Investigation of calcium disorders and sample preparation

Parathyroid hormone and ionised calcium measurements are the two most important parameters in calcium investigations, and their combined measurement should be considered in both hypo- and hypercalcaemic situations where the cause is not immediately obvious. Ionised calcium should always be used in the first instance to confirm actual calcium levels.

Total calcium measurement may not provide a true reflection of calcium status in all cases and ionised calcium, which is the biologically active part, only makes up 50 to 55 percent of the total calcium. Cases with renal dysfunction may therefore present with high total calcium when in fact ionised calcium is actually normal or low.

Sample preparation is critical for the accurate analysis of parathyroid hormone and ionised calcium

If ionised calcium is confirmed as high or low, then parathyroid hormone levels should be investigated to determine the cause of the abnormality. When measuring parathyroid hormone levels, ionised calcium should be measured at the same time since parathyroid hormone reflects the minute-to-minute changes in calcium levels.

Sample preparation is critical for the accurate analysis of parathyroid hormone and ionised calcium. Separated EDTA plasma should be shipped frozen for parathyroid hormone, and separated serum should be used for ionised calcium.

Calcium regulation disorders

The combination of ionised calcium and parathyroid hormone measurement usually permits differentiation between the four main categories of calcium regulation disorders (Table 1).

1. Primary hyperparathyroidism

The first category is primary hyperparathyroidism, or parathyroid-dependent hypercalcaemia. Here we include functional parathyroid neoplasia (occasionally hyperplasia).

In these cases, the ionised calcium is high and the parathyroid hormone is either high or in the top two-thirds of the reference range.

2. Parathyroid-independent hypercalcaemia

The second category is parathyroid-independent hypercalcaemia. It includes hypercalcaemia of malignancy, often associated with lymphoma and apocrine gland adenocarcinoma; vitamin D toxicosis, for example due to ingestion of calciferol-containing rodenticides or calcitriol; Addison’s disease; feline idiopathic hypercalcaemia; and granulomatous disease processes.

In these cases, the ionised calcium is high and the parathyroid hormone is low or in the lower third of the reference range.

3. Primary hypoparathyroidism

The third category is primary hypoparathyroidism or parathyroid-dependent hypocalcaemia. This is usually idiopathic and relates to parathyroid gland destruction which is thought to involve immune mechanisms or inflammation. Other causes include surgery or spontaneous infarction.

In these cases, the ionised calcium is low, and the parathyroid hormone is either low or in the lower third of the reference interval.

4. Secondary hyperparathyroidism

Category four is secondary hyperparathyroidism, or parathyroid-independent hypocalcaemia. Here we include renal failure, as well as calcium losses (eg eclampsia, pancreatitis, equine diarrhoea and colic), dietary deficiency, rickets, hyperadrenocorticism and hyperthyroidism.

In these cases, the ionised calcium is normal or low with the parathyroid hormone usually high.

The role of vitamin D and its metabolites

Vitamin D3 is intimately involved in calcium regulation and two forms of vitamin D are measurable in veterinary serum samples: 25-hydroxyvitamin D3 (25(OH)D3 or “calcidiol”) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 or “calcitriol”). Vitamin D is metabolised to calcidiol in the liver, which is in turn metabolised to calcitriol in the kidneys.

Calcidiol concentration parallels that of available vitamin D which can be dietary in origin (cholecalciferol (D3) and ergocalciferol (D2)) or produced in the skin of certain animals under the influence of UV light.

Calcitriol increases serum calcium by increasing the intestinal absorption of calcium, mobilising calcium from bone and facilitating calcium reabsorption from the kidney.

Calcitriol is produced by renal tubular cells as a result of 1α-hydroxylase enzymatic action on calcidiol substrates. The rate of this process is controlled by parathyroid hormone concentrations and increasing parathyroid hormone causes increased 1α-hydroxylase activity. Phosphorus also has an effect on this process as increases in phosphorus decrease 1α-hydroxylase activity.

Please note that dogs and cats have very limited to no synthesis of vitamin D3 in the skin.

Vitamin D3 in investigating calcium disorders

Because the enzymatic hydroxylation of vitamin D3 in the liver depends almost entirely on the availability of the substrate, this test is an excellent marker of overall vitamin D3 status. It can be used to diagnose conditions of both vitamin excess and deficiency; therefore, it is valuable in the investigation of both hyper- and hypocalcaemic disorders.

Because the enzymatic hydroxylation of vitamin D3 in the liver depends almost entirely on the availability of the substrate, this test is an excellent marker of overall vitamin D3 status

Cases where abnormal calcidiol results may be encountered include dietary deficiencies, malabsorption syndromes, such as protein losing enteropathy (PLE) or exocrine pancreatic insufficiency (EPI), and calciferol rodenticide toxicities.

Calcitriol is the most biologically potent form of vitamin D and its main activities are directed at increasing serum calcium concentrations, including increased intestinal uptake of calcium. The failure of renal tubular cells to generate calcitriol in renal disease is one of the contributing causes of renal secondary hyperparathyroidism and “rubber jaw”. The measurement of calcitriol may be of some value in understanding renal tubular function and the effects of parathyroid hormone on the vitamin D system.

In dogs, cats and many veterinary species, very little passive intestinal absorption of calcium occurs, and intestinal calcium absorption is almost exclusively mediated by vitamin D. The situation is different in horses and rabbits (and the hippopotamus) as significantly more passive absorption of calcium occurs, and its status is controlled further by renal excretion. In these species, the effects of renal disease on calcium status will be different to those in most common mammalian species.

Parathyroid hormone-related peptide

Parathyroid hormone-related peptide (PTHrP) may be used for the differential diagnosis of hypercalcaemia of unknown origin where other diagnostic tests have not identified the aetiology of the hypercalcaemia and where parathyroid hormone and ionised calcium tests suggest parathyroid-independent hypercalcaemia.

Parathyroid hormone-related peptide (PTHrP) may be used for the differential diagnosis of hypercalcaemia of unknown origin

PTHrP is a hormone that can be produced by several different types of tumours in dogs, cats and horses and is considered to be the underlying cause of hypercalcaemia of malignancy in many, but not all, cases. Over 50 percent of dogs with apocrine gland adenocarcinoma of the anal sac are hypercalcaemic at the time of diagnosis. The majority of hypercalcaemic lymphomas and smaller percentages of myelomas and carcinomas are PTHrP-positive.

Circulating levels of parathyroid hormone-related peptide in normal dogs are almost undetectable (less than 0.5pmol/l) so levels greater than 1.5pmol/l are considered significant in dogs. Cats appear to show similar values.

It is important to note that a negative or low PTHrP does not exclude a neoplastic cause particularly when the parathyroid hormone is low normal or low.

Feline idiopathic hypercalcaemia

Feline idiopathic hypercalcaemia is a common cause of hypercalcemia in cats and it is the most common cause reported in the USA. Its pathogenesis is unknown, and it is not reported in dogs. Although there is a suggested association with urinary acidifying diets in some studies, this has not been proven.

Feline idiopathic hypercalcaemia is a common cause of hypercalcemia in cats and it is the most common cause reported in the USA

Some believe the condition to be relatively benign, while others believe that the condition promotes the onset of renal dysfunction. Low-dose glucocorticoids have been suggested as a therapy if there is concern about progressive disease. Other drugs including bisphosphonates are also currently used.

For feline idiopathic hypercalcaemia, parathyroid hormone is low, and it is not associated with excess vitamin D or calcitriol.

Sample preparation

The process of sample preparation is important to avoid incorrect or spurious results.

Parathyroid hormone

For parathyroid hormone, the sample must be frozen EDTA plasma or frozen aprotinin EDTA plasma, and for PTHrP, the sample must be frozen aprotinin EDTA plasma. The addition of aprotinin is essential. The aprotinin EDTA sample tube supplied with the freezer pack must be used to collect the sample.

The collection technique

1. Firstly, take the blood sample into the aprotinin EDTA tube supplied with the freezer pack and mix well
2. Next, decant into a cooled plastic EDTA tube, or tubes, kept on ice. A high concentration of EDTA is desirable if you have documented marked hypercalcaemia – it is possible to half fill several EDTA tubes
3. Mix well but gently and centrifuge as quickly as possible, ideally in a refrigerated centrifuge
4. Transfer the plasma into a cooled plastic, not glass, plain tube kept on ice
5. Immediately freeze the plasma sample at less than −10°C and keep frozen until dispatch in the transport pack

Ionised calcium

The initial sample for ionised calcium is whole clotted blood in a plain tube which must be filled to the brim to exclude air; however, the submitted sample must be separated serum and not whole clotted blood as this can damage the sample in transit.

EDTA plasma is not acceptable as the EDTA would chelate all the available calcium making it unavailable for analysis.

The collection technique

1. Obtain a blood sample in a plain, non-gel tube and fill to the brim with blood to exclude air
2. Leave the tube to sit for half an hour to separate out or, alternatively and ideally, centrifuge the whole blood, aspirate the serum and decant into a further plain, non-gel tube as this will prevent in vitro haemolysis which can lead to an erroneously low estimation of the ionised calcium concentration
3. Close the lid firmly and mail the sample to a laboratory as soon as possible. The laboratory will apply a correction formula to take account of the change in pH that will have occurred due to exposure to air. This will result in an estimated ionised calcium at a standardised pH of 7.4

Rapid PTH analysis service from NationWide Specialist Laboratories (coming soon)

NationWide Specialist Laboratories will soon be offering a same day (less than 24 hours after sample’s arrival) service for the analysis of canine, feline and equine parathyroid hormone (PTH).

At present, the samples for PTH and PTHrP are assayed once a week on a Wednesday with results available on the Thursday. Going forward, clients will be asked to submit two separate frozen EDTA plasma samples if they want the rapid PTH analysis service as well as PTHrP. PTHrP will continue to be analysed once a week on a Wednesday.