Thyroid stimulation with recombinant human thyrotropin in healthy cats, cats with non-thyroidal illness and in cats with low serum thyroxin and azotaemia after treatment of hyperthyroidism

Abstract

This study investigated the recombinant human thyrotropin (rhTSH) stimulation test in healthy cats (group 1), cats with non-thyroidal illness (group 2) and cats with low serum total T₄ (TT₄) and azotaemia after ¹³¹I treatment (group 3). Serum TT₄ responses and thyroidal pertechnetate uptake after administration of 25 μg rhTSH IV were assessed. Baseline serum TT₄ was significantly lower in group 3 compared with group 1, but not between other group pairs. Serum TT₄ increased significantly in groups 1 and 2 but not in group 3 after rhTSH administration. Post-rhTSH serum TT₄ concentrations differed significantly between groups 1 and 3 and groups 2 and 3, but not between groups 1 and 2. Thyroid/salivary gland uptake ratio (T/S uptake ratio) differed only significantly between groups 1 and 3. Stimulation with rhTSH is valuable to differentiate euthyroidism from iatrogenic hypothyroidism in cats.

Radioiodine (¹³¹I) is the treatment of choice for feline hyperthyroidism, but iatrogenic hypothyroidism can occur in 6–30% of the cases. ^1–3 Another complication is that up to 39% of treated cats have chronic kidney disease (CKD) diagnosed after treatment of hyperthyroidism. ^4–7 When a low circulating serum total T₄ (TT₄) concentration is found in cats treated with ¹³¹I, non-thyroidal illness (NTI) must be considered, especially CKD, before a diagnosis of iatrogenic hypothyroidism can be confirmed. ⁸ Several NTIs such as diabetes mellitus (DM), hepatic insufficiency, gingivostomatitis and CKD can decrease serum TT₄ concentrations. ⁹

Cats with serum TT₄ below the reference range, and post-treatment azotaemia after ¹³¹I therapy, therefore represent a diagnostic challenge. Further, because hypothyroidism can also decrease renal function, ^10–13 definitive diagnosis of hypothyroidism or NTI is warranted.

Thyroid function can be assessed with serum free T₄ after equilibrium dialysis, which has a low specificity, ¹⁴ or endogenous serum thyroid stimulating hormone (TSH) concentration. However, feline TSH measurement is not available. A canine TSH assay was recently validated for use in cats ¹⁵ but accuracy of this assay remains unclear in the higher range of TSH concentrations as would be expected in hypothyroid cats. An alternative method which could prove valuable for measuring thyroid function, is stimulation of thyroid with TSH. The possibility of prolonged storage of rhTSH ¹⁶ and its biological activity in cats ¹⁷ enables the use of rhTSH in clinical feline medicine. Recent studies have described the stimulatory effect of 25 μg recombinant human TSH (rhTSH) on serum TT₄ concentration and thyroid scintigraphy in healthy cats and uptake of radioiodine ¹²³I in hyperthyroid cats. ^17–19

Following TSH administration, thyroid function can be assessed with serum TT₄ concentrations as a representative of thyroid hormone synthesis and release of preformed hormones. The metabolic activity of the thyroid gland can also be measured with technetium pertechnetate (^99mTcO₄^–) uptake which is concentrated in the thyroid but not incorporated in thyroid hormones. With computer software, regions of interest (ROI) can be drawn around thyroid lobes and salivary glands and the calculated thyroid/salivary gland (T/S) uptake ratio of pertechnetate is the commonly used parameter to determine functional status of the thyroid in healthy and hyperthyroid cats. ^20,21 When thyroid scintigraphy is performed after TSH administration, in healthy cats, a marginal but significant increase in T/S uptake ratio has been described. ¹⁸

The objectives of this study were to evaluate thyroid function following rhTSH stimulation assessing changes in TT₄ and by thyroid scintigraphy, in cats with a serum TT₄ below reference range combined with azotaemia after ¹³¹I treatment, and compare these with cats with NTI and a group of healthy control cats.

Materials and Methods

Animals

This study was conducted according to guidelines for animal care, with consent of the Ethical Committee of the Faculty of Veterinary Medicine from Ghent University, Belgium, and with informed owner consent.

Six healthy euthyroid female spayed cats, with an average age of 4 years and body weight (BW) range of 4.0–5.2 kg (mean±standard deviation [SD] 4.7±0.4), were included (group 1). Screening of these cats included physical and routine laboratory examinations (complete blood count, biochemistry and serum TT₄ concentration) and urinalysis (dipstick tests, microscopic analysis, protein/creatinine ratio and urine specific gravity). Cats were included in the study when these examinations showed no clinically significant abnormalities.

Five cats with NTI (group 2), all patients from the Veterinary Clinic of Small Animal Medicine, Ghent University, were included. Abnormal findings on physical and routine laboratory examination had to be compatible with the established disease and no other significant abnormalities could be present. The diseases in the cats with NTI were CKD (IRIS [International Renal Interest Society, www.iris-kidney.com/guidelines/en/staging_ckd.shtml] stage II, n=1), DM (n=1) and severe chronic gingivitis and stomatitis (n=3). The cats had an average age of 9 years and BW range of 2–6 kg (mean±SD 4.2±1.8).

The group of cats with low serum TT₄ concentration and azotaemia (group 3) were included from a larger group of hyperthyroid cats that were evaluated at 1, 3 and 6 months after ¹³¹I treatment. At evaluation, serum TT₄ and creatinine concentration, as well as urine specific gravity (USG) were measured. Glomerular filtration rate (GFR) was measured before and 1, 3 and 6 months after treatment with the plasma exo-iohexol clearance test (PexICT) as described previously. ²² Four cats with a documented serum TT₄ concentration below reference range and post-treatment renal azotaemia at 3 and 6 months after treatment, and at 7–8.5 months after ¹³¹I treatment when the study was performed, were included. Average age was 14.5 years and BW range of 3.0−8.0 kg (mean±SD 5.8±2.3).

Renal azotaemia was defined as serum creatinine concentration 140−249 μmol/l (IRIS stage II) or 250–439 μmol/l (IRIS stage III).

Experimental design

A 25 μg dose of rhTSH (Thyrogen; Genzyme corporation, Naarden, The Netherlands) was administered intravenously. The rhTSH had been dissolved in sterile water, divided in aliquots containing 25 μg rhTSH and frozen at −20°C for a maximum of 8 weeks as described by De Roover et al. ¹⁶ Aliquots were allowed to thaw at room temperature shortly before injection. Six hours later, a pertechnetate scan was performed as described previously. ¹⁸ A dose of 74 MBq (2 mCi) pertechnetate was injected intravenously (5.5 h after administration of rhTSH) and static images with a set number of 200,000 counts were acquired 30 min after injection, with a gamma camera (Toshiba GCA 901A, Exalto SA/NV, Saintes, Belgium) equipped with a low energy high resolution (LEHR) collimator. Cats were fasted for at least 10 h before the thyroid scan. Cats were maintained under light anaesthesia with propofol (PropoVet: Propofol 10 mg/ml, Abbott Logistics BV, The Netherlands) and placed in ventral recumbency over the camera. ROI were manually drawn over the left and right thyroid lobes and ipsilateral salivary glands by the same co-investigator (E Vandermeulen) in order to calculate the T/S uptake ratio in thyroid glands.

Two blood samples were collected by jugular venepuncture, less than 5 min before injection of the rhTSH and the pertechnetate scan, respectively. Serum was collected after centrifugation, aliquoted and frozen at–20°C until radioactivity had decayed for measurement of TT₄. The TT₄ was measured with a validated chemiluminescent immunoassay (Chemiluminescent Immulite 2000, DPC, Los Angeles USA [nmol/l]). Intra- and inter-assay coefficient of variation were 4 and 8% for the lower range and 6 and 7% for the higher range of serum TT₄, respectively. A serum sample with high TT₄ concentration was diluted with a serum sample with low TT₄ concentration, which showed a linear dilution curve.

Statistical analysis

Statistical analysis was performed with SAS version 9.1 (SAS Institute, Cary, USA). Analysis was based on analysis of variance (ANOVA) with group as categorical fixed effect. Differences in serum T₄ concentration between baseline and after rhTSH administration were analysed with a paired sample t-test for each of the three groups. Groups were compared pairwise with Tukey's multiple comparisons technique for BW, age and dose/kg BW, serum TT₄ concentration pre- and post- rhTSH administration, absolute and relative increase in serum TT₄ concentration after rhTSH administration and T/S ratio. Global significance level was 5%. Results were expressed as mean±SD.

Results

There was no significant difference between groups for BW (P=0.33) or dose/kg BW (P=0.26). There was a significant difference in age between all groups (P<0.001). All cats in group 3 had signs suggestive of hypothyroidism when the study was performed (increase in BW, poor hair coat, lethargy). All cats from group 3 had renal azotaemia stage II at 3 and 6 months after treatment with radioiodine. At the time of the study, cats from group 3 had renal azotaemia IRIS stage II (n=3) or III (n=1). The cat with CKD from group 2 had renal azotaemia stage II.

The results of serum TT₄ and T/S ratio for the three groups are shown in Table 1. Serum TT₄ concentration increased significantly in group 1 (P<0.001) and group 2 (P<0.001) but not in group 3 (P=0.926) after rhTSH administration. There was a significant difference in pre-rhTSH serum TT₄ concentration between groups 1 and 3 (P=0.005) but not between groups 1 and 2 (P=0.310) or groups 2 and 3 (P=0.076). There was a significant difference between groups 1 and 3 and between groups 2 and 3 in post-rhTSH serum TT₄ (P<0.001), absolute increase in serum TT₄ concentration (P=0.001) and relative increase in serum TT₄ concentration (P<0.001), though there was no difference for these variables between groups 1 and 2. There was only a significant difference between groups 1 and 3 (P=0.008) for T/S uptake ratio after rhTSH administration.

Table 1.

Serum TT₄ concentration pre- and post-rhTSH administration, absolute and relative increase in serum TT₄ concentration and T/S ratio after rhTSH administration in healthy cats (group 1), cats with NTI (group 2) and cats with serum TT₄ below reference range and azotaemia after ¹³¹I treatment (group 3).

	Group 1	Group 2	Group 3
Serum TT4 pre-rhTSH (nmol/l)	19.1±4.6a (12.9–25.8)	15.5±3.0a,b (11.6–18.1)	9.1±3.7b (5.9–12.9)
Serum TT4 post-rhTSH (nmol/l)	54.4±5.9a (49.0–65.8)	55.0±23.1a (33.5–92.9)	9.7±4.4b (5.9–14)
Serum TT4 absolute increase (nmol/l)	35.3±5.1a (25.8–40)	39.5±21.2a (21.9–74.8)	0.6±0.7b (0–1.3)
Serum TT4 relative increase (%)	195.8±64.1a (111–300)	250.0±103.9a (146–414)	5.0±5.8b (0–11)
Post-/pre- T4 ratio	2.8±1.3 (2.1–4.0)	3.6±7.6 (2.9–5.1)	1.1±1.2 (1.0–1.1)
T/S uptake ratio post-rhTSH	1.2±0.2a (1.0–1.4)	1.1±0.3a,b (0.8–1.5)	0.8±0.1b (0.6–0.9)

rhTSH=recombinant thyroid stimulating hormone; Post-/pre- t₄ ratio=post-rhTSH stimulation/pre-rhTSH stimulation serum TT₄ concentration ratio; T/S uptake ratio=thyroid/salivary gland uptake ratio. If superscripts (a, b, c) differ between columns for a variable, a significant difference was noted (P values are provided in the text).

Based on the marginal increase in serum TT₄ concentration, compared to the healthy cats and cats with NTI, it was concluded that all four cats from group 3 had iatrogenic hypothyroidism. Treatment with levothyroxin 10–20 μg/kg/day (Forthyron; Eurovet Animal Health BV, The Netherlands) was started. Cats were re-evaluated when euthyroidism was reached. One owner declined more than one follow-up visit, and, therefore, this cat could not be re-evaluated when euthyroid. Serum concentration of TT₄, creatinine, GFR and USG of cats in group 3 before and 1, 3 and 6 months after radioiodine treatment, as well as after levothyroxin supplementation, is presented in Table 2.

Table 2.

Mean±SD (range) of serum concentration of TT₄ (nmol/l), creatinine (μmol/l), GFR (ml/min/kg) and USG in cats from group 3 before (0), 1, 3 and 6 months after treatment with ¹³¹I, and after euthyroidism was re-established.

Group 3	0	1 month	3 months	6 months	Euthyroidism (n=3)
Serum TT4	83.4±26.4 (58.1–120.0)	8.4±9.7 (0–18.1)	5.8±6.8 (0–12.9)	4.5±5.2 (0–9.0)	37.4±9.7 (28.4–47.7)
Serum creatinine	83±22 (51–101)	136±26 (101–158)	162±18 (151–188)	183±38 (152–236)	161±32 (126–187)
GFR	2.7±0.6 (2.1–3.3)	1.3±0.3 (1.0–1.8)	1.2±0.2 (0.9–1.4)	1.1±0.1 (0.9–1.2)	1.1±0.1 (1.1–1.2) *
USG	1.030±0.02(1.022–1.060)	1.030±0.01 (1.018–1.047)	1.040±0.02 (1.015–1.048)	1.030±0.02 (1.012–1.060)	1.040±0.02 (1.014–1.060)

GFR=glomerular filtration rate; USG=urine specific gravity; UPC=urinary protein/creatinine ratio.

^*n=2.

Discussion

In this study we investigated the rhTSH stimulation test for the evaluation of thyroid function in cats suspected of iatrogenic hypothyroidism and showing azotaemia after treatment of hyperthyroidism with ¹³¹I, by comparing them to healthy cats and cats with NTI without history of thyroid problems. The cats with NTI were included in the study to evaluate the effects of NTI on the results of the rhTSH stimulation test.

There was no difference in serum TT₄ concentration pre-rhTSH stimulation between groups 1 and 2. Although chronic gingivitis and periodontal disease have been described as NTI, it was expected that these diseases would cause less severe NTI compared to CKD and DM in the other two cats. ⁹ This, combined with the small group sizes, could have lead to a smaller and, therefore, non-significant difference in serum TT₄ between groups 1 and 2.

There was, however, also no significant difference between groups 2 and 3 in basal serum TT₄ concentration. This underlines the need for further evaluation of thyroid function in cats suspected of iatrogenic hypothyroidism and renal azotaemia after ¹³¹I treatment. A diagnosis of hypothyroidism cannot be made based solely on the basal serum TT₄ concentration.

In several species, hypothyroidism can affect kidney function. ^10–13 Our group showed that GFR is markedly decreased in canine hypothyroidism. ¹³ Although this has not yet been investigated in cats, the presence of kidney disease and potential deleterious effects of the previous hyperthyroidism on kidney function merit attention. An early diagnosis of hypothyroidism in these cats is, therefore, important as untreated hypothyroidism can have long term effects on kidney function and is associated with congestive heart failure in humans. ^23,24 Stimulation with rhTSH has not yet been investigated as a diagnostic test in cats suspected of iatrogenic hypothyroidism. A recent study evaluated rhTSH stimulation in healthy dogs, euthyroid sick dogs and hypothyroid dogs and established the following criteria: dogs were classified as euthyroid if either post-TSH serum TT₄ concentration was equal to or exceeded 40 nmol/l or if the change in serum TT₄ concentration post-TSH was greater than or equal to 20 nmol/l. ²⁵ All of the cats from group 1 and four cats from group 2, though none of the cats from group 3, met these criteria. These criteria are arbitrary in cats and need further validation but the results in the study using these criteria are promising.

Thyroid stimulation can be indexed by the post-/pre-TT₄ ratio. ^26,27 In dogs with hypothyroidism or euthyroid sick syndrome, this ratio has an approximate value of 1. ^25,28 This is comparable to the results in our study for the cats from group 3, but lower than the results for the cats from group 2. However, the dose of rhTSH used in these studies was lower (4 μg/kg BW) compared to the average dose of around 5 μg/kg BW in our study. This suggests a higher biological activity of rhTSH in cats compared to dogs, but this needs further investigation.

In the study described here, there was no difference in post-rhTSH T/S uptake ratio between groups 2 and 3 or 1 and 2. This suggests that assessment of thyroidal function with thyroid scintigraphy after rhTSH stimulation is not accurate in cats with low serum TT₄ concentration combined with renal azotaemia. However, this does not exclude the possibility of a difference in T/S uptake ratio before stimulation with rhTSH between the groups. When different methods of thyroidal function assessment such as TSH stimulation combined with serum TT₄ concentration followed by thyroid scintigraphy are being evaluated in cats, the influence of rhTSH on thyroid scintigraphy must be taken into account. In a recent study by the same group, a marginal but significant effect of rhTSH on T/S uptake ratio by the thyroid gland was described in healthy cats. ¹⁸ It remains possible that basal T/S uptake ratios differed between groups but that this difference was masked by possible effects of rhTSH on remaining functional thyroid tissue in the glands of cats from group 3. Indeed, in dogs thyroid scintigraphy had the highest discriminatory power between primary hypothyroidism and NTI in one study. ²⁹

After the study, treatment with levothyroxin was started in all four cats from group 3 with iatrogenic hypothyroidism and azotaemia (Table 2). After euthyroidism was reached, serum creatinine decreased and GFR increased in one cat. The other two cats showed increased serum creatinine levels, and the diagnosis of concurrent CKD in these cats was confirmed based on the worsening kidney function despite improvement of serum TT₄ concentration. Hypothyroidism can cause glomerular lesions such as thickening of the basement membrane and increased mesangial matrix. ^30,31 Indeed, treatment of hypothyroidism in a human patient with progressive renal failure can lead to significant improvement of renal function. ³² Evaluation of kidney function in cats with iatrogenic hypothyroidism has not yet been described.

Limitations of this study are the small number of cats investigated and the age differences in the three groups of cats. Healthy control cats were selected younger than 6 years of age to minimise possible thyroid problems. However, results showed significant differences between the groups and suggests TT₄ measurement before and after rhTSH is a clinically useful test in cats and can reliably evaluate thyroid function and differentiate euthyroidism from iatrogenic hypothyroidism in cats.