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:

• Question 5 from the first paper of 2017
• Question 1 from the second paper of 2016
• Question 4 from the first paper of 2011
• Question 7 from the first paper of 2008

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. Unhinged rambling about the hormonal control of plasma calcium can also be found in the endocrine section, but the curious reader is warned that it contains a non-zero amount of palaeontology, among other self-indulgent garbage.

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. 

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.

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 (calbindinD_9k 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 a carbon instead of an oxygen 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; 

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