bound to albumin, of which the interstitial compartment has less. Thus, in your 10 or so litres of extracellular fluid, you have about 10-15mmol of mainly ionised calcium, and then a further 7.5-10 mmol of calcium in the plasma of the blood.

In the circulation 40-45% of calcium circulates bound to albumin, 10-15% is bound to other anions such as lactate and phosphate, and 40-45 % is free and ionised, which is the only biologically useful form. 

calcium distribution

Regulation of calcium intake

Most serious review literature on this subject starts with some kind of statement about how much calcium enters the adult human body, and how much leaves, and by which means it does so. Classically, this is represented in the form of a diagram where a length of bowel, a bone and a kidney are depicted as sources reservoirs and final destinations for calcium. Often, these diagrams are labelled Americanly, describing the flux of calcium in milligrams. In the Australian healthcare system, the ICU registrar and resident tasked with the duties of replacing calcium will usually do so by ordering calcium in millimoles, and so the author here takes liberties with some classical anatomy art to reframe calcium flux into this familiar metric scheme.

calcium homeostasis with millimolar metrics

Unlike potassium and sodium, which enter the body uninvited and just lounge around until the kidneys eliminate them, calcium is a carefully selected guest. Calcium absorption takes place in the duodenum by some active transcellular process, and passively along the rest of the gut (Bronner, 2003).  Because the duodenum is a brief and unremarkable pitstop along a food boluses' tour of the bowel, the transcellular uptake only plays a major role when dietary calcium intake is very low; which in the ICU probably correlates with periods of prolonged fasting or ileus. When calcium uptake is high or normal, paracellular passive uptake accounts for the majority of the gastrointestinal absorption. In addition to unabsorbed dietary calcium, some additional calcium is excreted as part of digestive secretions and shed gut mucosa (Davies et al, 2004); some of this becomes trapped in the gut by binding to dietary phosphate to form an irreversible bond and an insoluble compound. The net absorbed calcium (about 3.75mmol/day) is matched by the daily renal excretion of calcium (again 3.75 mmol/day). 

As you might have noticed, the pool of mobile calcium is small. The exchange of ionised calcium between the extracellular fluid, the soft tissue and the bone therefore has to be very finely controlled, as changes that might appear small will have a significant impact on the extracellular calcium concentration. Whenever you give somebody a 6.8 mmol ampoule of calcium chloride, you are essentially giving them two days worth of dietary calcium intake, rapidly increasing their total extracellular calcium content by one-third. Similarly, small changes in calcium binding to protein which make it more or less bioavailable can produce observable clinical change. 

Factors which influence the binding of calcium to protein

This is a topic large enough for a whole chapter all to itself. In brief summary: 

Albumin

increased albumin = decreased ionised calcium

pH

increased pH = decreased ionised calcium

Lactate

increased lactate = decreased ionised calcium

Phosphate

increased phosphate = decreased ionised calcium

Bicarbonate

increased bicarbonate = decreased ionised calcium

Citrate

increased citrate = decreased ionised calcium

Heparin

Presence of heparin in the sample = decreased ionised calcium

Free fatty acids

Increase in free fatty acids = decreased ionised calcium

Regulation of intestinal calcium absorption

The paracellular diffusion of calcium is not something you can control. However the transcellular mechanism is tightly regulated. This mechanism has three steps:

  • Apical entry into the cells via epithelial calcium channels TRPV5 (previously named ECaC1 or CaT2) and TRPV6 (previously named ECaC2 or CaT1); these are divalent cation channels which are probably also responsible for the absorption of other things, like magnesium
  • Diffusion along the cell bound to the  cytosolic calcium-binding protein (calbindinD9k or CaBP). Obviously, one cannot just let one's enterocytes get filled with ionised calcium every time one has a glass of milk, as it is a second messenger and would probably cause apoptosis or something. In order to protect these cells from the toxic effects of calcium, specialised proteins like CaBP exist, shuttling safely bound calcium through the cytosol.
  • Extrusion into the bloodstream is mediated largely by basal CaATPase and the Na+/Ca2+ exchange protein NCX1. Both ultimately use ATP, as the sodium which ends up exchanged for calcium will need to be removed by  Na+/K+ ATPase. Of the two of these, CaATPase does most of the work, and the NCX1 protein accounts for only 20% of the total calcium extrusion. 

Apparently, almost 90% of this occurs in the ileum, mainly because the residence of the intestinal content is longer. The following regulatory influences are involved:

  • Vitamin D, i.e. 1,25-OH-cholecalciferol, or calcitriol. Bronner (2003) reports that the intracellular transporter CaBP and the apical channels are highly dependent on vitamin D. In the absence of calcitriol (the active metabolite of Vitamin D), these critical steps in calcium absorption are downregulated. Calcitriol, therefore, is the main mechanism of control over calcium absorption in the gut.
  • Parathyroid hormone influences calcium absorption only indirectly by increasing the production of calcitriol in the kidney, through the stimulation of renal 1α-hydroxylase.
  • Calcitonin  appears to antagonise the calcitriol-indiced intestinal calcium absorption (Olson et al, 1972)

Regulation of calcium exit and entry from bone

Bone represents a massive and essentially unavailable locked reservoir of mineral calcium, from which (and into which) there is a constant trickle of calcium exchange.  The job of controlling this calcium trickle is performed by osteoblasts and osteoclasts which form or destroy bone, respectively. Bruzzaniti & Baron (2006) do an excellent job of discussing the detail of this, so that we do not need to revisit it here. The most important examinable elements are the hormonal influences:

  • Calcitonin has a potent inhibitory effect on osteoclast activity. When calcitonin binds it G-protein coupled receptors, a series of intracellular events takes place which disrupts the osteoclast cytoskeleton and eliminates their polarity, deacidifying their resorptive surface and turning them into basically useless jellyfish (Yamamoto et al, 2005). In short, calcitonin decreases calcium availability from the bone pool.
  • Parathyroid hormone has a direct effect on osteoblasts and osteocytes, and an indirect cytokine effect on osteoclasts, which stops bone synthesis and stimulates the release of calcium by increasing the rate of bone reabsorption. It is probably unnecessary to get carried away with the details of that indirect cytokine pathway; all you need to know is that you can't activate your osteoclasts by rubbing PTH all over them (nearby osteoblasts need to be involved).
  • Calcitriol also stimulates osteoclastic bone resorption, though things are not completely straightforward. It certainly can make osteoblasts turn into osteoclasts by means of the receptor activator of nuclear factor κB ligand (RANKL). This definitely happens in the lab. Interestingly, it can also prevent osteoporosis, i.e. there is some in vivo effect which is opposite to what is seen in vitro. This definitely happens in the field, i.e we routinely treat bone density loss with vitamin D supplements. The two conflicting actions are discussed by Takahashi et al (2014), who go into way more detail than is absolutely required here (tl;dr: we still do not know).

Action of bisphosphonates

Question 7 from the first paper of 2008 asked the candidates to "outline the mechanism of action of bisphosphonates". These drugs are analogs of pyrophosphate, containing an oxygen instead of a carbon atom. Their main effects are described in more detail by Fleisch et al (2002) and Rogers et al (2020). In a form condensed for a short written exam answer:

  • Inhibition of osteoclast and osteoblast activity
    • Bisphosphonates act locally on osteoclasts:
      • Binding to bone apatite crystals and local release during bone resorption
      • Thus, preferential accumulation under osteoclasts
    • Multiple effects:
      • inhibition of osteoclast recruitment and adhesion;
      • shortening of the life span of osteoclasts by being metabolised into toxic ATP analogues
      • inhibition of osteoclast activity by inhibiting several essential parts of the cholesterol synthesis pathway
    • The result is an inhibition of bone resorption as well as new bone formation
  • Inhibition of calcification by inhibiting the formation of calcium phosphate salts
    • Mainly seen in high doses
    • A totally physicochemical effect: they bind to the calcium of calcium phosphate
    • The result is inhibition of formation and aggregation of calcium phosphate crystals and inhibition of the transformation of amorphous calcium phosphate into hydroxyapatite.
    • This is useful for diseases of ectopic calcification
    • This also means mineralisation of normal bone (eg. during fracture healing) can be impaired

Regulation of calcium elimination via the kidneys

Renal handling of calcium consists of three main processes:

  • Only the ionised calcium is filtered
  • 70% of this is reabsorbed in the proximal tubule
  • 20% is reabsorbed in the thick ascending limb, which is autoregulated by the calcium-sensing receptor (CaSR) in the basolateral membrane of the thick ascending limb
  • 10% is reabsorbed in the distal convoluted tubule by a mechanism that is regulated by PTH and calcitonin

The transcellular reabsorption in the distal convoluted tubule is where all the fine control of plasma calcium concentration occurs. Like with the intestinal absorption, there are the same three steps here:

  • Apical transport proteins (TRPV5, TRPV6) allow calcium to enter the cell;
  • Calbindin bind it and allow it to traffic safely through the cell
  • Ca2+ATPase extrudes the reabsorbed calcium into the bloodstream

The regulatory influences on renal reabsorption, from Jeon (2008), consist of:

  • Calcitonin decreases the reabsorption of calcium and phosphate at the distal convoluted tubule, probably by altering the function of the basolateral Ca2+ATPase (Sexton et al, 1999).
  • Parathyroid hormone increases the reabsorption of calcium and phosphate at the distal convoluted tubule by increasing the transcription and apical membrane insertion of TRPV5. Calbindin activity also increases 
  • Calcitriol increases the activity of calbindin, TRPV5 and TRPV6; 

A brief digression about parathyroid hormone

PTH is this 84-amino acid peptide that comes from the chief cells of the parathyroid gland. All tetrapods appear to have some sort of parathyroid glands (but fish do not, and their PTH-equivalent is secreted by their gills). The actual PTH hormone and its receptors are actually rather ancient, being traced genomically to some point before the protosome-deuterostome split (i.e. pre-Cambrian). This makes sense, because the need to control extracellular calcium is fairly fundamental to the secondary messenger systems which use it, and those are common to all good god-fearing multicellular organisms. 

Palaeontological digressions aside, PTH is probably the most important regulator of calcium homeostasis and ends up being the first test you do to investigate a deranged calcium value. Its secretion is regulated by extracellular calcium levels, but in a manner that is not completely linear. High calcium concentrations can never completely suppress PTH secretion, and in severe hypocalcemia, the parathyroid gland does not appear to react proportionally to the existential threat of the frighteningly low calcium concentrations. This is illustrated by the graph below, which was shamelessly misappropriated from Felsenfeld et al (2007) and lightly modified to address the transPacific gap in results reporting (mg/dL instead mmol/L) and the unholy use of "z" in "ionised".

PTH vs clacium graph from Felsenfeld et al (2007)

In short, PTH secretion is stimulated by falling calcium levels, and reaches some sort of peak at an ICU-able ionised calcium concentration of around 0.90 mmol/L. A sensible reaction to such hypocalcemia would be to mobilise endogenous calcium stores, prevent its loss in urine and increase its absorption in the gut. All of these are effects produced by PTH. Within minutes of the onset of hypocalcemia, the rising level of PTH causes osteoclasts to pump calcium ions out of bone fluid and into the extracellular fluid pool. Then, over hours, changes in gut absorption and renal excretion increase the calcium levels further. Lastly, osteoclastic digestion of existing bone contributes more and more calcium from the massive (roughly 1.0kg) skeletal stores of calcium.

So, PTH ends up being the first test you order, when investigating calcium disturbances. If the calcium is low, one might expect PTH to be high, and so when one finds that it is not, the blame falls on the parathyroid gland. Similarly, when the serum calcium is high, one would not expect the PTH to also be high, unless something is fundamentally broken in the parathyroid glands. Conversely, when the PTH is reacting to the calcium level appropriately, that virtually rules out the parathyroid glands as the cause of the derangement. In those situations, one ends up looking at the Vitamin D level next.

Physiological role of calcium

It seems like a shame to waste the reader's time with something as pointless as the "physiological role" of an intrinsic building block of life. Unfortunately "the diverse roles of calcium" were 70% of the marks in Question 4 from the first paper of 2011.

  • Structural roles
    • Component of the hydroxyapatite matrix​​​​​​​
  • Functional roles
    • ​​​​​​​Intracellular second messenger, essential to:
      • Skeletal and smooth muscle contraction/relaxation
      • presynaptic release of neurotransmitter
      • endocrine/exocrine hormone secretion
      • apoptosis
      • mitochondrial energy production
      • Cell proliferation
      • Cytoskeletal changes which precede phagocytosis
    • Autocrine and paracrine extracellular messenger
    • Co-factor in extracellular enzymatic reactions:
      • Clotting cascade

References

Mundy, Gregory R., and Theresa A. Guise. "Hormonal control of calcium homeostasis." Clinical chemistry 45.8 (1999): 1347-1352.

Bronner, Felix. "Mechanisms of intestinal calcium absorption." Journal of cellular biochemistry 88.2 (2003): 387-393.

Davies, K. Michael, Karen Rafferty, and Robert P. Heaney. "Determinants of endogenous calcium entry into the gut." The American journal of clinical nutrition 80.4 (2004): 919-923.

Seldin, Donald W. "Renal handling of calcium." Nephron 81.Suppl. 1 (1999): 2-7.

Jeon, Un Sil. "Kidney and calcium homeostasis." Electrolyte & Blood Pressure 6.2 (2008): 68-76.

On, Jason SW, Billy KC Chow, and Leo TO Lee. "Evolution of parathyroid hormone receptor family and their ligands in vertebrate." Frontiers in endocrinology 6 (2015): 28.

Russell, John, Deborah Lettieri, and Louis M. Sherwood. "Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells." The Journal of clinical investigation 72.5 (1983): 1851-1855.

Felsenfeld, Arnold J., Mariano Rodríguez, and Escolástico Aguilera-Tejero. "Dynamics of parathyroid hormone secretion in health and secondary hyperparathyroidism." Clinical journal of the American Society of Nephrology 2.6 (2007): 1283-1305.

Bikle, Daniel D. "Vitamin D metabolism, mechanism of action, and clinical applications." Chemistry & biology 21.3 (2014): 319-329.

Bruzzaniti, Angela, and Roland Baron. "Molecular regulation of osteoclast activity.Reviews in Endocrine and Metabolic Disorders 7.1 (2006): 123-139.

Yamamoto, Yohei, Toshihide Noguchi, and Naoyuki Takahashi. "Effects of calcitonin on osteoclast.Clinical calcium 15.3 (2005): 147-151.

Chambers, T. J., et al. "The effects of calcium regulating hormones on bone resorption by isolated human osteoclastoma cells." The Journal of pathology 145.4 (1985): 297-305.

OLSON JR, BURDETTE E., HECTOR F. DELUCA, and JOHN T. POTTS JR. "Calcitonin inhibition of vitamin D-induced intestinal calcium absorption." Endocrinology 90.1 (1972): 151-157.

Takahashi, Naoyuki, Naoyuki Udagawa, and Tatsuo Suda. "Vitamin D endocrine system and osteoclasts." BoneKEy reports 3 (2014).

Fleisch, Herbert, et al. "Bisphosphonates: mechanisms of action." Principles of bone biology (2002): 1361-XLIII.

Rogers, Michael J., Jukka Mönkkönen, and Marcia A. Munoz. "Molecular mechanisms of action of bisphosphonates and new insights into their effects outside the skeleton." Bone 139 (2020): 115493.

Sexton, P. M., D. M. Findlay, and T. J. Martin. "Calcitonin.Current medicinal chemistry 6.11 (1999): 1067-1093.

Lips, Paul. "Vitamin D physiology." Progress in biophysics and molecular biology 92.1 (2006): 4-8.