Nutritional secondary hyperparathyroidism in a kitten, supported by immunoenzymatic measurement of feline intact parathyroid hormone

Abstract

A 6-mo-old, intact male, domestic shorthair cat was referred with a history of poor growth, reluctance to move, and deformation of the nasal profile. The kitten had been fed a diet composed almost exclusively of a complementary pet food and tuna, which was similar to an all-meat diet. We detected osteopenia and hypocalcemia associated with severe parathyroid hormone (PTH) and calcitriol increases; we measured PTH concentrations with an immunoenzymatic method that has been validated in cats. Dietary correction, consisting of a complete and balanced wet pet food formulated for growth, resulted in normalization of calcium and PTH concentrations within 2 mo.

A 6-mo-old, intact male, domestic shorthair cat was presented to the Internal Medicine service of the Veterinary Teaching Hospital (University of Milan, Lodi, Italy) because of a 2-mo history of poor growth, reluctance to move, and manifestation of discomfort when picked up by the owner, despite the cat having a good appetite since adoption. Moreover, ocular and nasal discharge, respiratory stertor, and deformation of the nasal profile were reported. During the history collection, the owner reported a dietary plan based on feline commercial pet food, but after further questioning, it was discovered that only a complementary wet pet food or natural tuna were fed.

Complementary pet food is defined as “pet food that has a high content of certain substances but which, by reason of its composition, is sufficient for a daily ration only if used in combination with other pet foods.” ⁶ According to the nutrient profile (Table 1), the wet pet food administered was similar to an all-meat diet. The declaration of nutritional contents of calcium (Ca), phosphorus, and vitamins D2 and D3 is not mandatory for complementary diets in the European Union, as reported by European Regulation 767/2009. ⁵ The biochemical investigation done by the referring veterinarian reported hypocalcemia (1.65 mmol/L; RI: 1.98–2.83 mmol/L). A test for antibody to feline immunodeficiency virus and for feline leukemia virus antigen (Idexx) was negative. An abdominal ultrasound evaluation revealed no abnormalities.

Table 1.

Ingredients and analytical constituents of the complementary wet pet food fed to a kitten that developed nutritional secondary hyperparathyroidism.

Nutrient	As fed, %	DM, %	g/100 kcal
Crude protein	16	76.2	19
Fat	0.5	2.3	0.6
Crude fiber	0.1	0.4	0.1
Ash	1	4.7	1.2
Moisture	79	21
Metabolizable energy,* kcal/100 g	84	400

Ingredients of the complementary wet pet food: chicken 75%; rice 2%; water sufficient for processing.

DM = dry matter.

^* Calculated according to NRC 2006. ¹²

At clinical examination, the kitten was quite reluctant to be handled; body condition was poor; muscle condition was normal (body weight [BW] 1.6 kg; body condition score 3 of 9); kyphosis was severe; limbs were deformed, particularly at the carpal joints; the nasal profile was deformed; and deciduous dentition persisted. Cephalic vein blood was drawn for a CBC, blood gas analysis, and a biochemical profile, including the determination of calcitriol (1,25-dihydroxycholecalciferol) and parathyroid hormone (PTH). Also, the cranium, vertebral column, and limbs were radiographed.

PTH was measured with a 2-site immunoenzymatic assay (ST AIA-PACK intact PTH; Tosoh Bioscience) in an automated immunoassay analyzer (AIA-360; Tosoh). The test has been validated in humans and in dogs and cats.^21,22 The test is performed entirely in a test cup; “intact PTH (iPTH) present in the sample is bound to a first polyclonal antibody immobilized on a magnetic solid phase and a second enzyme-labeled polyclonal antibody. The magnetic beads are washed to remove unbound enzyme-labeled polyclonal antibody and are then incubated with the fluorogenic substrate 4-methylumbelliferyl phosphate (4MUP). The amount of enzyme-labeled polyclonal antibody that binds to the beads is directly proportional to the iPTH concentration in the sample. A standard curve is constructed” using 6 calibrators, and unknown sample concentrations are calculated using this curve. ²¹ This immunoenzymatic assay offers several advantages over radioimmunoassay: simple to perform without dedicated staff, total assay time of ~20 min, limited space requirement, and no need for radioactive materials. Calcitriol was quantified with a radioimmunoassay at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI, USA).

The main laboratory findings at presentation (T0) were severe ionized hypocalcemia, coupled with total Ca (tCa) close to the lower limit of the RI, and severe increases of PTH and calcitriol concentrations (Tables 2, 3). Radiography revealed severe generalized reduction in bone density, with decreased cortical thickness, poor delineation of the skull, and lack of visualization of the lamina dura; vertebral column deformity; and widening of physeal plates and metaphyseal ends of the radius, ulna, tibia, and metacarpal and metatarsal bones, bilaterally (Fig. 1). No signs of pathologic folding fractures were visible.

Table 2.

Analytes measured at different times in a kitten with nutritional secondary hyperparathyroidism.

Analyte, unit	Result		RI
	T0	T2
Urea, mmol/L	10	14.6	7.1–21.4
Creatinine, µmol/L	61.9	79.6	<159
Total calcium, mmol/L	1.6	2.5	1.6–2.6
Ionized calcium, mmol/L	0.88	1.38	1.25–1.50
Inorganic phosphorus, mmol/L	1.94	2.29	1.45–2.62
Parathyroid hormone, ng/L	134	3.4	NA
1,25-dihydroxyvitamin D, pg/mL	327	NA	38–142*

NA = not available; T0 = first examination; T2 = after 2 mo.

^* Based on minimum and maximum concentrations from 22 healthy cats.

Table 3.

Published values of feline intact parathyroid hormone (PTH) in healthy cats.

Reference	Method	No. of cats	PTH, ng/L
			RI	$\overline{x}$  ± SD	Min–max
1	IRMA	40	NA	10.9 ± 5.3	3.3–22.5
2	IRMA	31	7.7–43.3	NA	NA
8	IRMA	21	NA	27.4 ± 3.58	3.15–58.9
11 , 20	CLEIA	27	8–25	NA	NA
14	IRMA	52	NA	9.1 ± 0.7	1.6–23.7
18	IRMA	7	<3–28	NA	NA
19	IRMA	28	<5.2–17.3	NA	NA

CLEIA = chemiluminescence enzyme immunoassay; IRMA = immunoradiometric assay; NA = not applicable.

Figure 1.

Radiographs of the right forelimb and the lumbar spinal column. Bone density and cortical bone thickness increased over time, with thinning of physeal plates of the radius and ulna and persistence of the spinal deformity. A, C. First examination. B, D. 3 mo later.

Based on nutritional history and clinical, clinicopathologic, and radiographic abnormalities, a diagnosis was made of nutritional secondary hyperparathyroidism (NSHP), likely as a result of insufficient Ca and an inappropriate Ca:P ratio in the diet. However, concurrent rickets resulting from nutritional hypovitaminosis D, as discussed below, could not be excluded, especially in light of the suggestive radiographic findings, such as the widening of physeal plates in the absence of evident folding fractures. Vitamin D–dependent rickets type II (VDDRII), an autosomal recessive disease, has been reported in cats and is also characterized by secondary hyperparathyroidism and increased calcitriol concentrations as a result of a defect in the vitamin D receptor gene. ¹³ The measurement of 25-hydroxyvitamin D before and after the dietary correction and knowledge of all nutrient contents of the diet would have helped in understanding the pathogenesis.

After the diagnosis, the nutritional recommendation was to choose a complete and balanced diet formulated for growth. The kitten then received an amount of wet pet food based on ideal body weight, according to the energy growth requirements recommended by the European Pet Food Industry (Fédération Européenne de l’Industrie des Aliments pour Animaux Familiers; FEDIAF) nutritional guidelines [1.75 or 2 × (100 kcal × BW kg)^0.67] and the U.S. National Research Council (NRC).^6,12 The specific dietary plan was to feed 230–270 g/d (277–320 kcal ME/d) divided into 4 meals, with fresh water always available (Table 4).

Table 4.

Ingredients and analytical composition of the feline complete wet pet food formulated for growth.

Nutrient	As fed, %	DM, %	g/100 kcal
Crude protein	13	52	11.7
Fat	7	28	6.3
Crude fiber	0.05	0.2	0.04
Ash	3.2	12.8	2.8
Moisture	75	25	NA
Calcium	0.45	1.8	0.4
Phosphorous	0.45	1.8	0.4
Vitamin D	48.3	193.2	43.5
Metabolizable energy, kcal/100 g*	111	444	NA

Ingredients of the feline complete wet pet food: meat and meat by-products (chicken 14%), fish and fish by-products, minerals, vegetable by-products.

DM = dry matter; NA = not applicable.

^* Calculated according to NRC 2006. ¹²

After dietary correction, periodic checkups were scheduled. Radiographic examinations were done by the same operator with the same instrument with the same settings at each checkup. The owner noted gradual improvement of physical activity and the disappearance of discomfort. After 2 mo (T2), clinical examination revealed improvement of general health with an increase of BW (2.1 kg), although partial limb and spine deformities persisted. The concentrations of both ionized Ca (iCa) and tCa were within RIs at T2, and both were higher compared to T0. The PTH concentration was also decreased dramatically, confirming the resolution of NSHP and hypocalcemia (Table 2). Although the RI of the immunoenzymatic method is unavailable, the comparison with RIs reported for iPTH and values detected in healthy cats confirmed that the PTH was unequivocally high at T0, supporting the diagnosis of NSHP (Table 3). Radiographic examinations performed 3 mo after the first presentation (Fig. 1) showed an increase in bone density and cortical bone thickness; skull bones were more evident, with persistent severe deformity of the nasal bone. Metaphyseal ends of long bones were still widened, but physeal plates appeared thinner. Forelimb deformity was also evident, with bilateral radius curvature. No changes were seen in the spinal deformity. The persistence of the deformity of the nasal profile was caused partially by a nasal mucocele. Vertebral column deformities likely persisted because of their severity before diagnosis.

Feline hyperparathyroidism consists of primary and secondary hyperparathyroidism, mainly as the result of nutritional disorders or chronic kidney disease. ¹³ Nutritional secondary hyperparathyroidism in cats is an uncommon but well-described disease caused by imbalances in dietary Ca, phosphorus, and/or vitamin D, or can also result from genetic defects affecting vitamin D metabolism, leading to increases in PTH concentrations.^4,9,11,18 PTH is the principal regulator of Ca concentration through effects on renal tubular reabsorption, intestinal absorption mediated via calcitriol, and bone resorption. ¹ PTH is highly sensitive to fluctuations in iCa, and its concentration increases dramatically when iCa is low, as occurs in NSHP, which results in rapidly increasing renal reabsorption and bone resorption and secondary activation of vitamin D; a sigmoidal curve with a steep slope represents the relationship between PTH and iCa. ¹⁴ Total Ca does not reliably predict active iCa, as demonstrated in cats. ¹⁶

In growing kittens, the metabolic demand for Ca for bone growth and limited Ca storage increase the risk of NSHP. ¹⁷ A minimum Ca concentration of 0.52 g/100 g dry matter (DM) or 1 g/100 g DM, according to NRC ¹² and FEDIAF ⁶ nutritional guidelines respectively, must be provided in cat food. This discrepancy in recommendations is the result of a safety margin, considering the bioavailability of Ca in the raw material. ⁶ Moreover, a Ca:P ratio of 1:1 is recommended in a kitten diet. These requirements are easily achievable with a complete and balanced commercial diet; conversely, diets poor in Ca and rich in phosphorus, such as an all-meat diet, result in an inappropriate Ca:P ratio and increase the risk of metabolic diseases such as NSHP. In our case, the diet was Ca-deficient, which led to the development of NSHP.

A decrease of calcifediol (25-hydroxycholecalciferol, 25-hydroxyvitamin D₃, 25(OH)D₃) and concurrent increase of calcitriol (1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin D₃) concentrations are reported in cats affected with NSHP, reasonably resulting from the effect of PTH on the increased conversion and activation of 25(OH)D₃ to calcitriol. In our case, a dietary deficiency of cholecalciferol or ergocalciferol could have contributed to a decrease of 25(OH)D₃, the substrate for calcitriol production. The increased calcitriol, which is far less abundant than 25(OH)D₃, detected here and in other published cases, does not eliminate a potential contribution of dietary vitamin D deficiency to the clinical picture.^13,18 VDDRII was considered unlikely in view of the optimal response to dietary change, but cannot be ruled out considering that 25(OH)D₃ was not measured, and the determination of calcitriol was not repeated at T2.

Despite the undoubted utility of PTH measurement, methods for determination of feline PTH concentrations are limited. We successfully used a validated PTH assay for the measurement of feline PTH allowing us to confirm the diagnosis and follow the disease progression. ²² PTH is an 84 amino acid single-chain polypeptide produced by the parathyroid glands and is highly conserved among mammalian species. ¹⁵ There are various circulating fragments of PTH: the intact active form (1-84)-PTH is rapidly inactivated by hepatic and renal metabolism, producing fragments of various lengths, the exact composition and biological function of which are still unknown. Overall, these fragments, produced mainly after the cleavage of the 1-34 N-terminal region, are considered predominantly biologically inactive and undergo renal excretion, the accumulation and effects of PTH fragments on bone, kidney, and the cardiovascular system in patients affected by CKD has been reported in human medicine. ³

Three generations of assays have been developed for the measurement of human PTH to avoid the measurement of the aforementioned fragments. First-generation PTH radioimmunoassays used a single polyclonal antibody directed against the PTH C-terminal part or midterminal part, but lacked specificity because of cross-reactivity with many biologically inactive C-terminal fragments, and have been abandoned. Second-generation assays, for iPTH, which include the method used in our case, use a second antibody directed at the N-terminal region, and although they should measure only full-length PTH, these assays also measure some C-terminal fragments. A third-generation immunoassay, for whole or bio-intact PTH, detects only full-length PTH using an N-terminal antibody directed against only the first 4 amino acids and a C-terminal antibody.^3,10,13,17

In both human and veterinary medicine, second-generation iPTH assays are used most commonly for measuring PTH concentrations, although use is limited in veterinary medicine given the availability of validated methods. Specifically in cats, a long-used validated second-generation human iPTH immunoradiometric assay (Allegro intact PTH; Nichols Institute Diagnostics) with appropriate RIs is no longer available. ¹ Similar assays available from other suppliers are no longer distributed by the manufacturers (e.g., Diagnostic Systems Laboratories; DPC), including a validated method (Total intact PTH immunoradiometric assay, coated bead version, 3KG600; Scantibodies).^1,7,8,19 A third-generation assay (Duo PTH IRMA kit; Scantibodies) has been validated and seems to be the better choice compared to second-generation assays given better identification of the biologically active PTH; however, irrefutable supporting data are not available. ¹⁴ Finally, a chemiluminescence immunoassay has been used successfully by Japanese authors, but without complete validation.^11,20