Diagnosing Hypothyroidism in Dogs

I have a 7-year old spayed Golden Retriever who weighs about 110 lbs. No signs of hypothyroidism except for being overweight with a borderline high serum cholesterol concentrations (256 mg/dl). The serum T4 value was slightly low at 0.9 µg/dl (reference interval, 1.0-4.0 µg/dl).

Is this slightly low serum T4 concentration diagnostic for hypothyroidism? Would you start her on levothyroxine (L-T4) and retest T4 levels in a month?

I'd appreciate your thoughts and recommendations.

My Response: 

I would never base the diagnosis on only a resting serum T4 value alone. We commonly find low values that fluctuate in and out of the reference range in dogs that are clinically normal and never develop hypothyroidism. In addition, most non-thyroidal illness will lower the total T4 values in dogs, and these dogs would not benefit from thyroid hormone supplementation (1). It certainly doesn't sound like your dog is sick or acting ill in any way, so this latter explanation probably doesn't apply here.

For your dog, I'd recommend that your veterinarian collect more sera to do a complete thyroid panel, which should include the following tests (2-4):

• Serum T4 concentration
• Serum T3 concentration
• Serum free T4 by dialysis
• Serum TSH concentration
• Serum levels of thyroglobulin autoantibodies

If you have it available in your area, thyroid scintigraphy (nuclear medicine scan) is actually the best and most accurate way to diagnose hypothyroidism in dogs (4.5).

 References: 

1. Kantrowitz LB, Peterson ME, Melian C, et al. Serum total thyroxine, total triiodothyronine, free thyroxine, and thyrotropin concentrations in dogs with nonthyroidal disease. J Am Vet Med Assoc 2001;219:765-769. 
2. Peterson ME, Melian C, Nichols R. Measurement of serum total thyroxine, triiodothyronine, free thyroxine, and thyrotropin concentrations for diagnosis of hypothyroidism in dogs. J Am Vet Med Assoc 1997;211:1396-1402.  
3. Nachreiner RF, Refsal KR, Graham PA, et al. Prevalence of serum thyroid hormone autoantibodies in dogs with clinical signs of hypothyroidism. J Am Vet Med Assoc 2002;220:466-471. 
4. Diaz Espineira MM, Mol JA, Peeters ME, et al. Assessment of thyroid function in dogs with low plasma thyroxine concentration. J Vet Intern Med 2007;21:25-32.  
5. Shiel RE, Pinilla M, McAllister H, et al. Assessment of the value of quantitative thyroid scintigraphy for determination of thyroid function in dogs. J Small Anim Pract 2012;53:278-285. 

What's the Human Risk Associated with Handling of Thyroid Medications?

Maybe I am missing something here, but I have a question about the human risk associated with handling thyroid medications. Why do you state that owners should be careful when handling and cutting methimazole tablets (Tapazole of Felimazole) because of the human safety concerns (1,2), when I can find no mention that we should use any precautions when handling or cutting L-thyroxine (L-T4) tablets?  I've heard that we should wear gloves when handling methimazole tablets or applying methimazole transdermally to the cat's ear, but no one has ever mentioned the need for gloves with L-T4 tablets.

So why is handling L-thyroxine any less of a concern than handling methimazole? Do they not both have the same potential to affect human thyroid levels? If handling methimazole can lower a human's thyroid levels, why wouldn't handling an L-thyroxine preparation potentially lead to hyperthyroidism?

My Response: 

Methimazole and L-thyroxine are totally different drugs, with different patterns of absorption and metabolism. Methimazole is a potent antithyroid drug whereas L-thyroxine is a synthetic form of natural thyroid hormone.

Methimazole
As you state, we must be careful when cutting methimazole tablets or applying transdermal methimazole since either form of methimazole could potentially be absorbed via human skin (1,2). Methimazole is a human teratogen (i.e., the drug may cause birth defects) and crosses the placenta concentrating in the fetal thyroid gland. Besides being a safety issue in pregnant women or women who may become pregnant, the drug should not be handled by lactating women because methimazole is transferred in breast milk at a high rate.

The same precautions pertain regardless of what methimazole product is used, i.e., the veterinary brand-name Felimazole preparation or a human brand-name or generic product. The methimazole tablets should not be cut with our bare hands. We want to wash our hands after administering the medication to minimize skin and oral contamination; repeated and chronic exposure could pose a risk.

L-Thyroxine
As compared to methimazole, L-thyroxine is not absorbed well from the skin. Results of two studies found that the hormone is not absorbed into the systemic circulation to have an effect on all tissues of the body (3,4). In support of that, of the few dogs that I have evaluated on transdermal L-T4, none have responded to the transdermal replacement therapy with a rise in serum T4 concentrations (5). I know that some compounded pharmacies will sell transdermal preparations of L-T4 but I do not believe that they actually work. Ask for some actual data and you will get none!

So go ahead and cut all the L-T4 pills that you want. It will not get absorbed unless you ingest the hormone supplement so you should still wash your hands to remove any residual L-T4 residue from your hands.

References:

1. Peterson ME. Methimazole-Handling Precautions for Cat Owners. Blog post, Animal Endocrine Clinic, September 2, 2012.
2. Felimazole-Coated Tablets. Product insert. Available at: www.dechra-us.com/files/dechraUSA/downloads/Product%20inserts/Felimazole.pdf 
3. Padula C, Pappani A, Santi P. In vitro permeation of levothyroxine across the skin. Int J Pharm 2008 12;349:161-165. 
4. Padula C, Nicoli S, Santi P. Innovative formulations for the delivery of levothyroxine to the skin. Int J Pharm 2009;372:12-16.
5. Peterson ME. Alternative Dosage Forms of L-Thyroxine for Hypothyroid Dogs. Blog post, Insights into Veterinary Endocrinology, March 28, 2012.

Is a High Serum T4 or Free T4 Level Always Diagnostic for Hyperthyroidism in Cats?

Thyroid scan (scintigraphy) in a cat suspected of hyperthyroidism. Both thyroid lobes are of normal size and shape. Uptake of the radionuclide by the thyroid is also normal, with a thyroid/salivary ratio of 0.8 (normal <1.5). This is a normal study and rules out hyperthyroidism.
Scintiscan provided by Dr. Michael Broome, Advanced Veterinary Medical Imaging 

I've read on your blog "we almost never see false-positive results with the total T4 test." Since you say "almost," I'm wondering what, if anything, might cause a false-positive result? 

I have a 16-year old, male domestic long hair cat who currently has a total T4 value of 6.0 µg/dl (reference range, 0.8-4.0 µg/dl) after being off of methimazole for 5 weeks. He has a long history of chronic diarrhea associated with inflammatory bowel disease (IBD). 

About a year ago, we suspected hyperthyroidism based on a high-normal total T4 with a high free T4 concentration. He had thyroid scintigraphy at that time to confirm hyperthyroidism and help with his radioiodine dose calculation (we wanted to ensure that he wasn't overdosed — I don't want to end up with a hypothyroid cat!). Surprisingly, the results of this thyroid scintigraphy were negative (see his thyroid scintiscan above), though he was getting iodine supplements at the time. 

About 6 months ago, his serum T4 was a bit higher (and his free T4 remained high) so we started treatment with methimazole. He was on methimazole for about 6 months, which did lower his T4, but the drug seemed to significantly worsen his chronic diarrhea, and he didn't gain any weight.  My veterinarian has never been able to palpate an enlarged thyroid tumor in my cat.

I am interested in I-131 treatment, but would like to be as certain as possible he is hyperthyroid before making arrangements. Could there be any other explanation for high total and free T4 concentrations Do you see false-positive results?

His primary veterinarian and his internist both say they know of no other explanation for high total T4 concentration other than hyperthyroidism. But when I read "almost never" on your blog, that seems to imply it's possible (though unlikely) to get a false-positive test result.  Since I know that you are the expert on feline hyperthyroidism, I thought I would try asking for your advice. I don't want to pursue radioiodine treatment if he isn't truly hyperthyroid. 

My Response:

In older cats that develop clinical signs of hyperthyroidism, confirming a diagnosis of thyroid disease is usually straightforward (1-3). However, the potential for false-negative and false-positive results exists with all thyroid function tests (4,5) — especially in the context of routine screening of asymptomatic cats or cats sick with nonthyroidal disease (such as the IBD in your cat). This can lead to clinical dilemmas and misdiagnosis.

To avoid unnecessary treatment and potentially adverse effects in a euthyroid cat (i.e., normal thyroid status), thyroid function test results must always be interpreted in the light of the cat’s history, clinical signs, and other laboratory findings.

Palpating the thyroid nodule 
All cats with hyperthyroidism have a thyroid nodule (goiter) affecting one or both thyroid lobes, since in all cases there is underlying thyroid pathology leading to the disease (e.g., thyroid adenomatous hyperplasia, adenoma or carcinoma) (6).

Because all hyperthyroid cats must have at least one (if not two and sometimes more) thyroid nodules in order to develop the disease, the finding of a thyroid nodule, either by cervical palpation or by imaging (e.g., thyroid scintigraphy – see later), plays a crucial role in diagnosis in these cats (1-5).

If the veterinarian cannot palpate a thyroid nodule in the cat suspected of having hyperthyroidism, diagnosis becomes much more difficult to confirm, especially in cats with mild or borderline disease or in cats with concurrent illness.

Measuring the total T4 concentration 
A high circulating total T4 concentration is the biochemical hallmark of hyperthyroidism and is extremely specific for its diagnosis (1–5).

False-positive results (i.e., a high T4 in a cat without hyperthyroidism) are relatively rare but are being seen with increasing frequency, especially with the automated T4 immunoassays and in-house testing kits. In agreement with that statement, we recently reported that 1–2% of all cats diagnosed as hyperthyroid based on the finding of a high serum T4 concentration turn out to be euthyroid based on results of thyroid scintigraphy (4,5).

Therefore, if a high serum T4 value is found in a cat that lacks clinical signs of hyperthyroidism, especially if no thyroid nodule is palpated or when concurrent disease is present, one should never hesitate to repeat the serum T4 test using a different technique. In these cases, T4 measured by radioimmunoassay (RIA) or chemiluminescence (Immulite) is preferred (4,5). Alternatively, additional thyroid function testing (complete thyroid profile, thyroid scintigraphy) may also be recommended (4,5,7-10).

Measuring the free T4 concentration 
Many veterinarians believe that determination of a serum free T4 concentration is more reliable than measuring total T4 alone, since free T4 values are more consistently elevated in hyperthyroid cats than are total T4 concentrations (3,11). Although free T4 is a more sensitive diagnostic test than total T4 for diagnosing hyperthyroidism, the test specificity for free T4 is poor, with up to 20% of sick (and some clinically normal) euthyroid cats having false-positive free T4 results (3,11-13).

Caution is, therefore, advised in using serum measurements of free T4 as the sole diagnostic test for hyperthyroidism. As always, it is important to combine these thyroid test results with the cat’s clinical features and the presence of a palpable thyroid nodule to make the correct diagnosis.

More important, however, is the situation in cats that maintain reference interval serum T4 values and are diagnosed as hyperthyroid on the basis of a high serum free T4 concentration. Up to 30% of these cats presenting with a mid- to high–normal total T4 value, together with high free T4 concentrations by dialysis, will turn out to be euthyroid based on results of thyroid scintigraphy (4,13).

It is unclear how much additional useful information is truly gained by the use of free T4 assays over the use of total T4 estimations alone. Many veterinarians mistakenly believe that the finding of high free T4 in a cat is completely diagnostic for hyperthyroidism, especially when total T4 is within the middle to upper half of the reference interval. However, since up to a third of these cats may turn out to be euthyroid (4,13), it is clear that the free T4 test can never be considered a "gold standard" diagnostic test for thyroid disease in cats.

Use of free T4 testing can lead to more confusion than clarity in some hyperthyroid cats; certainly, reliance on free T4 results risks misdiagnosis of hyperthyroidism in many euthyroid cats, especially those with non- thyroidal illness.

Careful monitoring may be best diagnostic approach
So what is the next step if we find a high serum T4 in an asymptomatic cat in which no goiter is palpable? The first steps should always be to repeat the cervical palpation looking for a thyroid nodule and to verify the high T4 concentration. Again, the veterinarian should never hesitate to repeat the serum T4 test using a different technique, with RIA or CEIA preferred in such cats. Again, if we have any doubt about the diagnosis, thyroid scintigraphy should be considered (1,2,4,5).

In many of these asymptomatic cats in which the diagnosis is unclear (e.g., borderline or only slightly high T4 concentration, no obvious thyroid nodule), the best approach is to use close observation rather than start any treatment (4,5). With this cautious approach, one should recheck the cat at 2- to 3-month intervals, and at each visit monitoring the cat’s body weight, carefully re-palpating for a thyroid nodule, and repeating the serum T4 concentration doing a complete serum thyroid panel (T4, free T4, T3 and TSH) (4,5). If the cat is truly hyperthyroid, the thyroid tumor will continue to grow, the serum T4 will eventually rise into the hyperthyroid range, and the cat will lose weight.

My Bottom Line: 

But what's up with your cat? Is he really hyperthyroid? Well, the finding of a T4 value of 6.0 µg/dl is certainly consistent with hyperthyroidism, but I'm bothered by a number of issues with the case.

First of all, his thyroid scan done a year ago was completely normal. Of course, a thyroid tumor could have developed during this period, but the clinical signs a year ago appear to be similar to those your cat is showing at this time.

Secondly, medical treatment made the diarrhea worse, not better. If hyperthyroidism was responsible for your cat's gastrointestinal signs (the main problem), lowering the serum thyroid values should have helped, not made the situation worse.

Thirdly, remember that all cats (as well as all humans) likely have their own individual "reference range," so it's possible that a T4 value slightly outside of the lab's reference range could actually be normal for that individual (14,15). Could it be that your cat normally runs higher thyroid hormone levels than most other cats? Or is current high T4 simply a laboratory error?  That would certainly be the most common reason for the finding of a single "high" T4 concentration in a euthyroid cat.

Finally, you have seen a number of veterinarians (your regular veterinarian as well as internal medicine specialists). Despite multiple exams being performed by different vets, no one has been able to palpate a thyroid nodule.  If this was my cat, I would not rush into treatment without repeating the thyroid scan to document the presence of a hyperfunctioning thyroid tumor.

References:

1. Mooney CT, Peterson ME. Feline hyperthyroidism In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;92-110. 
2. Baral RM, Peterson ME. Thyroid gland disorders In: Little SE, ed. The Cat: Clinical Medicine and Management. St. Louis: Elsevier Saunders, 2012;571-592. 
3. Peterson ME, Melian C, Nichols R. Measurement of serum concentrations of free thyroxine, total thyroxine, and total triiodothyronine in cats with hyperthyroidism and cats with nonthyroidal disease. J Am Vet Med Assoc 2001;218:529-536. 
4. Peterson ME. Diagnostic testing for hyperthyroidism in cats: more than just T4. J Fel Med Surg 2013;15:765-777. 
5. Peterson ME. Diagnostic testing for feline thyroid disease: Hyperthyroidism. Compend Contin Educ Vet 2013:in press.
6. Gerber H, Peter H, Ferguson DC, et al. Etiopathology of feline toxic nodular goiter. Vet Clin North Am Small Anim Pract 1994;24:541-565. 
7. Mooney CT, Thoday JL, Nicoll JJ, et al. Qualitative and quantitative thyroid imaging in feline hyperthyroidism using technetium-99m as pertechnetate.Vet Radiol Ultrasound 1992;33:313-320. 
8. Daniel GB, Sharp DS, Nieckarz JA, et al. Quantitative thyroid scintigraphy as a predictor of serum thyroxin concentration in normal and hyperthyroid cats.Vet Radiol Ultrasound 2002;43:374-382. 
9. Broome MR. Thyroid scintigraphy in hyperthyroidism. Clin Tech Small Anim Pract 2006;21:10-16. 
10. Peterson ME, Broome MR. Thyroid scintigraphic findings in 917 cats with hyperthyroidism. J Vet Intern Med 2012;26:754.
11. Mooney CT, Little CJ, Macrae AW. Effect of illness not associated with the thyroid gland on serum total and free thyroxine concentrations in cats. J Am Vet Med Assoc 1996;208:2004-2008.
12. Wakeling J, Moore K, Elliott J, et al. Diagnosis of hyperthyroidism in cats with mild chronic kidney disease. J Small Anim Pract 2008;49:287-294. 
13. Peterson ME, Broome MR, Robertson JE. Accuracy of serum free thyroxine concentrations determined by a new veterinary chemiluminscent immunoassay in euthyroid and hyperthyroid cats. Proceedings of the 21st Annual European College of Veterinary Internal Medicine (ECVIM-CA) Congress; 2011.
14. Andersen S, Bruun NH, Pedersen KM, et al. Biologic variation is important for interpretation of thyroid function tests. Thyroid 2003;13:1069-1078. 
15. Andersen S, Pedersen KM, Bruun NH, et al. Narrow individual variations in serum T(4) and T(3) in normal subjects: a clue to the understanding of subclinical thyroid disease. J Clin Endocrinol Metab 2002;87:1068-1072. 

Hyperthyroid in Cats: Table of Contents

As you know if you have been following this blog, I've spent much of the last few months writing about issue of hyperthyroidism, the most common endocrine disorder of the cat.

Before I move on to the topics of treatment of this common feline condition, I thought I'd post links to the topics I've covered thus far:

1. Do All Hyperthyroid Cats Have a Thyroid Tumor? Is It Thyroid Cancer?
2. Why Has Hyperthyroidism in Cats Reached Epidemic Levels?
3. Top 10 Signs of Hyperthyroidism in Cats
4. Top 12 Physical Exam Findings in Cats with Hyperthyroidism
5. Diagnosing Hyperthyroidism in Cats: Routine Testing Procedures
6. Diagnosis of Hyperthyroidism in Cats: Serum T4 Concentrations
7. Diagnosis of Hyperthyroidism in Cats: Serum T3 Concentrations
8. Diagnosis of Hyperthyroidism in Cats: Serum Free T4 Concentrations
9. Diagnosis of Hyperthyroidism in Cats: Serum Free T4 (Part 2)
10. Diagnosis of Hyperthyroidism in Cats: Serum Free T3 Concentrations
11. Diagnosis of Hyperthyroidism in Cats: Serum TSH Concentrations
12. Diagnosis of Hyperthyroidism: T3 Suppression Test
13. Diagnosis of Hyperthyroidism: TRH Stimulation Test
14. Diagnosis of Hyperthyroidism: Thyroid Scintigraphy

I'm halfway through with this "book" on feline hyperthyroidism!

Confirming the Diagnosis of Hyperthyroidism: Thyrotropin-Releasing Hormone (TRH) Stimulation Test

In my last blog post, I discussed use of dynamic test, the T3 suppression test. Suppression testing can be helpful in confirming the diagnosis when we suspect that a cat has mild hyperthyroidism but serum concentrations of total and free T4 are either normal or borderline.

An alternative dynamic test that can be used to help diagnose cats with mild or occult hyperthyroidism is the thyrotropin-releasing hormone (TRH) stimulation test (1-3). Compared with the T3 suppression test, this test takes the opposite approach — now we are stimulating, rather than suppressing, the secretion of TSH from the pituitary, which in turn, would lead to increased T4 secretion.

Thyrotropin-Releasing Hormone (TSH) Stimulation Test

TRH is the hormone released by the hypothalamus that subsequently stimulates the release of thyrotropin (TSH) from the pituitary gland (4). When administered intravenously to normal cats, TRH causes an prompt increase in TSH secretion and serum T4 concentrations (see Figure 1, left panel).

Figure 1: TRH tests in normal cats (left) & cats with hyperthyroidism (right)

In contrast to the situation in normal cats, administration of large doses of TRH to hyperthyroid cats has little or no effect on TSH or T4 secretion. The reason for this that pituitary TSH secretion has already been completely and chronically suppressed by the higher-than-normal thyroid hormone secretion characteristic of hyperthyroidism (see Figure 1 above, right panel).

Administration of only a single dose of TRH is not enough to stimulate chronically suppressed TSH-secreting cells.

Testing Protocol for the TRH Stimulation Test

To perform the TRH stimulation test in cats, the following protocol is recommended:

1. The veterinarian draws a blood sample for determination of baseline serum concentrations of total T4 (and T3).
2. TRH (Protirelin) is administered to the cat at the dosage of 100 μg/kg, IV.
3. Four hours later, the veterinarian again draws a blood sample for serum T4 (and T3) determinations.

One may ask: but what about measuring TSH? Indeed, that would be a more direct measure and would shorten the testing time from 4 hours to less than an hour (4). However, because use of TSH measurements in cats with thyroid disease have only recently been advocated, studies looking the serum TSH response to TRH have yet to be published in cats.

Interpretation of Results of TRH Stimulation Testing

When the TRH stimulation test is performed in normal cats and sick cats without hyperthyroidism (1), there is a consistent rise in serum T4 concentrations (approximately 2-fold rise). In contrast, when the test is performed in cats with hyperthyroidism, even in cats with only slightly high or high-normal resting serum T4 concentrations, there is little, if any, rise in serum T4 values after the administration of TRH (see Figure 2, below).

Figure 2 — Box plots of the serum T4 concentrations before (A) and after (B) TRH stimulation in 31 clinically normal cats, 35 cats with hyperthyroidism, and 15 cats with nonthyroidal disease. The "box" represents the interquartile range from the 25th to 75th percentile (represents the middle one-half of the data). The horizontal bar through the box is the median. The "whiskers" represent the main body of data, which in most cases is equal to the range. Outlying data points are represented by open circles. To convert serum T4 concentrations from nmol/L to µg/dl, divide the given values by 12.87.

Regarding interpretation of the TRH stimulation test results, we found that the relative rise (per cent increase) in serum T4 concentration after administration of TRH was the best (most sensitive) criteria for predicting whether or not cats are hyperthyroid (1). A percent rise in serum T4 of less than 50 per cent is consistent with mild hyperthyroidism, whereas a value of greater than 60 per cent is seen in normal cats; values between 50 and 60 per cent are equivocal or borderline responses.

The serum T3 response to TRH is less helpful in separating normal from hyperthyroid cats, because many normal cats have only a small and inconsistent rise in serum T3 concentrations after TRH administration. Therefore, I do not recommend determining the serum T3 response as part of the TRH stimulation test.

In another study of 36 cats with severe critical illness (5), half of the severely sick cats showed an inadequate T4 response to TRH stimulation test (i.e., a percent rise in serum T4 of less than 50 per cent). Unfortunately, this could create a situation where mild hyperthyroidism is falsely diagnosed in a cat with critical illness.  Although the reason for this is unclear, critical nonthyroidal illness may render pituitary TSH secretion unresponsive to the stimulatory effects of TRH administration.

The bottom line: This test is not meant to differentiate normal and hyperthyroid cats from cats with severe critical illness. I do not recommend doing this test in severely ill cats, inasmuch as the test results may be invalid.

Which is Better: TRH Stimulation Test vs. the T3 Suppression Test?

Advantages of the TRH stimulation test over the T3 suppression test include the shorter time needed to perform the test (4 hours vs. 3 days), and the fact that the TRH stimulation test is not dependent upon the owner's ability to administer oral medication.

The major disadvantage of the TRH stimulation test in cats is that side effects almost invariably occur immediately after administration of the TRH (1, 6-8). These adverse effect include the following:

• Excessive salivation (see Figure 3)
• Vomiting
• Induced defecation
• Rapid breathing (tachypnea); open mouth breathing

Fortunately, all of the adverse side effects are transient and completely resolve by the end of the 4-hour test period. Because of these adverse effects, however, I generally do a T3 suppression test over the TRH stimulation test for dynamic testing of the pituitary-thyroid axis.

Figure 3: Cat showing increased salivation after TRH administration

Our studies show a close relationship between the presence (or absence) of suppressed serum T4 concentrations in response to T3 suppression and stimulated T4 values in response to TRH stimulation. Therefore, although the two tests evaluate the pituitary-thyroid axis in different ways, our findings indicate that the two screening tests provide similar information and can probably be used interchangeably for diagnosing mild hyperthyroidism in cats.

Future Investigations: Serum TSH Response to TRH Stimulation in Cats

In human beings, the TRH stimulation test is generally done by directly evaluating the change in serum TSH concentrations, not indirectly by measuring serum T4  values.  When this is done, the TRH stimulation test is one of the most sensitive tests in detecting early or mild hyperthyroidism in human patients. Again, because TSH production is completely suppressed in hyperthyroid patients, they will not show a rise in serum TSH when TRH is administered, whereas normal individuals will respond with a brisk rise in circulating TSH within 30 minutes of TRH injection. The new, ultra-sensitive human TSH assays have largely eliminated the need for TRH stimulation testing, but it still remains a useful and time-proven test.

Similar serum TSH responses to TRH have been reported in normal dogs — baseline serum TSH concentrations increase 2- to 3-fold when measured 30 minutes after administration of TRH at doses ranging from 1-10 μg/kg. There are two major advantages of evaluating the TSH response rather than the T4 response to TRH:

1. The test period is shorten from 4 hours to only 15-30 minutes.
2. A much lower dose of TRH is needed, greatly reducing or eliminating the adverse signs commonly seen with the higher doses needed to elicit a serum T4 response.

Measuring the serum TSH response to TRH has not been reported in cats with hyperthyroidism. Obviously, this is an area that needs to be investigated, now that we know that the canine TSH assay can be used to measure circulating TSH in cats.

Because the canine TSH assay is not designed to measure low values, basal TSH measurements are not very useful as a diagnostic test in feline hyperthyroidism (see my previous blog on TSH in cats). However, measuring the response to TRH may turn out to be a extremely useful and sensitive test for this disease.

References:

1. Peterson ME, Broussard JD, Gamble DA: Use of the thyrotropin releasing hormone stimulation test to diagnose mild hyperthyroidism in cats. Journal of Veterinary Internal Medicine 1994;8:279-286.
2. Peterson ME. Diagnostic tests for hyperthyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:2-9.
3. Peterson ME: Diagnostic testing for feline hyper- and hypothyroidism. Proceedings of the 2011 American College of Veterinary Internal Medicine (ACVIM) Forum, pp. 95-97, 2011.
4. Utiger RD: Tests of thyroregulatory mechanisms. In Ingbar SH and Braverman LE (eds): The Thyroid: A Fundamental and Clinical Text, pp 511-523. Philadelphia, JB Lippincott, 1986.
5. Tomsa K, Glaus TM, Kacl GM, et al. Thyrotropin-releasing hormone stimulation test to assess thyroid function in severely sick cats. Journal of Veterinary Internal Medicine 2001;15:89-93.
6. Holtman JR, Buller AL, Hamosh P, et al: Central respiratory stimulation produced by thyrotropin-releasing hormone in the cat. Peptides 1986;7:207-212.
7. Beleslin DB, Jovanovic-Micic D, Tomic-Beleslin N: Nature of salivation produced by thyrotropin-releasing hormone (TRH). Brain Research Bulletin 1987;18:463-465.
8. Beleslin DB, Jovanovic-Micic D, Samardzic R, et al: Studies of thyrotropin-releasing hormone (TRH)-induced defecation in cats. Pharmacology Biochemistry & Behavior 1987;26:639-641.
9. Yagi K, Ohashi E, Tanabe S, Uzuka Y, Sarashina T. Serum thyrotropin response to TRH administration in six healthy beagle dogs. Veterinary Record 2000;146:706-707.

Confirming the Diagnosis of Hyperthyroidism: The T3 Suppression Test

Yawning Cat at
the Hypurrcat Spa

In most cats with hyperthyroidism, the diagnosis is easily confirmed by measuring a single serum T4 concentration. Measuring serum free T4, T3, and TSH adds more diagnostic information that can be used to help confirm the diagnosis in cats with borderline hyperthyroidism.

Occasionally, cats suspected of having hyperthyroidism can be difficult to diagnose, even when repeated serum thyroid tests are run. Many of these cats have early or mild hyperthyroidism and show only mild clinical signs, whereas others appear to have more severe clinical features of hyperthyroidism but also have another obvious (or not so obvious) concurrent disease.  The finding of concomitant disease (kidney, liver, or gastrointestinal disease) is relatively common in hyperthyroid cats, which is not surprising given the fact that many of these cats are middle-aged to older.

In the majority of these hyperthyroid cats in which a normal total T4 concentration is found, simply repeating the total T4 analysis with simultaneous measurement of free T4, as well as ruling out any concurrent disease, will easily confirm the diagnosis. Further diagnostic tests for hyperthyroidism are rarely required. However, if we still still suspect that a cat has mild hyperthyroid but serum concentrations of total and free T4 are either normal or equivocal, dynamic testing can be used to help make the diagnosis.

Dynamic Testing for Diagnosis of Hyperthyroidism

Two dynamic tests can be used in cats with mild or "occult" hyperthyroidism — both of these tests  act on the hypothalamic-pituitary-thyroid axis (see Figure 1) to either suppress or stimulate pituitary TSH and thyroid hormone secretion.

• Triiodothyronine (T3) suppression test
• Thyrotropin-releasing hormone (TRH) stimulation test

Figure 1: Hypothalamic-Pituitary-Thyroid axis
Normally, TRH stimulates the secretion of TSH from the pituitary, which in turn, stimulates the thyroid to secrete T4 and T3. Both circulating T4 and T3 "feed-back" to the hypothalamus and pituitary to shut off the secretion of TRH and TSH respectively.

Triiodothyronine (T3) Suppression Test

The T3 suppression test operates on the principle that administration of relatively high doses of exogenous T3 will suppress (lower) the secretion of pituitary TSH in normal cats. Such inhibition of pituitary TSH secretion by high circulating concentrations of thyroid hormone is a characteristic feature of normal hypothalamic-pituitary-thyroid regulation (see Figure 1).  The fall in circulating TSH levels results in lowered T4 secretion from the normal feline thyroid gland (see Figure 2, below, left panel).

Because resting serum TSH concentrations are already very low in normal cats (see my previous post on serum TSH measurements in cats), it would not be possible to use TSH measurement as an endpoint for interpretation of this test.

Figure 2: T3 suppression tests in normal cats (left) & cats with hyperthyroidism (right)

In contrast to the situation in normal cats, when thyroid function is autonomous — as it is in hyperthyroid cats, administration of large doses of exogenous T3 has little or no effect on T4 secretion. The reason for this that pituitary TSH secretion has already been completely suppressed by the higher-than-normal thyroid hormone secretion characteristic of hyperthyroidism (see Figure 2 above, right panel).

Testing Protocol for the T3 Suppression Test

To perform the T3 suppression test in cats, the following protocol is recommended:

• One day 1, the veterinarian draws a blood sample is drawn for determination of baseline serum concentrations of total T4 and T3. This serum sample is not yet submitted to the laboratory but kept refrigerated (or frozen) until day 4.
• Owners are instructed to give 7 doses of a T3 pill (liothyronine sodium; Cytomel) to their cat, beginning on the following morning.
• On day 2 and 3, the owners administer the liothyronine at a dosage of 25 µg every 8 hours for 2 days (6 doses). 
• On the morning of day 4, a seventh 25-µg dose of liothyronine is given and the cat returned to the veterinary clinic within 2 to 4 hours.
• The veterinarian again draws a blood sample for serum T4 and T3 determinations. 
• Both the basal (day 1) and post-liothyronine (day 4) serum samples are submitted to the laboratory together to eliminate the effect of between assay variation in hormone concentrations.

Interpretation of Results of T3 Suppression Testing

When the T3 suppression test is performed in normal cats and sick cats without hyperthyroidism, there is a marked fall in serum T4 concentrations after exogenous T3 administration (Figures 2 and 3). In contrast, when the test is performed in cats with hyperthyroidism, even in cats with only slightly high or high-normal resting serum T4 concentrations, minimal, if any, suppression of serum T4 concentrations is seen.

Figure 3: Box plots of the serum T4 concentrations before (A) and after (B) administration of liothyronine to 44 normal cats, 77 cats with hyperthyroidism, and 22 cats with nonthyroidal disease (from data in Reference no. 4). Data is plotted as box plots, in which the "box" represents the interquartile range from the 25th to 75th percentile (represents the middle one-half of the data). The horizontal bar through the box is the median. The "whiskers" represent the main body of data, which in most cases is equal to the range. Outlying data points are represented by open circles. To convert serum T4 concentrations from nmol/L to µg/dl, divide the given values by 12.87.

Regarding interpretation of T3 suppression test results, we find that the absolute serum T4 concentration after liothyronine administration is the best means of distinguishing hyperthyroid cats from normal cats or cats with nonthyroidal disease. Cats with hyperthyroidism have post-liothyronine serum T4 values greater than 20 nmol/L (greater than 1.5 μg/dl), whereas normal cats and cats with nonthyroidal disease have T4 values less than 20 nmol/L (Figure 3B). There may be a great deal of overlap of the per cent decrease in serum T4 concentrations after liothyronine administration between the three groups of cats, but suppression of 50 per cent or more only occurs in cats without hyperthyroidism.

Serum T3 concentrations, as part of the T3 suppression test, are not useful in the diagnosis of hyperthyroidism per se. However, these basal and post-liothyronine serum T3 determinations can be used to monitor owner compliance with giving the drug. If inadequate T4 suppression is found, but serum T3 values do not increase after treatment with liothyronine, problems with owner compliance should be suspected and the test result considered questionable.

Disadvantages of the T3 Suppression Test

Overall, the T3 suppression test is very useful for diagnosis of mild hyperthyroidism in cats, but the test does come with disadvantages:

1. T3 suppression testing is a relatively long test (3 days)
2. Owners are required to give multiple doses of liothyronine
3. Cats must swallow the tablets if the test is going to be meaningful. 

If the liothyronine is not administered properly or the cat does not swallow the liothyronine tablet, circulating T3 concentrations will not rise to decrease pituitary TSH secretion, and the serum T4 value will not be suppressed, even if the pituitary-thyroid axis is normal. Failure of a cat to ingest the liothyronine could result in a false-positive diagnosis of hyperthyroidism in a normal cat or cat with nonthyroidal disease.

References:

1. Graves TK, Peterson ME: Occult hyperthyroidism in cats. In Kirk RW and Bonagura JD (eds): Current Veterinary Therapy XI, pp 334-337. Philadelphia, WB Saunders Co, 1992.
2. Peterson ME, Melián C, Nichols CE: Measurement of serum concentrations of total and free T4 in hyperthyroid cats and cats with nonthyroidal disease. Journal of Veterinary Internal Medicine 1998;12:211.
3. Utiger RD: Tests of thyroregulatory mechanisms. In Ingbar SH and Braverman LE (eds): The Thyroid: A Fundamental and Clinical Text, pp 511-523. Philadelphia, JB Lippincott, 1986.
4. Peterson ME, Graves TK, Gamble DA: Triiodothyronine (T3) suppression test. An aid in the diagnosis of mild hyperthyroidism in cats. Journal of Veterinary Internal Medicine 1990;4:233-238.
5. Refsal KR, Nachreiner RF, Stein BE, et al: Use of the triiodothyronine suppression test for diagnosis of hyperthyroidism in ill cats that have serum concentration of iodothyronines within normal range. J Am Vet Med Assoc 1991;199:1594-1601.
6. Peterson ME. Diagnostic tests for hyperthyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:2-9.
7. Peterson ME: Diagnostic testing for feline hyper- and hypothyroidism. Proceedings of the 2011 American College of Veterinary Internal Medicine (ACVIM) Forum. pp. 95-97, 2011
8. Peterson ME: Hyperthyroidism in cats, In: Rand, J (ed), Clinical Endocrinology of Companion Animals. New York, Wiley-Blackwell, in press.

Confirming the Diagnosis of Hyperthyroidism in Cats: Serum TSH Concentrations

Measuring TSH in Human Patients with Thyroid Disease

In human patients, measurement of circulating thyroid stimulating hormone (TSH; also called thyrotropin) is commonly used as a front-line test of thyroid function. This hormone is secreted by the pituitary gland and, as its name suggests, acts to stimulate the thyroid gland to secrete T4 and T3.

The pituitary gland constantly monitors the circulating levels of T4 and T3, and if it senses the slightest increase in serum thyroid hormone concentrations, it stops producing TSH. In contrast, if the pituitary senses even a slight decrease in circulating T4 and T4 concentrations, it increases the secretion of TSH in an attempt to increase thyroid hormone levels back to normal.

Consequently, the finding of a low to undetectable blood TSH value in a human patient is diagnostic for hyperthyroidism, whereas a high serum TSH concentration is diagnostic for hypothyroidism. When testing human patients, the finding of a normal TSH result excludes both hypo- or hyperthyroidism.

TSH Concentrations as a Diagnostic Test for Cats

A species-specific feline TSH assay has not yet been developed for use in cats, and human TSH assays cannot be used to measure feline TSH. However, assays for measuring canine TSH (cTSH) are widely available, and studies have investigated the use of cTSH measurements as a diagnostic test for cats with suspected thyroid dysfunction — both hyperthyroidism or hypothyroidism (1-3).

However, there are many problems with the use of the cTSH assay, both in dogs as well as in cats.

First Problem: The first issue or problem with the use of the canine TSH assay is that this test, although first developed in 1997 (now 14 years ago), is still a considered "first generation" assay. Overall, this assay is not considered very reliable for the following reasons:

• It has a diagnostic test sensitivity of only 60-75% in canine hypothyroidism, a very common endocrine problem in dog (4-6). Because 25% to 40% of dogs with confirmed hypothyroidism do not have the high serum TSH concentrations (> 0.6 ng/ml) that we expect to find with thyroid failure, it's possible that some isoforms of the TSH hormone are not being detected with this assay.
• In addition to this high incidence of false-negative results, cTSH levels appear to be falsely-high in 10% to 20% of dogs with normal thyroid function (4). Some of these dogs have other nonthyroidal illnesses; however, falsely high serum cTSH values have even been reported in clinically normal dogs that had completely normal total and free T4 concentrations.
• The high prevalence of false-negative and false-positive test results make this assay unreliable to use as a sole diagnostic test for dogs with suspected hypothyroidism.

Second Problem: It is important to remember that the current canine TSH assays only detects approximately 35% of the circulating feline TSH. In other words, the current cTSH assay does not completely cross-react with feline TSH; therefore, the assay is not measuring the total amount of TSH present in the cat's serum.

This poor cross-reactivity of feline TSH in the canine assay explains why the upper limit of the reference range for TSH is so much lower in cats (0.15-0.3 ng/ml) than it is in dogs (0.5-0.6 ng/ml). Again, the cTSH assay is only measuring about a third of the feline TSH present in the circulation.

Third Problem: A major problem with the canine TSH assay, being a first generation assay, is that its detection limit (assay sensitivity) is not very good.  In fact, it's really poor! Therefore, this assay does not perform well when we are trying to measure very low concentrations of TSH.

In cats this issue is again made even worse by the fact that only 35% of the cat's TSH will be detected by the cTSH assay. As a consequence of this poor cross-reactivity, the assay sensitivity, which is already considered poor at 0.03 ng/ml in the dog, equates to only 0.10 ng/ml of TSH in the cat (7).  With such a "high" detection limit, we cannot reliably distinguish a normal TSH concentration from a low or undetectable value in cats with this cTSH assay. In other words, this assay just cannot accurately measure low enough to distinguish between normal and low feline TSH concentrations.

All human TSH assays currently used are second or even third generation assays. Like the cTSH assays, the first generation human TSH assays were also unable to distinguish low-normal from low TSH concentrations. The major advantages of the second to third generation TSH assays is their 10- to 100-fold improvement in assay sensitivity (8); this much lower detection limit greatly improves their ability to accurately distinguish between normal and even partially suppressed TSH results.

Hopefully, we will also be moving on to a second generation of TSH assays for dogs (and cats), which should improve the sensitivity (detection limit) of the assay. This will also help make this a better test for hyperthyroidism, as well as hypothyroidism in cats.

Using TSH Concentrations as a Diagnostic Test for Hyperthyroidism in Cats

Of course, the poor detection limit of the current cTSH assay represents a major issue in cats with hyperthyroidism, where low suppressed values are expected. In one of the best studies of cTSH concentrations in cats (2), all of the hyperthyroid cats tested had cTSH concentrations at or below the limit of detection of the assay (0.03 ng/ml).  However, of the 40 cats without hyperthyroidism tested in that same study, 5 cats also had undetectable levels of TSH, indistinguishable from the values in the hyperthyroid cats (see Figure below).

TSH values in normal cats, hyperthyroid cats, and cats with chronic kidney disease. Notice that  all hyperthyroid cats have very low TSH concentrations. However, there is much overlap between the TSH values in the hyperthyroid cats and the values in the cats without hyperthyroidism. Modified from data in reference 2.

Obviously, a better TSH assay for feline hyperthyroidism is needed— specifically, a feline-specific TSH assay that has adequate sensitivity to reliably distinguish a normal value from a low one. However until better TSH assays for cats are available, caution is advised in over interpreting values in cats since can be so difficult to distinguish normal values from the suppressed values expected in cats with hyperthyroidism. Perhaps the only use for TSH measurements using the cTSH assay would be to exclude hyperthyroidism, i.e., finding a mid- to high-normal value rather than a suppressed value (3).

Using TSH Concentrations as a Diagnostic Test for Hypothyroidism in Cats

At this time, the major use for the current cTSH assay is as a diagnostic test for hypothyroidism, where the low circulating thyroid hormone concentrations sensed by the pituitary gland leads to high serum TSH values. In accord with that, high cTSH values have been reported in cats with naturally occurring hypothyroidism, as well as cats with iatrogenic hypothyroidism, i.e., secondary to methimazole or radioiodine treatment (9).

Normal cats and cats with nonthyroidal illness generally maintain normal values for serum TSH. Therefore, the finding of a low total or free T4 in combination with a high TSH concentration greatly improves the diagnostic sensitivity for hypothyroidism in cats.

References:

1. Greco DS. Diagnosis of congenital and adult-onset hypothyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:40-44
2. Wakeling J, Moore K, Elliott J, et al. Diagnosis of hyperthyroidism in cats with mild chronic kidney disease. Journal of Small Animal Practice 2008;49:287-294.
3. Wakeling J. Use of thyroid stimulating hormone (TSH) in cats. Canadian Veterinary Journal 2010;51:33-34.
4. Peterson ME, Melian C, Nichols R. Measurement of serum total thyroxine, triiodothyronine, free thyroxine, and thyrotropin concentrations for diagnosis of hypothyroidism in dogs. Journal of the American Veterinary Medical Association 1997;211:1396-1402.
5. Scott-Moncrieff JC, Nelson RW, Bruner JM, et al. Comparison of serum concentrations of thyroid-stimulating hormone in healthy dogs, hypothyroid dogs, and euthyroid dogs with concurrent disease. Journal of the American Veterinary Medical Association 1998;212:387-391.
6. Dixon RM, Mooney CT. Evaluation of serum free thyroxine and thyrotropin concentrations in the diagnosis of canine hypothyroidism. Journal of Small Animal Practice 1999;40:72-78.
7. Ferguson DC, Caaffall Z, Hoenig M. Obesity increases free thyroxine proportionally to nonesterified fatty acid concentrations in adult neutered female cats. Journal of Endocrinology 2007;194:267-273.
8. Dunlap DB. Thyroid Function Tests. In: Walker HK, Hall WD, Hurst JW (eds). Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd ed. Boston, 1990.
9. Baral R, Peterson ME: Thyroid Diseases, In: Little, S. (ed), The Cat: Clinical Medicine and Management. Philadelphia, Elsevier Saunders, in press.

Confirming the Diagnosis of Hyperthyroidism in Cats: Serum Free T3 Concentrations

As I discussed in my recent posts on diagnostic testing for hyperthyroidism, the feline thyroid gland makes two active thyroid hormones, thyroxine (T4) and triiodothyronine (T3). T4 makes up 90% of the circulating thyroid hormones, while T3 makes up less than 10%. In cats, all circulating T4 originates from thyroid secretion but almost all T3 in the cat is produced extrathyroidally from T4 deiodination (1).

Although only 10% of the circulating thyroid hormone is T3, this thyroid hormone is 3-10 times more active than T4; T4 can be considered a prohormone whose main function is only to be converted into active T3. In order for the body’s cells and tissues to use more of this active form of thyroid hormone, T4 (which contains 4 iodine molecules) is converted to T3 (which contains 3 iodine molecules) by losing an iodine molecule. This function is performed in peripheral tissues (such as the liver and kidney) by deiodinases (enzymes that act to remove an iodine group from the thyroid hormone molecule). See my previous blog post on total T3 for more information.

Despite the fact that T3 is more potent than T4, use of total T3 concentrations are not a good diagnostic test for hyperthyroidism in cats. Over 30% of hyperthyroid cats have a normal serum T3 concentration even when they have clearly high total and free T4 values (2-4).

But what about free T3 concentrations? Would measuring free T3 be a better indicator of what's happening at this tissue level?

Physiology of T3, Total T3, and Free T3 in the Body

What's the difference between total and free T3? It's the same situation as with total T4 and free T4, which I discussed in a recent post.

When we measure a serum T3 level, we are checking the total amount of T3 hormone circulating in the blood—both the bound and unbound T3 molecules (5). More than 99% of T3 hormone in the circulation is “bound,” meaning that it is attached to thyroid-binding proteins in the bloodstream. When bound, this T3-thyroid binding protein complex is too large to enter the body's tissues (see Figure).

For circulating T3 to do its functions and regulate metabolism, the hormone must first break loose from its binding proteins (i.e., become “free” T3) in order to leave the bloodstream and enter the body’s tissues and cells. Only then can intracellular free T3 have its effect on the body’s metabolism (see Figure).

Total T3 circulates mostly bound to thyroid binding protein, with a small proportion  being unbound or "free." It is only the free T3 that can pass into the tissues and cells to complete its functions to regulate metabolism.

Serum free T3 represents the tiny fraction (less than 1%) of T3 hormone that is unbound and therefore is biologically active. It is important to realize that a dynamic equilibrium exists between free and protein-bound T3 that depends on the amount of thyroid-binding proteins in the blood stream, as well as the affinity of the thyroid binding proteins for T3. In other words, a T3 molecule circulating in the bloodstream may be free (unbound) one minute, protein-bound the next, and free again shortly thereafter.

Again, it is only the free T3 portion of the total T3 measured in the blood that can pass into the cells and act on the body’s tissues to influence metabolism (see Figure).

Serum Free T3 Concentration as a Diagnostic Test in Hyperthyroidism

The use of total T3, as noted above, is not a very useful diagnostic test for hyperthyroidism in cats (2-4). Similarly, total T3 are rarely useful in the routine diagnosis of human patients with hyperthyroidism either (6-8). Rarely, an occasional human patient with hyperthyroidism will develop a syndrome called "T3 toxicosis" in which circulating total and free T4 concentrations remain normal but serum concentrations of total and free T3 are very high (6-8). Such a syndrome of T3 hyperthyroidism has never been reported and does not appear to develop in cats (2-4).

In hyperthyroidism, the increases in free T4 and free T3 concentrations are usually more marked than the increases in total hormone concentrations. In human patients, progressive increases in serum total T4 can eventually exceed the limited binding capacity of thyroid binding globulin, the major serum binding protein for T4 and T3 in people (5), This leads to disproportionate increases in the free serum concentrations of T4 (9) and T3 (10). Although similar studies have not been reported in cats, similar findings could be expected.

The questions remains — would determination of free T3 be of any added benefit over the use of T4 and free T4 concentrations for diagnosis of hyperthyroidism in cats? No one knows the answer for sure, primarily due to the fact that most diagnostic laboratories do not offer free T4 measurements. However, one large diagnostic laboratory, the Michigan State University's Diagnostic Center for Population and Animal Health (DCPAH) does offer complete feline thyroid profiles that include T4, T3, free T4, and free T3). Click this link to see their lab submission form.

Recently, I called Dr, Kent Refsal, an endocrinologist who has worked at the DCPAH for many years and a leading expert in this field of diagnostic endocrinology. I asked Dr. Refsal if he had any evidence that adding free T3 test as part of the "Feline Thyroid Profile" increased the diagnostic accuracy of their thyroid panel for cats with hyperthyroidism. The short answer is no — adding free T3 might help in other situations, such as monitoring thyroid hormone replacement, but it does NOT appear to add any diagnostic advantage of the use of total and free T4 concentrations in cats.

Bottom Line: Free T3 determinations, at least by themselves, do not appear to be a useful test for hyperthyroidism in cats.

References: 

1. Foster DJ, Thoday KL, Beckett GJ. Thyroid hormone deiodination in the domestic cat. Journal of Molecular Endocrinology 2000;24:119-126.
2. Peterson ME, Melian C, Nichols R. Measurement of serum concentrations of free thyroxine, total thyroxine, and total triiodothyronine in cats with hyperthyroidism and cats with nonthyroidal disease. Journal of the American Veterinary Medical Association 2001;218:529-536.
3. Peterson ME. Diagnostic tests for hyperthyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:2-9.
4. Peterson ME: Diagnostic testing for feline hyper- and hypothyroidism. Proceedings of the 2011 American College of Veterinary Internal Medicine (ACVIM) Forum. pp. 95-97, 2011
5. Stockigt JR. Free thyroid hormone measurement. A critical appraisal. Endocrinology and metabolism clinics of North America 2001;30:265-289.
6. Dunlap DB. Thyroid Function Tests. In: Walker HK, Hall WD, Hurst JW (eds). Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd ed. Boston, 1990.
7. Klee GG. Clinical usage recommendations and analytic performance goals for total and free triiodothyronine measurements. Clinical Chemistry 1996;42:155-159.
8. Sapin R, Schlienger JL. Thyroxine (T4) and tri-iodothyronine (T3) determinations: techniques and value in the assessment of thyroid function. Annales de Biologie Clinique 2003;61:411-420.
9. Inada M, Sterling K. Thyroxine transport in thyrotoxicosis and hypothyroidism. The Journal of Clinical Investigation 1967;46:1442-1450.
10. Nauman JA, Nauman A, Werner SC. Total and free triiodothyronine in human serum. The Journal of Clinical Investigation 1967;46:1346-1355.

Confirming the Diagnosis of Hyperthyroidism in Cats: Serum Free T4 (Part 2)

As I discussed in a recent blog post, determining a cat's serum free T4 concentration is a very useful test in confirming the diagnosis of hyperthyroidism, especially when run together with a total T4 concentration (1-6).

Physiology of T4, Total T4, and Free T4 in the Body

Again, what's the difference between total and free T4? When we measure a serum T4 level, we are checking the total amount of T4 hormone circulating in the blood—both the bound and unbound T4 molecules.

More than 99% of T4 hormone in the circulation is “bound,” meaning that it is attached to thyroid-binding proteins in the bloodstream. When bound, this T4-thyroid binding protein complex is too large to enter the body's tissues (Figure 1).

For circulating T4 to do its functions and regulate metabolism, the hormone must first break loose from its binding proteins (i.e., become “free” T4) in order to leave the bloodstream and enter the body’s tissues and cells. Only then can intracellular free T4 be converted to T3 to have its effect on the body’s metabolism (Figure 1).

Serum free T4 represents the tiny fraction (less than 0.1%) of thyroxine hormone that is unbound and therefore is biologically active. It is important to realize that a dynamic equilibrium exists between free and protein-bound T4 that depends on the amount of thyroid-binding proteins in the blood stream, as well as the affinity of the thyroid binding proteins for T4. In other words, a T4 molecule circulating in the bloodstream may be free (unbound) one minute, protein-bound the next, and free again shortly thereafter.

Again, it is only the free T4 portion of the total T4 measured in the blood that can pass into the cells and act on the body’s tissues to influence metabolism (Figure 1).

Figure 1: T4 circulates mostly bound to thyroid binding proteins in the circulation. This bound T4 exists in a dynamic equilibrium with unbound or free T4, which accounts for less than 1% of the total T4 in the circulation. Only the free, nonprotein-bound moiety is able to pass into the body's cells, where it can be converted into T3, the most active thyroid hormone

Measuring Serum T4, T3, and Free T4 in Cats with Mild Hyperthyroidism

When we measure a serum free T4 concentration, we are checking only the free or unbound portion of the T4 hormone circulating in the blood. Since changes in the concentrations (or binding affinity) of the thyroid binding proteins does not affect the free T4 levels, this test is considered a more accurate test of true thyroid activity than determination of serum total T4.

Free T4 is much less likely to be influenced by nonthyroidal illness or drugs. However, the tests biggest advantage appears to be a more sensitive test for diagnosis of early or mild hyperthyroidism.

As might be expected, the free T4 test is a more sensitive diagnostic test for feline hyperthyroidism than is determination of the total T4 concentration.

In our study of 917 hyperthyroid cats (3), we found that determination of serum free T4 was diagnostic in 98.5% of the cases (for more information, see Figure 1 in my last blog post). This ability to confirm hyperthyroidism in cats with the disease was higher than the total T4 concentration, which was diagnostic in 91% of cats or T3, which was diagnostic on only 67%.

However, when we looked at the cats with mild hyperthyroidism in that study (3), the advantage of free T4 over of total T4 or T3 measurements was much more striking (Figure 2).

In that subgroup of 205 cats with mild hyperthyroidism, we found that determination of serum total T4 was diagnostic in 61% of the cases (left panel, yellow boxed data). Use of serum T3 concentrations were of almost no value in cats with mild disease, since they were diagnostic in only 20% of the cases (middle panel, blue boxed data). However, despite the poor test sensitivities of T4 and T3 measurements, determination of free T4 was still diagnostic for hyperthyroidism in 91% of these cats with mild hyperthyroidism (right panel, purple boxed data).

Figure 2: Box plots of serum total T4, T3, and free T4 concentrations in 205 cats with mild hyperthyroidism, defined as a serum T4 less than 5 μg/dl (66 nmol/L). The T-bars represent the main body of data. The box represents the interquartile range (25th percentile to 75th percentile or middle half of the data). The horizontal bar in the box is the median or 50% percentile. Outlying data points are represented by open circles. The green shaded area represents the reference interval (normal range). From reference 3.

Disadvantages of Free T4 as a Diagnostic Test

Although the free T4 is a very sensitive diagnostic, the main problem with free T4 assays is that the test is less specific than the total T4 value. In other words, many cats suffering from other illnesses NOT associated with hyperthyroidism can have false-positive results with the free T4 test (for more information, see this blog post).

In contrast, cats with nonthyroidal illness will never have high total T4 values. Rather, the will have corresponding total T4 values in the low-normal or subnormal range. For this reason, the total T4 test remains the diagnostic test of choice for cats with suspected hyperthyroidism because we almost never see false-positive results with the total T4 test. Measurement of free T4 alone can never be used to make a reliable diagnosis because of the chance that the result is not accurate.

However, in cats in which hyperthyroidism is suspected based on clinical features (e.g., weight loss despite a good appetite, palpable thyroid tumor), the finding of a high free T4 concentration can be considered diagnostic. This is especially true when the total T4 values are in the upper half of the reference range (see Figure 2: left panel, yellow boxed data) and all other diseases have been excluded. 

If the corresponding total T4 is in the lower half of the reference range, however, nonthyroidal disease is very likely. In our study (3), all 5 of the cats that had total T4 concentrations in the low-normal range had severe concurrent illnesses (see Figure 2: left panel, yellow boxed data).

References:

1. Baral R, Peterson ME: Thyroid Diseases, In: Little, S. (ed), The Cat: Clinical Medicine and Management. Philadelphia, Elsevier Saunders, in press.
2. Graves TK, Peterson ME. Diagnostic tests for feline hyperthyroidism. The Veterinary Clinics of North America: Small Animal Practice 1994;24:567-576.
3. Peterson ME, Melian C, Nichols R. Measurement of serum concentrations of free thyroxine, total thyroxine, and total triiodothyronine in cats with hyperthyroidism and cats with nonthyroidal disease. Journal of the American Veterinary Medical Association 2001;218:529-536.
4. Peterson ME. Diagnostic tests for hyperthyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:2-9.
5. Peterson ME: Diagnostic testing for feline hyper- and hypothyroidism. Proceedings of the 2011 American College of Veterinary Internal Medicine (ACVIM) Forum. pp. 95-97, 2011
6. Peterson ME: Hyperthyroidism in cats, In: Rand, J (ed), Clinical Endocrinology of Companion Animals. New York, Wiley-Blackwell, in press.

Confirming the Diagnosis of Hyperthyroidism in Cats: Serum Free T4 Concentrations

Thyroxine (T4) is the main thyroid hormone secreted by the thyroid gland. As discussed in my recent post about T4 measurement, checking a cat’s serum T4 level is a very good screening test for hyperthyroidism (1-6). Over 90% of hyperthyroid cats will have a high serum T4 concentration, thereby confirming the diagnosis.

When we measure a serum T4 level, it is important to understand that we are checking the total amount of T4 hormone circulating in the blood—both the bound and unbound T4 molecules. More than 99% of T4 hormone is “bound,” meaning that it attaches to proteins in the blood. For circulating T4 to do its functions and regulate metabolism, the hormone must first break loose from its binding proteins (i.e., become “free” T4) in order to leave the bloodstream and enter the body’s tissues and cells. Only then can intracellular free T4 have its effect on the body’s metabolism.

Therefore, measuring a total T4 concentration by itself can sometimes be misleading, because the total T4 is affected by changes in the amount of circulating thyroid binding proteins or the binding affinity of these proteins for thyroid hormone. Such changes in the binding of T4 can occur in cats (and dogs) treated with certain drugs (e.g., glucocorticoids), as well as with many nonthyroidal illnesses (e.g., kidney disease, liver, disease, intestinal disease, or cancer).

In hyperthyroid cats, administration of drugs or other concurrent illness can lower the total T4 concentration. In some hyperthyroid cats, the high serum T4 concentration may be suppressed enough to actually fall into the normal range. In this scenario, use of a total T4 determination would fail to diagnose hyperthyroidism. In addition, some cats with mild or early hyperthyroidism will also have normal serum T4 concentrations and can be difficult to diagnose.

Physiology of Free T4 in the Body

Serum free T4 represents the tiny fraction (less than 0.1%) of thyroxine hormone that is unbound and therefore is biologically active. Again, it is only the free T4 portion of the total T4 measured in the blood that can pass into the cells and act on the body’s tissues to influence metabolism.

When we measure a serum free T4 concentration, we are checking only the free or unbound portion of the T4 hormone circulating in the blood. Since changes in the thyroid binding proteins in the blood do not affect the free T4 levels, this test is considered a more accurate test of true thyroid activity than determination of serum total T4. Free T4 is much less likely to be influenced by nonthyroidal illness or drugs. And finally, it appears to be a more sensitive test for diagnosis of early or mild hyperthyroidism.

Measuring the Serum Thyroxine (T4) Concentration: Advantages as a Diagnostic Test  

As might be expected, the free T4 test is a more sensitive diagnostic test for feline hyperthyroidism than is determination of the total T4 concentration.

In our study of 917 hyperthyroid cats (3), we found that determination of serum free T4 was diagnostic in 98.5% of the cases (see Figure below; middle blue boxed data). This ability to confirm hyperthyroidism in cats with the disease was higher than the total T4 concentration, which was diagnostic in 91% of cats (see Figure 3 in my previous blog on T4 measurement).

The free T4 was high in most cats with mild or early hyperthyroidism, even when the total T4 was within the established normal range. In addition, in hyperthyroid cats with concurrent diseases or in those treated with drugs, the free T4 is not suppressed and remains high even when the total T4 has been suppressed. Therefore, use of the free T4 is a very useful diagnostic test, especially in hyperthyroid cats in which total T4 values are within reference range limits.

Figure 1: Box plots of Free T4 concentrations in 172 clinically normal cats, 917 cats with hyperthyroidism, and 221 cats with nonthyroidal disease. The T-bars represent the main body of data. The box represents the interquartile range (25th percentile to 75th percentile range or the middle half of the data). The horizontal bar in the box is the median. Outlying data points are represented by open circles. The shaded area indicates the reference interval (normal range). From reference 3.

Measuring the Serum Thyroxine (T4) Concentration: Disadvantages as a Diagnostic Test  

So if determination of the free T4 level is more diagnostic, why bother with running the total T4 at all? Why not measure just measure free T4 concentrations as the main diagnostic test for hyperthyroidism?

It turns out that there are 2 good reasons why free T4 concentrations cannot replace total T4 as the main diagnostic test for hyperthyroid cats.

First of all, although the free T4 is a very sensitive diagnostic, the main problem with free T4 assays is that the test is less specific than the total T4 value. In other words, many cats suffering from other illnesses NOT associated with hyperthyroidism can have false-positive results with the free T4 test (see Figure above; right purple boxed data).

Of the 221 cats with nonthyroidal disease in my study, 14 cats (6.3%) had a falsely high free T4 concentration (see Figure above; right purple boxed data; Notice all of the outlying data points above the normal range). Therefore, calculated specificity of measuring serum free T4 concentration as a diagnostic test for hyperthyroidism was significantly lower than that of the total T4 concentration.

Remember that these cats with nonthyroidal illness will NEVER have high total T4 values — rather, as expected, the have corresponding total T4 values in the low-normal or subnormal range (see Figure 3 in my previous blog on T4 measurement).

For this reason, the total T4 test remains the diagnostic test of choice for cats with suspected hyperthyroidism because we almost never see false-positive results with the total T4 test. Measurement of free T4 alone can never be used to make a reliable diagnosis because of the chance that the result is not accurate.

The second reason why free T4 concentrations should not be used as the initial screening test for hyperthyroidism is that the free T4 test is generally about 2 to 3 times as expensive as a T4 alone.

While most endocrinologists favor the standard equilibrium dialysis radioimmunoassay method for measuring free T4, newer analog and chemiluminescence assays now offer alternative and accurate methodologies. These new assays are also faster to run and are less costly.

No matter how free T4 is determined, however, the free T4 test must be run with a total T4, adding to the expense of diagnostic testing for hyperthyroid cats.

Bottom Line:  Caution is advised in using serum free T4 measurements as the sole diagnostic test for hyperthyroidism. This test is associated with a high rate of false-positive results in sick cats without hyperthyroidism. This test should never be run by itself but it is more reliable if interpreted with a corresponding total T4 value:

• High-normal total and free T4 concentrations are generally consistent with hyperthyroidism (especially is clinical features of disease present— i.e., thyroid nodule, weight loss despite good appetite)
• Low to low-normal total T4 concentrations together with a high free T4 are usually associated with non-thyroidal illness

References:

1. Baral R, Peterson ME: Thyroid Diseases, In: Little, S. (ed), The Cat: Clinical Medicine and Management. Philadelphia, Elsevier Saunders, in press.
2. Graves TK, Peterson ME. Diagnostic tests for feline hyperthyroidism. The Veterinary Clinics of North America: Small Animal Practice 1994;24:567-576.
3. Peterson ME, Melian C, Nichols R. Measurement of serum concentrations of free thyroxine, total thyroxine, and total triiodothyronine in cats with hyperthyroidism and cats with nonthyroidal disease. Journal of the American Veterinary Medical Association 2001;218:529-536.
4. Peterson ME. Diagnostic tests for hyperthyroidism in cats. Clinical Techniques in Small Animal Practice 2006;21:2-9.
5. Peterson ME: Diagnostic testing for feline hyper- and hypothyroidism. Proceedings of the 2011 American College of Veterinary Internal Medicine (ACVIM) Forum. pp. 95-97, 2011
6. Peterson ME: Hyperthyroidism in cats, In: Rand, J (ed), Clinical Endocrinology of Companion Animals. New York, Wiley-Blackwell, in press.
7. Peterson ME, Broome MR, Robertson J: Accuracy of serum free thyroxine concentrations determined by a new veterinary chemiluminescent immunoassay in euthyroid and hyperthyroid cats. Proceedings of 2011 European College of Veterinary Internal Medicine (in press)