Total serum calcium comprises three major forms:

  1. Free ionized calcium (about 50-55% of total)
  2. Protein bound (about 35-45% of total). Most of the protein-bound calcium is bound to albumin (80%, with the rest bound to globulins), likely to imidazole groups.
  3. Complexed with anions such as bicarbonate, citrate, lactate, and phosphate (about 5-10% of total).

Ionized or free ionized calcium (iCa2+) is the metabolically active form of calcium and this is the form that the body senses and responds to (e.g. by stimulating osteolysis if ionized calcium concentrations are low). Specific receptors on parathyroid chief and C cells sense and respond to ionized calcium concentrations through unique calcium-sensing receptors on their surfaces. Activation of the receptor (e.g. high free ionized calcium) inhibits PTH secretion, whereas inhibition of the receptor (through genetic mutations or inhibitory antibodies, low free ionized calcium) stimulates PTH secretion. Inactivating and activating mutations of the calcium-sensing receptor may be the underlying cause of hypercalcemia and hypocalcemia in neonatal animals. Free ionized calcium concentration can be measured by ion-selective electrodes but this usually is not part of the routine screening panel of serum chemistry tests. Note also that the free ionized calcium concentrations are affected by pH within the tube as part of in vitro changes. Decreased pH with cell metabolism and lactate production in the tube may increase free ionized calcium by decreasing protein and anion binding, while an increased pH with frozen storage or loss of carbon dioxide with exposure to air may decrease free ionized calcium.


About 99% of calcium is found as hydroxyapatite in bone, with the remaining 1% in serum or plasma. Intracellular calcium (which is critical for cell homeostasis) is a miniscule portion of total calcium, being around 10,000 fold lower in cells than that in serum. Calcium is particularly involved in muscle and nerve function. Specific voltage-dependent calcium channels are ion channels in muscle and neural membranes that allow the influx of calcium into cells, leading to calcium-mediated excitation (e.g. muscle contraction, nerve conduction), gene transcription and cell signaling.

The main organs involved in calcium homeostasis are the intestines and kidneys. Calcium is absorbed in the intestine with phosphate under the action of vitamin D. Calcium is stored in the body in bone and excreted through the kidneys and feces (to some extent). Normally, losses of calcium in the intestinal and renal systems are balanced by dietary calcium intake. If losses are in excess of intake, the bone serves as a source of calcium for the body. Intracellular calcium is vital for most cellular responses (many enzymes, signaling and transport processes rely on calcium). Calcium is usually sequestered within endoplasmic reticulum (which acts as a reservoir to release stores) and mitochondria. It is also bound to cellular proteins, e.g. troponin C, calmodulin. Leakage of calcium from mitochondria is a marker of mitochondrial injury and low intracellular stores of calcium result in cytotoxicity and cell death.

  • Absorption: The main site of absorption is the ileum. As indicated above, this is actively mediated by vitamin D, although passive absorption of calcium also occurs along a favorable concentration gradient, which is facilitated by vitamin D (indirectly alters the permeability of the intestinal epithelium) (Blaine et al 2015).  Vitamin D induces the production of calbindins (a family of calcium-binding proteins), particularly calbindin-D9k, in the intestinal mucosa. Calbindin-D9k facilitates the transport of calcium across the luminal surface of the intestinal epithelial cells through calcium channels and may also increase the activity of the sodium-calcium basolateral pump that transfers calcium into blood from the intestinal cell. Parathyroid hormone (PTH) indirectly promotes absorption by stimulating 1α-hydroxylase in the kidney to convert vitamin D (calcidiol or 25-hydroxycholicalciferol) to its most active form, calcitriol (1,25 dihydroxycholicalciferol). Note, that this does not occur in horses, which lack this hydroxylase enzyme. Thus intestinal absorption of calcium under physiologic conditions is largely independent of vitamin D in the horse (likely due to the high dietary content of calcium) and horses absorb much larger amounts of calcium in their diet compared to other species (up to 75% of dietary calcium) (Toribio, 2011). Absorption of calcium is inhibited by corticosteroids. Enteric calcium absorption is also influenced by acidity (acidifying substances promote absorption), presence of other components in the diet which may chelate calcium (e.g. oxalate, phosphates, phytates), and the health of the intestinal epithelium.
  • Release from bone: Calcium is stored with phosphate as hydroxyapatite in bone. Bone calcium is the largest source of calcium in the body, with more than 99% of calcium tied up within bone.  Calcium and phosphate are released from bone by the action of parathyroid hormone (PTH). PTH acts primarily via stimulating osteoblasts to secrete factors which promote osteoclasts to develop from monocytes, specifically RANKL (Receptor Activator of Nuclear Kappa B Ligand). RANKL  binds to its receptor (RANK) on osteoclasts triggering differentiation (with monocyte-colony stimulating factor or M-CSF), preventing osteoclast apoptosis and promoting bone resorption. RANKL is also produced in bone marrow stromal cells (and also activated T cells) and its production is also stimulated by dexamethasone, vitamin D and locally produced cytokines that have osteoclastogenic properties (e.g. interleukin-6, interleukin-11, tumor necrosis factor α). RANKL’s actions are blocked by a protein called osteoprotegrin (OPG), which acts as a soluble decoy receptor of RANKL. The RANKL/OPG ratio determines the level of osteoclast formation (a high ratio would favor osteoclast formation and osteolysis, whereas a decreased ratio would inhibit osteolysis). Vitamin D not only stimulates RANKL production but also facilitates the action of PTH. In contrast, calcitonin inhibits osteolysis by binding to receptors on osteoclasts, but this effect is transitory.
  • Renal excretion: Free ionized calcium or anion-bound calcium is filtered through the glomerulus. Most of the filtered calcium (around 60%) is reabsorbed in the proximal convoluted tubules along with sodium. This reabsorption is passive but can be stimulated by angiotensin II, which stimulates sodium absorption in the proximal tubules. Control of renal excretion occurs in the ascending limb of the loop of Henle (20-25%) and distal nephron (around 5-15%), which contains receptors for PTH, vitamin D, and calcitonin. In the thick ascending limb of the loop of Henle, calcium is absorbed by the paracellular pathway in response to a favorable electrochemical gradient established by absorption of NaK2Cl and back-excretion of potassium. A calcium sensing receptor in this segment also alters the permeability of the epithelium by modulating claudin expression (tight junction proteins). PTH stimulates calcium resorption and phosphate excretion transcellularly and actively in the loop of Henle and distal tubules, whereas vitamin D promotes calcium absorption by increasing calbindin in the distal tubules, but this is not the main mechanism of action for vitamin D (rather, it is stimulation of intestinal absorption). The renal resorption of calcium is so efficient, that <2% of calcium is lost through the kidneys. Corticosteroids promote renal excretion of calcium. In horses, renal excretion of calcium is a major mechanism for excreting excess dietary calcium; a much larger percentage of absorbed dietary calcium is excreted by horses than in other species. The same may be true of guinea pigs. Factors that promote or inhibit NaK2Cl in the ascending limb of the loop of Henle will increase or decrease calcium resorption, respectively (Blaine et al 2015).


Calcium is usually measured with colorimetric assays and dyes that bind to calcium. The o-Cresolphthalein  colorimetric method is used at Cornell University as indicated below:

Reaction type

Blanked end-point


In an alkaline solution calcium reacts with o-cresolphthalein complexone to form calcium-o-cresolphthalein complex, which produces a purple color that is measured photometrically and is proportional to the concentration of calcium in the sample. The reaction is shown below:

Calcium + o-cresolphthalein complexone alkaline solutionCa-o-cresolphthalein complex

Units of measurement

The concentration of calcium is measured in mg/dL (conventional units), mEq/L (conventional units), and mmol/L (SI units). The conversion formulas are shown below:

mg/dL x 0.2495 = mmol/L 

mEq/L x 0.5 = mmol/L

Sample considerations

Sample type

Serum, plasma, and urine


Exposure of blood to anticoagulants such as EDTA, citrate, and oxalate (the anticoagulant in the sodium fluoride tube) reduces calcium to an unmeasurable level. Since calcium less  than 2 mg/dL is not compatible with life, exposure to agents that chelate calcium are indicated by such a result. In general, calcium values in heparinized plasma and serum are usually similar, but this may not always be the case.  Internal studies in the Clinical Pathology Laboratory at Cornell University in bovine blood show that values in heparinized plasma range from 0.2 to 0.5 mg/dL higher than serum (Naeves and Stokol, unpublished data).


  • Human: Per reagent manufacturer product information sheet
    • Serum and plasma: 7 days at room temperature, 3 weeks refrigerated, and 8 months frozen (-20°C). Bovine serum or plasma is stable frozen at -80C for 12 months (Bach et al 2020).
    • Urine: 2 days at room temperature, 4 days refrigerated, and 3 weeks frozen (-20°C).
  • Bovine: Calcium is stable in whole blood, separated serum or plasma for 14 days when stored at 4C (Bach et al 2020). Internal studies in the Clinical Pathology Laboratory at Cornell University show that there are minimal differences in total calcium results (i.e. changes of 0.2 mg/dL or less) in whole blood stored in a red top or green top (heparin) tube (before separation) for up to 6 hours at 22°C (Naeves and Stokol, unpublished data). 


  • Lipemia: Severe lipemia may increase concentrations (>1000 lipemia index)
  • Hemolysis: Does not really affect calcium concentrations.
  • Icterus: Severe icterus may increase concentrations (>60 icteric index)
  • Drugs: None reported for animals.

Test interpretation

Increased calcium concentration (hypercalcemia)

Hypercalcemia is not common in any species but is encountered more often in dogs and horses than in cats and cows. Evaluation of a patient with a calcium result above or at the top of the reference interval should be done with consideration of albumin concentration. A calcium result near the high limit of the reference interval in a hypoalbuminemic animal indicates hypercalcemia and a need to consider causes of hypercalcemia or measure free ionized calcium values. Hypercalcemia can affect many different tissues, but the effects are often most pronounced on the heart, kidneys, GI tract, skeletal muscle, and nervous tissue.  The severity of the clinical signs not only depends on the severity of the hypercalcemia, but also on the speed of development. Signs will be more severe with rapid-onset hypercalcemia than they will be with a more slowly developing hypercalcemia of the same magnitude.

The deleterious effects of hypercalcemia are:

  • Nephrogenic diabetes insipidus: Calcium decreases the renal concentrating ability by inhibiting the response to antidiuretic hormone (ADH) and reducing medullary tonicity (via decreasing NaCl absorption in the loop of Henle, decreasing the permeability of the collecting ducts to urea and enhancing renal medullary blood flow). This results in polyuria and a compensatory polydipsia with a low urine specific gravity (< 1.030).
  • Renal tubule damage: This is due to decreased glomerular filtration rate from renal vasoconstriction. This can result in renal ischemia and renal tubular dysfunction. In addition, hypercalcemia can result in mineralization within the kidney. This results in ischemia of the renal tubules and urolithiasis (if severe). Animals with hypercalcemia often present with renal failure. After the cause of the hypercalcemia has been identified and corrected, renal function should be re-assessed. Renal failure can also result in hypercalcemia so it is not always clear which comes first – the renal failure or the hypercalcemia.
  • Urinary tract infections: Calcium predisposes to urinary tract infections, via unknown mechanisms.
  • All tissues: If the calcium phosphate product is > 60 – 80 mg/dL, soft tissue mineralization can result causing organ dysfunction. This is highly dependent on the disease process causing the high calcium and phosphate (e.g. common with renal failure and vitamin D toxicosis) amongst other unknown factors. Excess calcium actually blocks voltage gated ion channels (sodium and calcium channels) leading to decreased excitability of muscle and neural tissue. Hypercalcemia also raises the threshold required for excitation of nerves and muscles.
  • Muscular system: Decreased excitability of skeletal muscle leads to generalized weakness. Decreased contractility of smooth muscle in the gastrointestinal system can cause ileus and constipation.
  • Gastrointestinal system: Ileus and constipation are possible (see above). Hypercalcemia also stimulates gastrin and acid secretion, which can lead to gastric ulceration and vomiting.
  • Nervous system: Decreased excitability can lead to stupor, coma, and paresis.
  • Cardiovascular system: Hypercalcemia usually does not cause clinical signs associated with the heart, but can cause arrhythmias detectable by electrocardiogram, including prolonged PR and shortened QT intervals.  Rarely, the patient can develop serious arrhythmias such as ventricular fibrillation related either to the direct effects of hypercalcemia or to tissue damage from precipitation of excess calcium causing mineralization within cardiac tissue.

Pathophysiologic mechanisms of hypercalcemia include the following (note; multiple mechanisms may be operative in a single disorder):

  • Increased gastrointestinal absorption: Usually under the influence of vitamin D (except horses).
  • Increased osteolysis: Under the influence of PTH (PTH or parathyroid hormone-related protein under pathologic conditions)
  • Decreased renal excretion: Renal disease in various species, but particularly horses with chronic renal disease (not ruminants).
  • Increased protein or anion binding (under these situations, free ionized calcium is often normal, but total calcium is increased).

The most common causes of hypercalcemia in the various species are humoral hypercalcemia of malignancy and Addison’s disease in dogs, chronic renal disease in horses, and iatrogenic administration of calcium in cattle. Hypercalcemia is uncommon in cats, but can be seen mostly with idiopathic hypercalcemia and cancer (not lymphoma, more likely carcinomas) and less frequently with renal disease.

  • Iatrogenic: Thiazide diuretics can cause hypercalcemia in dogs, presumably by direct absorption through the distal tubules and through causing volume depletion with secondary production of angiotensin II (which stimulates sodium and concurrent calcium absorption in the proximal renal tubules). Systemic administration of calcium can result in hypercalcemia, particularly in down dairy cattle given calcium. This is usually transient.
  • Physiologic: Young, growing puppies can  have calcium levels slightly higher than the reference interval for adults of that species.
  • Pathophysiologic: Mechanisms include osteolysis (localized, humoral due to PTH or PTHrP), increased intestinal absorption (dietary excess, excess vitamin D), decreased renal excretion (e.g. horses with chronic renal disease) and increased protein binding (uncommon).
    • Osteolysis: Osteolysis can be due to hormones which are acting systemically (e.g. PTH, PTHrP) or localized osteolysis due to the production of osteoclastogenic cytokines (e.g. interleukin-1, interleukin-11) or RANKL (by tumor cells or stromal cells). Vitamin D also promotes osteolysis by stimulating RANKL production and enhancing the action of PTH, therefore hypervitaminosis D can also induce hypercalcemia through this mechanism.
      • Primary hyperparathyroidism: This has been reported in both dogs and cats and is due to parathyroid chief cell neoplasia (adenoma or carcinoma) or hyperplasia. It is very rare in other species. In dogs, it is inherited in Keeshonds and German Shepherd dogs. There is autonomous PTH secretion, with hypercalcemia (including free ionized), hypophosphatemia and isosthenuria. PTH may secondarily cause increased vitamin D (promotes conversion to calcitriol). Calcium uroliths may be observed in dogs but not usually cats. Clinical signs are vomiting, anorexia, polyuria, polydipsia, folding fractures (from osteolysis), and muscle weakness. Cats are often not polyuric or polydipsic. Note that phosphate may be normal or even high if the animal has a decreased glomerular filtration rate. Primary hyperparathyroidism is diagnosed by identifying a parathyroid adenoma by surgical exploration and biopsy and/or by measuring high or normal PTH concentration in conjunction with high free ionized calcium values. Primary hyperparathyroidism has also been reported infrequently in horses with hypercalcemia (Gorenberg et al 2020). Affected horses had high PTH and parathyroid adenomas. 
      • Humoral hypercalcemia of malignancy: Hypercalcemia is a paraneoplastic syndrome in domestic animals and usually due to systemic secretion of a hypercalcemia-inducing hormone, usually PTH-related proten or PTHrP (but it also can be due to tumor secretion of vitamin D or osteoclastogenic cytokines). Phosphate is frequently normal, but can be decreased if the hypercalcemia is due to increased PTHrP. This is the most common cause of persistent hypercalcemia in dogs and is associated with free ionized hypercalcemia. Lymphoid neoplasms of T cell origin (regulatory or helper T cell) are the most common tumors to cause hypercalcemia in dogs, followed by adenocarcinoma of the apocrine glands of the anal sac. Other tumors that have been associated with hypercalcemia are carcinomas originating in various tissues, thymoma (34% of 116 cases; Robat et al 2013) and histiocytic sarcoma (this could be due to production of vitamin D or PTHrP by the tumor and is an infrequent finding in dogs with histiocytic sarcoma). In horses, paraneoplastic hypercalcemia has been seen with lymphoma, ameloblastoma, gastric squamous cell carcinoma and an adrenal cortical carcinoma. There has been a single report of hypercalcemia with concurrent hypophosphatemia in a mammary carcinoma in a cow that resolved after surgical removal of the tumor (which is also rare in cattle). Tumor cells were weakly positive with a polyclonal anti-PTHrP antibody (Varvil et al 2019).
      • Localized osteolysis: Localized osteolysis can result in hypercalcemia but is uncommon. The most common cause of hypercalcemia (free ionized calcium may be increased) due to localized osteolysis is multiple myeloma. The bone lysis is due to the tumor either directly secreting osteoclastogenic factors (RANKL, interleukin-6, interleukin-11, monocyte inhibitory peptide-1) or inducing stromal cells/osteoblasts to release osteoclastogenic factors, including RANKL. This is why the punched-out osteolytic bone lesions (usually in vertebrae, sternum) are seen with multiple myeloma. These lesions represent sites where the tumor is located. Multiple myeloma can potentially also produce hypercalcemia due to increased binding to the monoclonal immunoglobulin (if it has increased negative charge; free ionized calcium may be normal). This is considered a less common mechanism than localized osteolysis. Primary bone tumors (e.g. canine osteosarcoma) and metastatic bone tumors may also induce localized osteolysis, but this is a rare cause of hypercalcemia. Most dogs with osteosarcoma have normal total calcium values.
    • Increased intestinal absorption: This is primarily mediated through vitamin D, therefore causes of increased vitamin D should be considered. Excess PTH can also induce intestinal absorption of calcium but this is through increased vitamin D production in the kidney and not due to direct effects of PTH on the intestine.
      • Addison’s disease: This usually affects dogs and is rare in other species. Up to 28-45% of dogs with hypoadrenocorticism have high total calcium (free ionized calcium can be normal or increased [Gow et al 2009]). The hypercalcemia is thought to be due to enhanced absorption of calcium in the gastrointestinal tract (corticosteroids inhibit absorption) or decreased renal excretion (increased tubular resorption) of calcium. Replacement therapy with corticosteroids returns the calcium to normal within a few days. Phosphate is usually normal unless there is concurrent pre-renal azotemia (vomiting) or secondary acute kidney injury or renal azotemia.
      • Hypervitaminosis D: Vitamin D promotes the absorption of calcium and phosphate in the intestine, so concurrent high concentrations of calcium (including free ionized) and phosphate (in the absence of decreased glomerular filtration rates, which will independently increase phosphate concentrations) are characteristic of disorders due to excess vitamin D. Causes of hypervitaminosis D are:
        • Ingestion of rodenticides containing cholecalciferol produces marked hypercalcemia (15 to 20 mg/dL) within 24 hours. Animals are concurrently hyperphosphatemic.
        • Excessive dietary supplementation or parenteral doses (a cause of hypercalcemia in cattle [Littledike and Horst 1982]).
        • Inadvertent ingestion of vitamin D containing drugs (e.g. antipsoriasis creams). Reported in dogs.
        • Ingestion of plants whose leaves contain cholecalciferol (Cestrus diurnum or Day-blooming Jessamine, Solanum species, and Trisetum flavescens, Nierembergia) (Schild et al 2021). Horses with hypervitaminosis D secondary to ingestion of these plants usually are hyperphosphatemic.
        • Humoral hypercalcemia of malignancy: Macrophages and lymphocytes can produce vitamin D, which may explain hypercalcemia in some dogs with lymphoma (and normal PTHrP) or histiocytic sarcoma.
        • Granulomatous disease: Vitamin D is thought to be responsible for the hypercalcemia secondary to granulomatous disease (e.g. fungal diseases such as blastomycosis, histoplasmosis, and coccidioidomycosis, parasitic infections, such as schistosomiasis) in dogs and possibly in cats. Macrophages contain 1α-hydroxylase and can produce vitamin D. Macrophages can also produce PTHrP (Fierer et al 2012 [single case report in a human with coccidiomycosis], Fradkin et al 2001 [2 dogs with schistosomiasis]), which may be an additional mechanism (through osteolysis) of hypercalcemia with granulomatous inflammation. There have been isolated case reports of hypercalcemia in horses with idiopathic granulomatous disease. Granulomas are found internally and in the skin, but horses usually present due to the skin lesions, which are located at mucocutaneous junctions and the coronary band (Sellers et al., 2001). It is possible that these granulomas are due to mycobacterial infections.
    • Decreased renal excretion: 
      • Renal disease: Usually in chronic renal disease in small animals, total calcium is normal or decreased. However, 10-20% of cases (especially in dogs with inherited renal disease), total calcium can be increased. However, free ionized calcium cannot be predicted from total calcium values. To emphasize this, studies in dogs and cats with renal disease have shown that free ionized calcium may be high (rarely), normal or low when total calcium is high or normal and free ionized calcium values are usually normal or low when total calcium is low. The mechanism for high total calcium is thought to be mostly  due to increased complexing with anions (e.g. phosphate, which is frequently high in renal disease). However, other hypotheses that have been put forward are autonomous PTH secretion with decreased PTH degradation and reduced calcium excretion by the kidneys (which may normalize or increase free ionized calcium even when anions, such as phosphate, are high). Hypercalcemia should be attributed to renal failure in dogs and cats only after other causes of hypercalcemia have been considered and ruled out. In dogs and cats, hypercalcemia is much more likely to be the cause of renal failure with signs of azotemia, polyuria/polydipsia, and poorly concentrated urine than the result of renal failure. In contrast, hypercalcemia is a frequent finding (with decreased phosphate) in horses with chronic renal disease, especially if on a high calcium diet, and is accompanied by hypophosphatemia (mechanism unknown). It can also be seen in horses with acute renal injury. Hypercalcemia is rare in ruminants, even those with renal disease.
      • Addison’s disease: Corticosteroids promote renal excretion of calcium, therefore the lack of corticosteroids may promote calcium retention. If dogs are concurrently azotemic, increased calcium may also be due to increased anion binding.
      • Primary hyperparathyroidism: PTH promotes calcium resorption in the kidneys (see above).
      • Humoral hypercalcemia of malignancy: Tumors secreting PTHrP will cause a hypercalcemia through combined osteolysis and increased renal absorption (PTHrP binds to the same receptors as PTH).
    • Increased protein binding: Causes of hyperalbuminemia (e.g. hemoconcentration) may increase total (but not free ionized calcium). Increases are generally mild. We do not recommend the use of correction formulae (to “normalize” calcium for changes in albumin concentration). Although immunoglobulins are more positively charged than albumin under normal conditions (and would not bind calcium), it is possible that abnormal immunoglobulins produced in multiple myeloma may contain amino acids with increased negative charge or be less positively charged than normal. This may contribute to high total (but not free ionized) calcium in animals with increased paraproteins (monoclonal immunoglobulins), however the main mechanism for hypercalcemia in such animals is localized osteolysis (even if not visible on radiographs).
    • Other causes:
      • Idiopathic hypercalcemia: This has been reported in cats, some of which have an associated calcium oxalate uroliths. The total calcium ranged between 10.4 and 14.1 mg/dL. A definitive cause for the hypercalcemia was not identified in cats (PTH was appropriately low and concentrations of calcidiol, PTHrP and phosphate were within reference intervals). Several cats in one study (Midkiff et al., 2000) were on acidifying diets (intestinal acidosis will promote calcium absorption). Some subsequently developed renal failure (which could be a consequence of persistent hypercalcemia). The hypercalcemia normalized after prednisolone treatment (Midkiff et al., 2000) or biphosphonate treatment (Hostutler et al., 2005), although numbers of treated animals were very low in both studies. There are anecdotal reports of a syndrome of hypercalcemia in foals, but the mechanism is unclear. Affected foals were consistently asphyxic (Toribio 2011).
      • Endometritis and retained fetus: A single case report documented hypercalcemia in a young dog with endometritis and a retained fetus. The hypercalcemia resolved upon ovariohysterectomy but was unresponsive to treatment with calcitonin, intravenous fluids, and diuretics (Hirt et al., 2000).
Conditions iPTH iCa2+ 25-Hydroxyvitamin D
Primary hyperPTH Normal or High High
Renal hyperPTH High Low
of malignancy
Low High Normal/high (PTHrP may be high)
Vitamin D intoxication Low High High


Measurement of intact parathyroid hormone (iPTH), free ionized calcium (iCa2+), and 25-hydroxyvitamin D (calcidiol) can help discriminate between the various causes of hypercalcemia in dogs. Guidelines for interpretation of these tests in combination are shown in the table at right. Note that PTHrP can also be measured and would only be anticipated to be high with humoral hypercalcemia of malignancy secondary to PTHrP production.

Decreased calcium concentration (hypocalcemia)

Hypocalcemia results in increased muscle and neural excitability, since the threshold for nerve and muscle depolarization is decreased (more sensitive). Clinical signs of hypocalcemia in dogs include muscle tremors, convulsions, ataxia, and weakness. In horses, hypocalcemia is associated with synchronous diaphragmatic flutter and signs of tetany including stilted gait, muscle tremors, flared nostrils, inability to chew, recumbency, convulsions, and cardiac arrhythmias. In cows, hypocalcemia is usually manifested as weakness and recumbency. Signs of hypocalcemia develop when free ionized calcium is too low for normal muscle and nerve function. Because of factors that influence free ionized and protein-bound calcium fractions, the total calcium result does not necessarily correlate with free ionized calcium and is not by itself always a reliable indicator of clinical hypocalcemia. The following are general pathophysiologic mechanisms of hypocalcemia:

  • Decreased protein binding
  • Abnormal PTH: Decreased, inhibited or altered setpoint (insufficient osteolysis, renal excretion)
  • Decreased absorption in the gastrointestinal system (e.g. oxalate toxicity). 
  • Excessive loss: This can occur via the kidneys (a common cause of hypocalcemia), gastrointestinal system (e.g. gastrointestinal disease in horses) or milk (increased demand for calcium cannot be compensated for by PTH – cannot respond quickly enough leading to clinical hypocalcemia – milk fever in cattle and eclampsia in other species)

The most common cause of hypocalcemia in all species is low albumin. Other common causes are renal disease (dogs, cats), pancreatitis (dogs), gastrointestinal disease (colic in horses) and milk fever (cattle).

Note, that although these are separated to some extent by mechanism, more than one mechanism is usually operative in many of these disorders, e.g. calcitonin inhibits osteolysis and promotes calcium excretion in the kidneys; lack of PTH results in inhibition of osteolysis and decreased renal resorption). Also, low magnesium exacerbates or can cause hypocalcemia due to decreased PTH secretion and resistance of end-organs to PTH.

  • Artifact:
    • Anticoagulant: EDTA or citrate (chelate calcium resulting in very low calcium values (partial chelation may only slightly drop calcium and will be more difficult to identify). With EDTA, very high K values are expected concurrently and with sodium citrate, high sodium values will be seen.
  • Iatrogenic: Sodium phosphate enemas in cats have resulted in hypocalcemia (presumably through binding calcium in the intestine). Administration of calcitonin (to treat hypercalcemia) could result in hypocalcemia.
  • Pathophysiologic: General mechanisms include decreased protein binding (the most common cause of low total calcium), decreased osteolysis (absent, abnormal or inhibited PTH), increased renal, mammary, or gastrointestinal losses (common) and decreased absorption of calcium (mineral imbalance).
    • Decreased protein binding: This is a consequence of hypoalbuminemia, of which there are different causes or mechanisms. Because such a large percentage of total calcium is protein-bound (particularly to albumin), hypoalbuminemia can decrease total calcium. Free ionized calcium concentration will be unaffected by decreased albumin alone (values will be normal). Therefore, the first step in interpreting low calcium is to look at the albumin result. A mild hypocalcemia in the presence of hypoalbuminemia usually does not indicate a disorder of calcium metabolism. This interpretation is probably applicable for all species. Formulas have been created to adjust the total calcium for the albumin concentration. For dogs, this formula has been used: Adjusted calcium = measured calcium – serum albumin concentration + 3.5. The assumption was that if the total calcium corrected to within reference intervals, then the calcium was not “truly” low. However, such assumptions are not necessarily correct and free ionized calcium concentrations can still be abnormal, therefore we do not recommend using these formula to correct for hypoalbuminemia.
    • Abnormal PTH:
      • Primary hypoparathyroidism: This has been reported mostly in dogs, some cats and rarely in horses. Clinical signs include seizures, ataxia, and lens cataracts. It is characterized by hypocalcemia (total and free ionized), normal or increased phosphate and normal magnesium. In dogs, it is usually due to lymphocytic infiltration, atrophy and fibrosis of the parathyroid gland (and may be immune-mediated). Low concentration of intact parathyroid hormone confirms primary hypoparathyroidism. Hypocalcemia is due to decreased absorption from bone, increased loss via kidneys (PTH is required for calcium absorption in the loop of Henle and distal tubules) and inadequate conversion of vitamin D to its active form (which would decrease intestinal absorption). Hyperphosphatemia may be seen because PTH causes phosphate excretion in the kidneys.
      • Pseudohypoparathyroidism: This is due to cellular unresponsiveness to PTH due to abnormal receptors. Unlike primary hypoparathyroidism, PTH will be increased in this rare disorder.
      • Resistance to PTH: This can occur in various disorders, including renal disease, sepsis, primary metabolic alkalosis, hypomagnesemia (e.g. grass tetany in cattle) and pancreatitis. Low magnesium may also result in less secretion of PTH by parathyroid chief cells.
    • Decreased absorption of calcium: Dietary lack of calcium or gastrointestinal disease can result in hypocalcemia.
      • Nutritional secondary hyperparathyroidism: This  can occur in all species with an imbalance of calcium and phosphate in the diet. The resulting low free ionized calcium stimulates PTH, which restores calcium but at the expense of bone calcium (osteolysis results). In horses, this occurs with diets low in calcium or which have an imbalanced phosphate to calcium ratio of greater than 3:1 (e.g. grain diets, hence so-called “bran” or “miller” disease) or grass diets high in oxalates (which bind calcium in the diet).  The typical signs of bran disease are lameness, bone pain, osteopenia (producing hyperostotic fibrous osteodystrophy – “big head”) and pathologic fractures.
      • Hypovitaminosis D: This can be secondary to dietary lack or lack of exposure to sun (rickets type I), renal disease (loss of calcidiol, FGF23- and phosphate-mediated inhibition or decreased conversion, tubular disease), gastrointestinal disease (lack of absorption, e.g. fat malabsorption), endorgan or receptor unresponsiveness to vitamin D (vitamin D dependent rickets, type II). Defects in receptors (e.g. cubilin, megalin)  that mediate uptake of filtered calcidiol can also result in deficiency – this has been identified in dogs). Low vitamin D can also be a consequence of hypoparathyroidism and hypomagnesemia (endorgan resistance to PTH).
      • Renal secondary hyperparathyroidism: This can be due to chronic renal disease in dogs, cats, and cattle (not horses, since they do not require vitamin D for intestinal absorption of calcium) and is thought to be due to low vitamin D. Tubule disease (1α-hydroxylase deficiency), decreased absorption of filtered calcidiol or vitamin D binding proteins, and increased GFR and hyperphosphatemia (which decreases vitamin D synthesis by inhibiting 1α-hydroxylase) are the postulated reasons behind the lack of vitamin D in renal disease. Recent data suggests that FGF23 may be the main inhibitor of 1α-hydroxylase and that FGF23 may be stimulated by decreased GFR before increased phosphate (Hardcastle and Dittmer 2015). Decreased free ionized calcium (from hyperphosphatemia or decreased absorption of calcium) stimulates PTH which then causes osteolysis in an attempt to maintain free ionized calcium values. The osteolysis can be quite severe in some animals, leading to the term “rubber jaw” (mandibular osteolysis). Total calcium values may actually be normal or increased due to anion complexing (e.g. phosphates, citrates). Free ionized calcium values cannot be predicted and may be low or normal.
      • Toxicosis: Ingestion of oxalate containing plants by grazing animals (e.g. rhubarb, purslane, sorrel, dock, foxtail grass, kikuyu grass) can cause hypocalcemia (oxalates bind calcium in the gastrointestinal tract, causing hypocalcemia).
      • Gastrointestinal disease: Hypocalcemia can be seen frequently in horses with gastrointestinal disorders (colic, enterocolitis, endotoxemia) and in some horses, may be severe enough to require treatment with calcium-containing solutions. Hypocalcemia can occur in some dogs with protein-losing enteropathy.
        • Protein-losing enteropathy: Severe hypocalcemia and hypomagnesemia has been reported in some dogs with protein-losing enteropathy. This syndrome has been particularly described in Yorkshire Terriers (Kimmel et al., 2000, Smmerson et al 2014). Clinical signs of hypocalcemia (despite very low values of total and free ionized calcium) are usually inapparent. The exact mechanism is unknown and the low calcium may be secondary to decreased vitamin D (lack of absorption [Mellanby et al 2005]), decreased absorption of calcium due to intestinal disease, and low magnesium (PTH resistance and decreased PTH secretion).
      • Hyperadrenocorticism: Some dogs with Cushing’s disease can have low values of calcium, presumably secondary to decreased intestinal absorption (corticosteroids inhibit this process) or increased renal excretion (promoted by corticosteroids). Horses with equine metabolic syndrome or pituitary pars intermedia dysfunction (PPID) have normal calcium.
    • Losses of calcium: This can occur via the kidney, gastrointestinal system (lack of absorption – see above), or milk. The fetus is another source of calcium demand in pregnant animals. Calcium can also precipitate in organs (e.g. fat with pancreatitis, renal tubules with ethylene glycol toxicity)
      • Renal losses: Increased renal loss of calcium can occur due to various mechanisms, including lack of PTH, resistance to PTH, or inability of tubules to resorb calcium. Calcitonin also causes increased loss of calcium in the renal tubules and is thought to contribute to hypocalcemia due to pancreatitis and C cell tumors. 
        • Pancreatitis: Mild hypocalcemia, usually without clinical signs referable to hypocalcemia, is fairly common in acute pancreatitis. The mechanism is unknown but postulated to be due to glucagon release from the inflamed pancreas. Glucagon stimulates calcitonin release, which decreases calcium absorption in the kidney and inhibits osteolysis. Another mechanism is thought to be precipitation of calcium by formation of salts with fatty acids released from peripancreatic fat by the action of lipase.
        • Hypercalcitonism: Calcitonin-secreting tumors of the medullary thyroid cells (C cell tumors) have been identified in dogs, horses and cattle (mostly older bulls). Calcium is lost through the renal tubules.
        • Ethylene glycol toxicity: Hypocalcemia is a common finding in the chemistry panel of dogs and cats poisoned with antifreeze. Precipitation of calcium by oxalate, one of the metabolites of ethylene glycol, in the renal tubules is thought to be the mechanism, although renal tubular damage preventing absorption of calcium may contribute.
        • Alkalemia due to a primary metabolic alkalosis: Administration of bicarbonate can result in hypocalcemia, which is thought to be secondary to bicarbonaturia (excretion of bicarbonate in urine) with consequent losses of calcium, which binds to the filtered bicarbonate. This is a rare cause of hypocalcemia. Alkalosis may also decrease endorgan responsiveness to PTH and suppresses PTH release.
      • Pregnancy, parturient or lactational hypocalcemia/eclampsia: Parturient hypocalcemia (milk fever) is most commonly seen in highly producing dairy cows and results in paresis. They have low serum calcium and phosphate (the latter is also lost in milk). Magnesium is often normal, but vitamin D may be increased (secondary to increased PTH from the free ionized hypocalcemia). It is caused by demand for calcium for milk production, in excess of that which can be absorbed or released from bone. Even though PTH is high, PTH resistance or unresponsiveness is thought to contribute to the syndrome. Eclampsia can also be seen in dogs, cats, ewes, sows, guinea pigs, mares and goats and produces tetany in these species. Inability of calcium homeostatic mechanisms to compensate for loss of calcium in milk is thought to be the cause of lactation-associated hypocalcemia in dogs (called puerperal tetany). The condition is seen most commonly in small breed dogs two to three weeks after whelping. Hypocalcemia associated with lactation in horses is called lactation tetany and is less common than in cows. In cats, hypocalcemia more frequently occurs before parturition and usually results in anorexia, depression and lethargy versus tetany).
      • Excess sweating: Since sweat contains calcium (and KCl), excess sweating in horses can result in hypocalcemia. The chief sign in this setting is synchronous diaphragmatic flutter, which is contraction of one or both flanks coincident with the heartbeat.
    • Unknown mechanisms
      • Sepsis: Sepsis has been associated with hypocalcemia in critically ill dogs and cats. Septic foals also have lower free ionized calcium (and higher PTH) than non-septic foals (Hurcombe et al., 2009). Experimental endotoxemia in the horse is associated with decreases in free ionized calcium and free ionized magnesium and total phosphate concentration. The mechanism is unknown but some animals had unexpectedly low PTH despite the hypocalcemia, suggesting endotoxin- or inflammatory cytokine-induced inhibition of PTH contributed to the hypocalcemia (Toribio et al., 2005). Hypocalcemia can be a risk factor for mortality. If severe, the hypocalcemia can result in neural or muscle excitability or cardiac dysfunction in dogs (Holowaychuk, 2013). The low calcium in sepsis may be partly due to increased sensitivity of calcium sensing receptors in the pituitary to free ionized calcium (they sense more calcium than is truly there, resulting in suppression of PTH or lack of PTH release in response to free ionized hypocalcemia).
      • Idiopathic hypocalcemia in foals: There is one report of idiopathic hypocalcemia in foals ranging from 4 days to 5 weeks old. They display tachycardia, sweating, muscle rigidity, recumbency, seizures and opisthotonus. The affected foals did not appear septic. The foals did not respond to calcium supplementation and all five affected foals died or were euthanized (Beyer et al., 1997). The cause was not identified.
      • Equine myopathy: Various types of muscle injury in horses, e.g. transport tetany, exertional rhabdomyolysis, vitamin E or selenium deficiency (Perkins et al., 1998), malignant hyperthermia, consequence of endurance racing, have been associated with hypocalcemia. The mechanism is unknown but could be due to decreased intake, increased renal loss, movement of calcium into damaged muscle, and excess loss through sweating (and generation of a metabolic alkalosis which increases resistance to PTH and may promote renal loss of calcium with bicarbonate).
      • Blister beetle toxicosis (cantharidin) in horses: This beetle can be associated with alfalfa hay and causes severe hypocalcemia and hypomagnesemia with associated clinical signs of  diaphragmatic flutter and sweating. The calcium is thought to be lost through the gastrointestinal tract, although acute renal injury can contribute (the toxin also causes renal injury).
      • Acute renal injury: This can also result in a mild hypocalcemia in horses, cattle and sheep. Urinary tract obstruction can sometimes cause hypocalcemia.
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