Test Options for Evaluating Calcium Metabolism
Test selection depends on the animal’s history, clinical signs, and suspected differential diagnoses. Ionized, or free, calcium is the physiologically active and hormonally regulated fraction of the total calcium concentration and is required to assess for the presence of calcium abnormalities (standalone ionized calcium is available (20026)). The basic parathyroid profile (20033) provides measurement of both ionized calcium and parathyroid hormone (PTH), the primary hormone involved in calcium regulation. In cases where malignancy is a possibility, the malignancy profile (20030) includes measurement of ionized calcium, PTH, and parathyroid hormone-related protein (PTHrP). In cases where there is concern for vitamin D excess or deficiency, measurement of 25-hydroxyvitamin D should be requested. The vitamin D profile (20035) includes measurement of ionized calcium, PTH, and 25-hydroxyvitamin D concentrations.
Total Calcium Versus Ionized Calcium
Serum total calcium concentrations obtained on biochemistry profiles do not always correlate to the ionized calcium concentration, especially in disease states. Serum total calcium is composed of three fractions: ionized, complexed, and protein bound. With fair consistency between dogs and cats, about 50% of total calcium is ionized (or free) calcium, 40-45% is protein-bound (80% to albumin and 20% to globulins), and the remaining 5-10% complexed fraction is bound to non-protein anions (citrate, sulfate, phosphate, others). Alterations in any one of these fractions can impact the total calcium concentration. Studies have shown that hypercalcemia is overestimated, and hypocalcemia is underestimated, when total calcium or “adjusted” total calcium is used to predict ionized calcium. “Correction” formulas for estimating or predicting ionized calcium status using adjusted total calcium concentrations are no longer recommended. Particularly in patients with renal disease, total calcium or adjusted total calcium are unreliable predictors of calcium status due to diagnostic discordance where the total calcium concentration is increased due to increased binding of calcium to non-protein anions. A discrepancy (total hypercalcemia with an ionized calcium concentration within reference interval or even decreased) is not uncommon in animals with renal disease.
Ionized Calcium and Parathyroid Hormone Measurement: Submit Serum
Parathyroid hormone (PTH) should be measured in serum, and the ionized calcium concentration should also be measured in serum. In some exotic species (such as birds or some reptiles), ionized calcium concentration can be measured in heparinized plasma, but care must be taken to standardize the collection procedure since the type and amount of heparin can impact the ionized calcium result. An ionized calcium concentration is often undetectable in EDTA and citrated plasma as both EDTA and sodium citrate prevent blood coagulation by chelating calcium.
Parathyroid Hormone-Related Protein (PTHrP) Measurement: Submit EDTA Plasma
EDTA plasma is required for parathyroid hormone-related protein (PTHrP) measurement. Studies at the Michigan State University Veterinary Diagnostic Laboratory evaluated PTHrP concentrations in paired EDTA plasma and serum samples. PTHrP was negative (undetectable) in approximately 50% of the serum samples when the EDTA plasma sample showed an increased concentration of PTHrP. False negative results obtained with serum is most likely due to degradation of PTHrP.
Interpretation of Ionized Calcium and Parathyroid Hormone (PTH) Results
First, determine the animal’s calcium status based on the ionized calcium measurement. Is there a clinically significant hypercalcemia? A clinically significant hypercalcemia is typically defined as an ionized calcium concentration that is ≥ 0.1 mmol/L above the upper limit of the reference interval. Is ionized calcium within the reference interval? Is there a clinically significant decrease in ionized calcium? Interpretation of the parathyroid hormone (PTH) concentration is made in conjunction with the ionized calcium concentration, and these must be measured in the same sample. The patient’s history, clinical signs, and information obtained from additional diagnostic tests are also important for interpretation.
Normal Calcium Metabolism
Concentrations of both ionized calcium and parathyroid hormone (PTH) that are within reference interval are suggestive of normal calcium metabolism. Additionally, when the ionized calcium concentration is within reference intervals but the PTH concentration is low, normal calcium homeostasis is still likely; a low concentration of PTH is of questionable significance in the absence of hypercalcemia or hypocalcemia.
Increased Ionized Calcium Concentration: Pathologic Causes
When a clinically significant ionized hypercalcemia has been identified, the parathyroid hormone (PTH) concentration is necessary to determine if the hypercalcemia is likely due to primary parathyroid disease (primary hyperparathyroidism), or if the hypercalcemia is likely due to disease unrelated to parathyroid dysfunction (parathyroid independent hypercalcemia).
The combination of a clinically significant ionized hypercalcemia and a parathyroid hormone (PTH) concentration above the upper limit of the reference interval is consistent with a diagnosis of primary hyperparathyroidism. The most common cause of primary hyperparathyroidism is a parathyroid adenoma, and typically only one of the four parathyroid glands is affected. In dogs, it is also recognized that a mid-normal or higher concentration of PTH with a clinically significant ionized hypercalcemia represents inappropriate lack of suppression of PTH and is consistent with a diagnosis of primary hyperparathyroidism. This mid-normal convention for determining primary hyperparathyroidism is not well defined in other species including cats and horses.
Parathyroid Independent Hypercalcemia
The combination of a clinically significant ionized hypercalcemia and a parathyroid hormone (PTH) concentration below the lower limit of the reference interval, or low-normal, is consistent with parathyroid independent hypercalcemia. In dogs, the most common cause of parathyroid independent hypercalcemia is malignancy. In cats, the most common causes of parathyroid independent hypercalcemia are idiopathic hypercalcemia and hypercalcemia of malignancy. The next most likely additional differentials for parathyroid independent hypercalcemia include vitamin D excess, granulomatous disease, and hypoadrenocorticism (in dogs).
Hypercalcemia of Malignancy
An increased (positive) parathyroid hormone-related protein (PTHrP) result with ionized hypercalcemia and concurrent suppression of parathyroid hormone (PTH) adds support for humoral hypercalcemia of malignancy. In dogs, neoplasms most often associated with hypercalcemia are lymphoma and apocrine gland adenocarcinoma of the anal sac. Other tumors reported with increased PTHrP results include thymoma, carcinoma (thyroid, bronchogenic, pancreatic, skin) and multiple myeloma. Increased PTHrP concentrations have been reported in cats with carcinoma (pulmonary, renal, and thyroid), osteosarcoma, and occasionally lymphosarcoma. It is important to remember that a normal (or negative) concentration of PTHrP does not exclude the possibility of malignancy. PTHrP is only one of a number of factors that tumors may secrete that can result in an ionized hypercalcemia (e.g. interleukins, tumor necrosis factor, calcitriol). In addition to humoral factors, local mechanisms underlying hypercalcemia are also possible. Malignancy may still be present even if the PTHrP concentration is not increased.
Inconclusive Parathyroid Hormone (PTH) Concentrations
- CANINE: The combination of ionized hypercalcemia and a parathyroid hormone (PTH) concentration that is between low- and mid-normal is equivocal with regard to distinguishing between primary hyperparathyroidism and parathyroid independent causes of hypercalcemia. In these dogs, clinical signs and additional diagnostics may provide additional clues to determine the most likely cause of hypercalcemia. In some cases, the parathyroid profile may need to be repeated before a definitive diagnosis becomes apparent.
- FELINE: The combination of ionized hypercalcemia and a PTH concentration between the lower third and the upper limit of the reference interval is equivocal with regard to distinguishing between primary hyperparathyroidism and parathyroid independent causes of hypercalcemia. However, primary hyperparathyroidism is an uncommon diagnosis in cats. Some cats with idiopathic hypercalcemia may have a PTH concentration in the equivocal range.
The combination of a low or normal ionized calcium concentration and a parathyroid hormone (PTH) concentration above the upper limit of the reference interval is consistent with a diagnosis of secondary hyperparathyroidism. This condition is typically associated with chronic renal disease, a nutritional deficiency of vitamin D, or both. Measurement of 25-hydroxyvitamin D, as the best indicator of dietary intake and absorption of vitamin D, may be useful to help differentiate. Chronic gastrointestinal disease can be a cause of vitamin D deficiency and/or calcium malabsorption. In dogs, secondary hyperparathyroidism can also be seen with hyperadrenocorticism.
The combination of a clinically significant ionized hypocalcemia with a low or low-normal (within the lower half of the reference interval) parathyroid hormone (PTH) concentration is consistent with a diagnosis of primary hypoparathyroidism. These animals usually have a history of clinical signs associated with hypocalcemia, low total calcium, and high-normal or increased serum phosphorus concentrations.