Control of plasma calcium

This chapter is related to Section U1(vii) from the 2017 CICM Primary Syllabus, which asks the exam candidate to  "describe the control of plasma calcium". It is unclear why calcium was singled out, considering that body water, plasma sodium and plasma potassium are also under tight endocrine control, and the hormonal systems involved in their regulation are surely also of some interest to the intensivist. Weirdly, a much more inclusive syllabus item does exist in the section on body fluids and electrolytes, where the college refers to "the function, distribution, regulation and physiological importance of sodium, chloride potassium, magnesium, calcium and phosphate ions". Rather than to meekly accept this poor structure, the author has chosen to have all the electro-regulatory chapters gathered together in one place, and a detailed review of the distribution and regulation of calcium in the body fluids is among them. 

Calcium-related SAQs in the First Part exam have also been resettled in the body fluid section, even though many of them have a strongly endocrine tone. "Describe the regulation of plasma calcium concentration", they usually demand. Distribution and functional importance of calcium are also asked about, and occasionally one encounters some discussion of calcium supplement pharmacology. These SAQs consisted of:

• 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

Mundy & Guise's "Hormonal Control of Calcium Homeostasis" (1999) is the best resource for the casual reader, as it covers all the hormonal systems, but if anybody needs to look into each element individually, they can be referred to Goltzman (2018) for parathyroid hormone, Lips (2006) for Vitamin D and Masi & Brandi (2007) for calcitonin.

Control of plasma calcium

The concentration of the calcium in the blood is controlled by the balance between input and output, where the input is a combination of absorbed dietary calcium and reabsorbed bone calcium, and output is renal elimination and bone deposition. Thus, there are three main regulatory factors: intestinal intake, renal excretion, and the flux of calcium in and out of bone. 

• 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

Additionally, the binding of calcium to albumin is an important factor in its biological availability, and this is influenced by a host of factors, but you couldn't really call this "control" of plasma calcium, as it is not a regulatory mechanism. 

Parathyroid hormone

PTH is a polypeptide hormone secreted by the chief cells of the parathyroid glands. It is a large 84-amino-acid peptide, cleaved from an even larger prohormone (as all of these peptide hormones tend to be).  The interested reader can review the biosynthesis of PTH in Kumar & Thompson (2011)- spoiler, it is stored in granules which undergo fusion with the cell membrane to mediate exocytosis, a predictable mechanism which is of little interest to the CICM trainee because we do not have a convenient method of manipulating it. 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. PTH release is not stimulated, but rather disinhibited: in other words, most of the regulatory influences on PTH are inhibitory influences, and PTH secretion only increases when they are lifted.  The binding of calcium to the parathyroid calcium-binding receptor is the most important inhibitor of its release, i.e. the release of PTH increases with falling calcium (ionised calcium, of course, is the most important variable, rather than the total calcium). Other inhibitory modulators of PTH secretion include Vitamin D (specifically calcitriol), cations such as lithium, and various cytokines and prostaglandins. Inorganic phosphate is the only proper stimulatory release factor, as high phosphate levels tend to stimulate PTH secretion (by acting directly on the parathyroid calcium sensing receptor).

The relationship of calcium levels and PTH secretion 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".

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.

The actions of PTH are to increase plasma calcium by increasing its renal reabsorption and osteoclast activity (as well as increasing Vit D conversion into calcitriol). It exerts these effects by binding to PTH receptors, of which there are two, unimaginatively named PTH1R and PTH2R. These are G_s-protein coupled receptors, i.e they produce their intracellular effects by increasing the availability of cAMP. Thing 1 is distributed widely across bone, renal tubules, intestine and skin, whereas Thing 2 is mainly seen in the pancreas and central nervous system. In detail, the effects of PTH are:

• Osteoclastic:
  • Direct effect on decreasing osteoblast activity
  • Increased osteoclast activity (indirect cytokine effect)
  • Thus, increased release of calcium and phosphate from bone, and decreased bone deposition
• Renal:
  • Decreased reabsorption of inorganic phosphate at the proximal tubule
  • Increased reabsorption of calcium at the thick ascending limb of the loop of Henle
  • Increased production of production of calcitriol in the kidney, through the stimulation of renal 1α-hydroxylase.
• Intestinal:
  • Indirect effect, by increasing the the production of calcitriol in the kidney, which in turn increases the 

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.

Vitamin D

Vitamins D₂ and D₃ (ergocalciferol and cholecalciferol) are secosteroids, made from a broken steroid structure that only contains three rings. Chemistry weirdos are referred to page 1814 of this steroid nomenclature document for more information about their naming conventions. Even IUPAC had decided that "proper" names for these chemicals would be too cumbersome and proposed to call them something functional but short, which is how we have ended up with calciol calcidiol and calcitriol (the di- and tri- indicate the locations of hydroxyl groups). Not everybody dances to the IUPAC tune, and the terms "calciol" and "calcidiol" are basically unknown. Calcitriol, on the other hand, is used fairly often, as the alternative would be to twist your tongue around 1α,25-dihydroxycholecalciferol. This metabolite is the most important of the group, as it is the active form of the hormone, and is responsible for the majority of its physiological effects.

Cholecalciferol is produced when 7-dehydrocholesterol is exposed to UV light, and this reaction requires no enzymes. Apparently, this was the original purpose of vitamin D - to be an inert byproduct of radiation defence, acting as a radiation scavenger and protecting primitive unicellular life at the dawn of time. Hanel & Carlberg (2020) put its evolutionary origin at roughly 1.2 billion years ago, around the time when increasing oxygen levels had made the synthesis of cholesterol possible for the earliest algae. Only later, closer to the Cambrian explosion, did we start using it properly - it appears the nuclear vitamin D receptor was "invented" by some of the earliest chordates around 550 million years ago (which begs the question, what the hell were these things doing with it, being creatures without a calcified skeleton). 

Anyway: in the bone-filled calcium-loving vertebrate, vitamin D controls not only important aspects of calcium homeostasis, but aspects of innate and adaptive immunity, energy metabolism (mainly of carbohydrates), cell proliferation and differentiation, and probably a million other things. David Bikle's 2009 article on the nonclassical actions of vitamin D is a classic for those who want to know more about these shadowy activities, but for the CICM exam candidate, the calcium effects will be more than enough. 

Synthesis of calcitriol from raw ingredients occurs in three sites, an example of the sort of inefficiency you might run into if your metabolic pathways arose in the course of millions of years of evolution spanning between unicellular and multicellular life. A substrate is processed in the skin, then again in the liver, and finally in the kidney, to become an active form of the hormone. Observe:

• 7-dehydrocholesterol, an unsaturated sterol which is also used a precursor for cholesterol synthesis, turns into previtamin D₃ when UVB light of wavelengths between 295 and 300 nm hits the double bound between C7 and C8. The bond breaks and opens the ring, creating the secosteroid structure.
• Previtamin D₃ is the name given to the unstable breakdown product of this reaction, and it isomerises spontaneously at body temperature to form cholecalciferol.
• Cholecalciferol can also be acquired through diet. "Oily fish" is the comment usually thrown by medical physiology textbooks, as if everybody's inner Inuit will immediately nod knowingly at the concept. For the person whose fish experience is limited to suspiciously discounted cans, "oily" literally means the fish are full of oil, to the point where you could literally use some of them to fuel a fire. These are sprats, herring, mackerel, haddock, and salmon, among others. Wherever it comes from, cholecalciferol in the circulation binds to a chaperone protein (unimaginatively named vitamin D binding protein), and it travels the bloodstream until it is hydroxylated in the liver, where CYP2R1 or CYP27A1 turn it into 25-hydroxyvitamin D₃. 
• 25-hydroxyvitamin D₃ is also known as 25-hydroxycholecalciferol, calcifediol or calcidiol. It is also entirely inactive, and also bound to vitamin D binding protein in the circulation. It then gets to circulate pointlessly until it is processed by 1α-hydroxylase in the proximal tubule. This enzyme is the main regulatory lever used to control the activity of vitamin D; for example its activity is stimulated by PTH and inhibited by hyperphosphataemia.
• 1,25-dihydroxyvitamin D₃, or 1α,25-dihydroxycholecalciferol, or calcitriol, is the active form of the hormone.

For some of us, is perhaps easier to visualise this as a flowchart:

From there, this hormone does predictable steroid things. The calcitriol receptor is a nuclear transcription factor, and most of the effects mediated by calcitriol are genomic effects. They are broadly the same as those of PTH, in the sense that the whole process ultimately leads to the increase of plasma calcium, but the two hormones do slightly different things. Calcitriol has a lot of influence on intestinal absorption of calcium, whereas PTH has little direct control over it; and both of them increase the renal reabsorption and reclamation of calcium from bone. In an exam-like structure, the effects of calcitriol could be summarised like this:

• Osteoclastic:
  • Weak direct effect on increasing osteoclast activity
  • Potent effect on osteoclast precursor differentiation
  • That should be expected to produce bone resorption, but in fact bone formation is also one of the possible effects, and the detailed mechanisms of these conflicting effects remain to be established (Takahashi et al, 2014).
• Renal:
  • Increased reabsorption of inorganic phosphate at the proximal tubule (the opposite effect to the effect of PTH)
  • Increased reabsorption of calcium at the thick ascending limb of the loop of Henle, but also at other sites (by increasing the transcription of all the transport proteins involved in this process)
• Intestinal:
  • Direct effect on enterocytes, where the transcription of an intracellular transport protein is increased (calcium-binding protein, CaBP)
  • Intestinal absorption of phosphate is also increased
  • Most of this happens in the ileum (Bronner, 2003) 

That intestinal effect is basically the only way to increase the entry of calcium into the body, which makes calcitriol rather important. By comparison, PTH merely shuffles it around between different compartments. Both act together to increase the recovery of calcium from the renal tubule, and both can promote the reabsorption of bone, but they do opposite things to the renal elimination of phosphate. But then one stimulates the release of the other. Ureña Torres & De Brauwere (2011) tried to disentangle this knot of endocrine feedback loops:

• PTH increases intestinal phosphate absorption, but also increases its renal elimination, which should theoretically be phosphate-neutral. It also stimulates the synthesis of calcitriol.
• Calcitriol increases both intestinal absorption and renal absorption, which means it should promote the accumulation of phosphate.
• But: the accumulation of phosphate directly increases the release of PTH, which increases the activity of 1α-hydroxylase, which then produces more calcitriol, which increases the absorption of phosphate, which should lead to some kind of out of control positive feedback loop of endless phosphate hoarding.
• Fortunately, raised phosphate stimulates a third regulatory system, which is FGF23 (Fibroblast Growth factor 23, a circulating 32-kDa peptide secreted by osteocytes). This regulatory molecule directly decreases the reabsorption of phosphate in the renal tubule by acting on the proximal tubule transport proteins, and also decreases the synthesis of calcitonin by inhibiting the activity of 1α-hydroxylase in the proximal tubule.

Calcitonin

Calcitonin is a 32-amino-acid polypeptide hormone secreted from parafollicular cells of the thyroid. It is thought by some to be a vestigial hormone, and the people who think so point to the fact that in humans there are no known cases of calcitonin deficiency (whereas when you run out of PTH or vitamin D, all calcium hell breaks loose). On the other hand, from an evolutionary perspective it goes back to the earliest vertebrates, and when you look at modern animals it appears they have all kept the same basic structure, i.e. it is highly conserved. That would suggest that it is important, because unimportant sequences tend to become garbled over millions of years, and only essential peptides preserve their structure across species. For calcitonin the preservation has such high fidelity to the original arrangement that your human calcitonin receptors will gladly bind to salmon calcitonin for a basically identical physiological effect. It is true that its role in calcium metabolism is not dominant, but it probably has a lot of other regulatory functions, which are discussed in the excellent articles by Hirsch et al (2001) and Davey & Findlay (2012).

For this calcium-centric chapter, the role of calcitonin in calcium regulation is the focus. Calcitonin is made from procalcitonin, a much larger peptide, in the Golgi apparatus of parafollicular cells, and is then stored as vesicles. Its release is stimulated by the increase of plasma calcium, but also by other humoral actors such as oestrogen and gastrin (for example, calcitonin will increase after a meal, and during pregnancy and lactation). Its calcium-stimulated release is mediated by same calcium-sensing receptor (CaSR) as is responsible for the release of PTH, except in the case of PTH the secretion of the hormone is suppressed instead. 

Calcitonin receptors are scattered pretty widely across the human organism. Osteocytes are obviously the main target, but there are receptors in the kidney, the CNS, placenta, testes, lung, and in the cells of the immune system. They are a family of G-protein coupled receptors which have cAMP-mediated effects, but they also dabble in phospholipase C, D, and other signalling pathways. 

The physiological effects of calcitonin are:

• Osteoclastic:
  • Direct effect on decreasing osteoclast activity; in fact their motility is affected within one minute, which is followed by a more gradual retraction
  • This decreases the resorption of bone, and therefore limits the entry of bone calcium and phosphate into the blood
• Renal:
  • Calcitonin acts as a weak diuretic, increasing the elimination of sodium, chloride, phosphate and calcium. The effect on calcium is mainly due to inhibited reabsorption.
  • It also increases production of production of calcitriol in the kidney, through the stimulation of renal 1α-hydroxylase.
• Intestinal:
  • Calcitonin increases gastric acid and pepsin secretion and decreases pancreatic amylase secretion.
  • It has no direct effect on calcium absorption in the intestine, but it can increase it indirectly by stimulating renal calcitriol synthesis.