Canine Endocrine Testing: Interpretation and Diagnostic Strategy

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Key Takeaways

• Endocrine testing in dogs requires a structured, pretest probability-driven approach, as hormone concentrations are dynamic and influenced by stress, illness, and medication, leading to potential misinterpretation. Initial screening tests like total T4 for hypothyroidism and urine cortisol:creatinine ratio for hypercortisolism are crucial, but require confirmatory testing due to their limitations in specificity.
• Hypothyroidism diagnosis relies on integrating total T4, free T4 by dialysis, and canine TSH results with clinical signs; a low total T4 alone is insufficient and can be affected by non-thyroidal illness or drug administration. Free T4 by dialysis is less influenced by binding protein changes common in sick dogs, making it a more reliable confirmatory test.
• Hypercortisolism diagnosis involves confirming excess cortisol production, often with a low-dose dexamethasone suppression test, followed by differentiation between pituitary-dependent disease and adrenal neoplasia using endogenous ACTH concentration or high-dose dexamethasone suppression. Ultrasonography of the adrenal glands provides complementary anatomical information.
• Hypoadrenocorticism diagnosis is definitively established by an ACTH stimulation test, which assesses the adrenal glands' reserve capacity; a normal baseline cortisol effectively excludes the disease, but a low baseline necessitates further stimulation testing. A low sodium-to-potassium ratio is a supportive but not diagnostic indicator.
• Monitoring treated endocrine diseases involves specific parameters: total T4 4-6 hours post-levothyroxine for hypothyroidism, ACTH stimulation tests for trilostane-treated hypercortisolism, and electrolyte concentrations for hypoadrenocorticism, all balanced with clinical assessment.
• Assay interference from hemolysis, lipemia, or hyperbilirubinemia, along with preanalytical errors like delayed sample processing, are common failure modes in endocrine testing, necessitating careful sample handling and communication with the diagnostic laboratory.

Endocrine testing in dogs is a core component of internal medicine practice, yet it carries a higher risk of misinterpretation than most other laboratory disciplines. Hormone concentrations fluctuate with stress, concurrent illness, drug administration, and circadian rhythm, and no single test provides a perfect sensitivity and specificity pair for any canine endocrinopathy. This article provides a framework for selecting tests, interpreting results in context, and avoiding the diagnostic traps that produce false positives and false negatives. It is written for the practicing veterinarian who needs a practical, reasoning-based approach instead of a catalogue of assays.

The central question this article answers is straightforward: when a dog presents with polyuria, polydipsia, alopecia, weakness, or weight loss, which endocrine tests should be run, in what order, and how should ambiguous results be handled? The answer depends on pretest probability, the availability of assays, and the recognition that endocrine diagnosis is often a process of exclusion and confirmation instead of a single laboratory event. The sections that follow cover the physiological basis of hormone testing, the major adrenal and thyroid disorders, and the interpretation strategies that distinguish disease from laboratory artefact.

At a Glance

Physiological Basis of Endocrine Testing

Endocrine assays measure static hormone concentrations or dynamic responses to stimulation or suppression. The interpretation of any result depends on understanding the feedback loops that govern hormone secretion. The hypothalamic-pituitary-adrenal axis and the hypothalamic-pituitary-thyroid axis both operate on negative feedback principles, and disruption at any level produces characteriztic patterns on testing.

For the adrenal axis, corticotropin-releasing hormone from the hypothalamus stimulates pituitary secretion of ACTH, which in turn drives cortisol production from the zona fasciculata and zona reticularis. Cortisol then suppresses further release of both CRH and ACTH. Disease can arise from the pituitary, the adrenal glands, or exogenous glucocorticoid administration, and each produces a distinct biochemical signature. For the thyroid axis, thyrotropin-releasing hormone stimulates pituitary TSH release, which drives thyroxine production. Circulating T4 and triiodothyronine suppress TSH secretion. Primary thyroid failure therefore produces low T4 with high TSH, while pituitary or hypothalamic disease produces low T4 with low or inappropriately normal TSH.

The diagnostic laboratory serves as the arbiter of these pathways, but the quality of the result depends on preanalytical factors. Sample handling, assay methodology, and the reference population all influence interpretation. A testing laboratory perspective on canine thyroid evaluation emphasizes that each assay has strengths and weaknesses and that both false positive and false negative results are possible with every test. This reality underpins the interpretive approach taken throughout this article.

Assay Principles and Limitations

Immunoassays remain the standard platform for veterinary endocrine testing. These assays use antibodies directed against the hormone of interest, with detection systems that vary from radioisotopic to chemiluminescent. The choice of platform matters because assay performance differs between manufacturers, and reference intervals are not transferable between laboratories.

Free hormone measurements, such as free T4 by equilibrium dialysis, are technically more demanding than total hormone measurements. The dialysis step separates protein-bound hormone from the free fraction, which is the biologically active component. This method is less influenced by changes in binding protein concentrations, a feature that becomes clinically relevant in dogs with non-thyroidal illness. The trade-off is cost and turnaround time, which is why total T4 remains the initial screening test in most practices.

Dynamic testing, whether stimulation or suppression, adds information that static measurements cannot provide. Stimulation tests assess the functional reserve of an endocrine gland, while suppression tests assess the integrity of negative feedback. Both approaches require attention to timing, drug preparation, and sample handling, and the results must be interpreted against published thresholds from the specific assay in use.

Diagnostic Reasoning in Endocrine Disease

Endocrine testing should never be performed as a blind panel. The pretest probability of disease, derived from signalment, history, and physical examination, determines the predictive value of any test result. A low total T4 in a dog with classic hypothyroid signs carries different weight than the same result in a dog with pruritic skin disease, because the prevalence of true hypothyroidism differs between these populations.

The same logic applies to hypercortisolism and hypoadrenocorticism. Testing every dog with polyuria and polydipsia for all three conditions wastes resources and generates false positives that lead to unnecessary further testing. A staged approach, starting with the most sensitive screening test and progressing to confirmatory testing only when screening is positive, is the most efficient strategy. This approach mirrors the diagnostic reasoning used in other areas of internal medicine, where expert consensus statements provide structured guidance on test selection and interpretation.

Reference Standards and Professional Guidance

Veterinary endocrine diagnosis operates within a framework of published guidelines and professional standards. The American College of Veterinary Internal Medicine publishes consensus statements that synthesise available evidence into practical recommendations for diagnosis and management of endocrine disorders. These documents are regularly updated and should be the first reference consulted when clinical uncertainty arises.

General veterinary references, including the MSD Veterinary Manual, provide species-specific information on endocrine physiology, testing protocols, and disease management. Professional organizations such as the American Veterinary Medical Association offer practice resources that address laboratory standards and quality assurance. International standards for animal health, such as those published by the World Organization for Animal Health, are more relevant to regulatory and population-level testing than to individual patient care, but they establish the framework within which diagnostic laboratories operate.

The evidence base for some endocrine tests is stronger than for others. Validation studies for antemortem endocrine testing have historically relied on postmortem histologic confirmation as the gold standard, but this approach has limitations. A study of pituitary histopathology in aged horses found only fair agreement among pathologists in identifying disease, which raises questions about the validity of histology as the reference standard for endocrine diagnosis. This finding, while from equine medicine, illustrates a broader principle: the gold standard against which endocrine tests are judged may itself be imperfect, and clinicians should interpret test validation data with appropriate caution.

Initial Assessment and Test Selection

The diagnostic sequence begins with a directed history and physical examination, then proceeds to confirmatory testing only when the pretest probability of endocrine disease is meaningful. Indiscriminate endocrine panels generate false positives that misdirect therapy and erode client confidence. A practical framework matches each suspected disorder to a minimum data set, then adds tests only when the initial results are equivocal.

Thyroid Testing in the Hypothyroid-Suspect Dog

Hypothyroidism remains the most common endocrine submission to commercial laboratories, yet it is also the most overdiagnosed condition in canine practice. The clinical signs of lethargy, weight gain, and dermatopathy overlap substantially with nonendocrine disease, and no single test distinguishes all affected from unaffected dogs. The testing laboratory perspective offered by Kemppainen and Behrend emphasizes that total T4, free T4 by equilibrium dialysis, and canine TSH each carry both false positive and false negative potential, and that interpretation requires integration of all three results with the clinical picture.

The recommended sequence is a total T4 as the initial screening test. A total T4 within the reference interval effectively excludes hypothyroidism in a dog with compatible signs, provided the dog is not receiving medications known to suppress thyroid hormone concentrations. A low total T4 is nonspecific, however, because any systemic illness, glucocorticoid administration, or sulfonamide therapy can suppress it. When total T4 is low, add free T4 by dialysis and endogenous canine TSH. A low free T4 with a high TSH supports hypothyroidism. A normal free T4 with a normal TSH makes the diagnosis unlikely, and the low total T4 is attributed to nonthyroidal illness or drug effect. The difficult case is the dog with low total T4, low-normal free T4, and normal TSH. This pattern occurs in euthyroid sick dogs and in early hypothyroidism, and repeat testing after resolution of concurrent illness is the most practical next step.

Thyroglobulin autoantibody testing adds little to the diagnostic algorithm. Positive autoantibodies support an immune-mediated pathogenesis but do not confirm current hypothyroidism, and many euthyroid dogs are seropositive. The diagnosis of hypothyroidism is ultimately a clinical judgment supported by laboratory data, and the response to levothyroxine supplementation is the final arbiter in ambiguous cases.

Adrenal Axis Testing

Hypercortisolism: Confirming the Diagnosis

The diagnostic approach to hypercortisolism follows a two-step logic. First confirm that hypercortisolism exists, then differentiate pituitary-dependent disease from adrenal neoplasia. The urine cortisol to creatinine ratio is the most sensitive screening test, but it is also the least specific. A normal ratio excludes hypercortisolism with high confidence. An elevated ratio confirms only that further testing is warranted, because stress, illness, and even a car ride to the clinic can elevate it. The low-dose dexamethasone suppression test is the preferred confirmatory test in most practices. A dog that fails to suppress serum cortisol below the laboratory threshold 8 hours after dexamethasone administration has confirmed hypercortisolism.

The ACTH stimulation test is less sensitive than the low-dose dexamethasone suppression test for pituitary-dependent hypercortisolism, but it is the test of choice when iatrogenic hypercortisolism from exogenous glucocorticoid administration is suspected. It also provides prognostic information in that dogs with marked post-ACTH cortisol concentrations tend to have more severe disease. The choice between screening tests depends on the clinical presentation. A dog with classic dermatologic and polydipsic signs and a high urine cortisol to creatinine ratio can proceed directly to differentiation testing. A dog with equivocal signs and a borderline ratio should have a low-dose dexamethasone suppression test before any differentiation testing is performed.

Differentiation Testing

Once hypercortisolism is confirmed, the endogenous ACTH concentration distinguishes pituitary-dependent disease from adrenal neoplasia. A suppressed endogenous ACTH supports an adrenal tumor, while a normal or elevated concentration supports pituitary disease. The low-dose dexamethasone suppression test can also differentiate the two forms, because approximately 60% of pituitary-dependent cases show partial suppression at 4 hours. The choice between endogenous ACTH measurement and the high-dose dexamethasone suppression test depends on laboratory access and cost. Endogenous ACTH requires careful sample handling with chilled plasma and rapid centrifugation, and the assay is not offered by all commercial laboratories.

Ultrasonography of both adrenal glands provides complementary information. Bilateral enlargement supports pituitary disease, while one enlarged gland with a contralateral small or normal gland supports adrenal neoplasia. Imaging is particularly valuable when biochemical differentiation is equivocal or when surgical adrenalectomy is planned. The combination of endogenous ACTH and adrenal ultrasonography resolves most cases without the need for high-dose dexamethasone suppression testing.

Hypoadrenocorticism Testing

The diagnosis of hypoadrenocorticism rests on the ACTH stimulation test, which remains the gold standard. A baseline cortisol below the reference interval with a blunted or absent response to exogenous ACTH confirms the diagnosis. The test is robust, but it requires injectable ACTH, which has periodic availability problems. The baseline cortisol alone is a useful screening tool. A baseline cortisol above the laboratory's cutoff for hypoadrenocorticism effectively excludes the disease, while a low baseline cortisol requires ACTH stimulation for confirmation.

The sodium to potassium ratio is a rapid bedside indicator. A ratio below 27:1 supports hypoadrenocorticism, but it is neither sensitive nor specific enough to confirm the diagnosis. Atypical hypoadrenocorticism, in which glucocorticoid deficiency occurs without mineralocorticoid deficiency, presents with normal electrolytes and requires a high index of suspicion. These dogs often have chronic intermittent gastrointestinal signs, and the diagnosis is made only when an ACTH stimulation test is performed. The endogenous ACTH concentration is not helpful in diagnosing hypoadrenocorticism, because it is elevated in primary disease and suppressed in secondary disease, and the distinction does not alter the acute management.

Pancreatic and Other Endocrine Testing

Diabetes Mellitus and Diabetic Monitoring

The diagnosis of diabetes mellitus is straightforward when classic clinical signs are accompanied by persistent fasting hyperglycemia and glucosuria. Endocrine testing beyond glucose measurement is rarely needed for diagnosis. Fructosamine provides a 2 to 3 week average of glycaemic control and is useful when stress hyperglycemia is suspected or when a single glucose measurement does not match the clinical picture. Serial fructosamine measurements every 6 to 8 weeks track the response to insulin therapy more reliably than single glucose readings, because they are unaffected by the stress of the clinic visit.

Parathyroid and Calcium Disorders

Ionised calcium is the physiologically active fraction and the preferred test for suspected hyperparathyroidism or hypoparathyroidism. Total calcium measurements can mislead when serum albumin or protein concentrations are abnormal. Parathyroid hormone should be measured concurrently with ionised calcium, because the interpretation of parathyroid hormone depends entirely on the ambient calcium concentration. A high parathyroid hormone with high ionised calcium confirms primary hyperparathyroidism. A high parathyroid hormone with low ionised calcium indicates secondary hyperparathyroidism, most commonly from chronic kidney disease. The parathyroid hormone assay requires careful sample handling, and the laboratory should be consulted for specific collection requirements.

Monitoring Treated Endocrine Disease

Monitoring parameters differ by disorder and by the treatment used. For hypothyroidism, the total T4 concentration is measured 4 to 6 hours after levothyroxine administration, targeting the upper half of the reference interval. Clinical response is assessed over 4 to 8 weeks, and the dose is adjusted based on both the T4 concentration and the resolution of clinical signs. Overtreatment is detected by a T4 concentration above the reference interval, which may cause polyphagia, weight loss, or anxiety.

For hypercortisolism treated with trilostane, the ACTH stimulation test performed 4 to 6 hours after medication administration guides dosing. The post-ACTH cortisol should fall within the therapeutic target range established by the reference laboratory. Clinical signs, particularly water intake, improve within 2 weeks, and the dose is adjusted based on both the cortisol response and clinical improvement. For hypoadrenocorticism, monitoring focuses on clinical signs, body weight, and serum electrolyte concentrations. Electrolytes are rechecked 1 to 2 weeks after initiating mineralocorticoid therapy, then at increasing intervals as stability is confirmed.

The frequency of monitoring reflects the stability of the patient. A newly diagnosed diabetic dog requires weekly adjustments during insulin dose titration, then rechecks every 2 to 3 months once stable. A well-regulated hypothyroid dog on a stable dose can be rechecked every 6 to 12 months. The monitoring schedule should be documented in the medical record, along with the specific laboratory parameters used to assess control, so that trends are visible over time.

Recognized Complications and Failure Modes

Endocrine testing fails in predictable patterns. Assay interference, sampling error, and misinterpretation of dynamic test results account for most diagnostic errors. The laboratory should be contacted whenever a result conflicts with the clinical picture, because repeat analysis on a fresh sample frequently resolves the discrepancy.

Hemolysis, lipaemia, and hyperbilirubinaemia interfere with immunoassays to varying degrees. Free thyroxine by equilibrium dialysis resists these effects more than total hormone assays, but no assay is completely immune. Samples that sit unseparated for hours at room temperature allow cellular uptake of thyroid hormones, lowering measured concentrations. Cortisol is more stable, yet delayed separation still risks bacterial degradation of steroid hormones.

Dynamic testing introduces additional failure modes. Incomplete urine collection invalidates a urine cortisol-to-creatinine ratio. Vomiting after oral dexamethasone administration produces falsely low suppression. Stress during sampling elevates cortisol and can obscure suppression. The ACTH stimulation test is robust, but expired or mishandled synthetic ACTH produces spuriously low post-stimulation cortisol values.

The table below summarizes common failures and the discriminating checks.

Common Errors and Corrective Action

Less experienced clinicians often over-interpret a single low total T4 concentration. This test has poor specificity in sick dogs, and a low value alone does not establish hypothyroidism. The corrective approach is to confirm with free T4 by dialysis and endogenous TSH, as described in laboratory-based guidance on thyroid testing Kemppainen and Behrend, diagnosis of canine hypothyroidism. Conversely, a normal total T4 does not exclude mild thyroid dysfunction when clinical suspicion is strong.

Another frequent error is diagnosing hypercortisolism from a single positive screening test without considering pretest probability. A dog with polyuria, polydipsia, and a pendulous abdomen has high pretest probability, whereas the same test result in a dog with only alopecia is more likely false positive. The same principle applies to the low-dose dexamethasone suppression test, where stress and concurrent illness reduce specificity.

Clinicians also misinterpret the ACTH stimulation test in early hypoadrenocorticism. Electrolyte changes may be absent in the early phase, and a normal baseline cortisol does not exclude the disease. The test must be performed when clinical suspicion is moderate or high, not deferred until the dog is critically ill.

Limitations of Current Evidence

The evidence base for canine endocrine testing is uneven. Thyroid testing has been studied extensively, and laboratory-based interpretive frameworks are well established Kemppainen and Behrend, diagnosis of canine hypothyroidism. Adrenal testing is supported by clinical experience and consensus guidance, but direct comparative studies of diagnostic accuracy are limited. Expert opinion still differs on the optimal screening test for hypercortisolism, with some favouring the urine cortisol-to-creatinine ratio for its high sensitivity and others preferring the low-dose dexamethasone suppression test for its specificity.

The ACVIM consensus statements provide structured guidance where evidence is sufficient, but they also identify areas where data are lacking ACVIM consensus statements. Clinicians should recognize that reference intervals vary between laboratories and assays, and that published cut-offs may not transfer directly to a different laboratory's platform.

Referral and Escalation

Referral to a specialist is warranted when the diagnosis remains uncertain after appropriate testing, when dynamic testing cannot be performed safely in practice, or when the patient fails to respond to treatment despite a confirmed diagnosis. Specialist consultation is particularly useful for differentiating atypical hypercortisolism, managing brittle diabetes mellitus, and investigating calcium disorders that do not fit a clear pattern.

Laboratory involvement should occur early, not as a last resort. Most endocrine laboratories employ veterinary specialists who can advise on test selection, sample handling, and interpretation of discordant results. They can also flag assay interference and recommend alternative testing strategies.

Regulatory reporting applies to specific circumstances. Reportable endocrine diseases in dogs are uncommon, but clinicians should be aware of local requirements. The World Organization for Animal Health maintains international standards for disease surveillance and reporting, and veterinarians should consult these standards when a notifiable condition is suspected WOAH terrestrial animal health standards. Professional practice resources from the AVMA can also clarify obligations in specific jurisdictions AVMA practice resources.

Frequently Asked Questions

How Should I Prioritize Endocrine Testing When the Owner Has a Limited Budget?

Start with the test that most directly changes management. For a dog with polyuria, polydipsia, and a pendulous abdomen, a urine cortisol-to-creatinine ratio is inexpensive and has high sensitivity for hypercortisolism, though a normal result does not exclude the disease. For suspected hypothyroidism, a total T4 is the cheapest first step, but a low value is not diagnostic on its own. If the total T4 is clearly within the reference interval, hypothyroidism is effectively excluded. Reserve confirmatory testing, such as free T4 by dialysis or ACTH stimulation, for cases where the screening result is equivocal or the clinical suspicion remains high despite a normal screen. Document the financial discussion in the record.

What Can I Do When Reference Intervals From My Laboratory Do Not Match the Published Literature?

Laboratory-specific reference intervals should take precedence over textbook values because assays vary between platforms and between batches. When an individual result falls near the boundary of the laboratory interval, interpret it in light of the pretest probability and the clinical picture. For example, a total T4 at the lower end of the reference interval in a dog with strong clinical signs of hypothyroidism still warrants further testing, whereas the same value in a clinically normal dog does not. If you suspect an assay problem, ask the laboratory about their calibration and whether they participate in external quality assurance. The ACVIM consensus statements provide guidance on assay interpretation across common endocrine disorders.

How Do I Interpret Endocrine Test Results in a Dog Receiving Medications That Interfere With the Assay?

Glucocorticoids, whether exogenous or endogenous, suppress the hypothalamic-pituitary-adrenal axis and can lower cortisol and ACTH concentrations. Phenobarbital can alter thyroid hormone concentrations through hepatic enzyme induction, producing low total T4 with normal free T4 by dialysis. Sulfonamides can cause falsely low total T4 and free T4 values in some assays. When possible, collect samples before starting these medications or after a washout period. If that is not feasible, interpret results with the drug effect in mind and choose assays less affected by the interference. The MSD Veterinary Manual summarizes known drug effects on endocrine assays, and the laboratory should be contacted directly for assay-specific interference data.

When Should I Repeat an Endocrine Test instead of Act on a Single Result?

Repeat testing is indicated when the first result is discordant with the clinical picture, when the result falls in an equivocal zone, or when the test has known day-to-day variability. For example, a single normal urine cortisol-to-creatinine ratio in a dog with strong clinical signs of hypercortisolism should be repeated because cortisol secretion is episodic. A single low total T4 in a dog with non-thyroidal illness should be followed by a free T4 by dialysis or a canine TSH measurement before committing to lifelong therapy. The diagnostic approach from a commercial testing laboratory emphasizes that no single endocrine test is perfect and that false positives and false negatives occur with every assay.

How Should I Document Endocrine Test Results and the Reasoning Behind My Interpretation?

Record the specific assay used, the laboratory that performed it, the reference interval, and the result with units. Note the timing of sample collection relative to medication administration and any recent stress or illness. Write a brief interpretation that links the result to the clinical findings and states the next diagnostic step or the treatment decision. If a test is repeated, record both values and the interval between them. This documentation supports continuity of care and is valuable if the case is referred. The AVMA practice resources include guidance on medical record content and professional communication standards.

How Do I Explain an Equivocal Endocrine Test Result to the Owner?

Use plain language that separates the test result from the diagnosis. Explain that the test is a piece of information, not the whole answer, and that the result falls in a grey zone where it neither confirms nor excludes the disease. Describe the next step, whether that is a different test, a repeat test after a defined interval, or a therapeutic trial with a scheduled reassessment. Be explicit about the cost and time involved. Avoid giving a false sense of certainty, and acknowledge that some cases remain diagnostically challenging despite appropriate testing. This approach mirrors the caution advised in equine pituitary pars intermedia dysfunction guidelines, where disparate diagnostic criteria and mild clinical signs can make interpretation difficult.

Related Clinical & Scientific Guides

• Feline Hepatic Lipidosis: Nutritional and Medical Management
• Canine Respiratory Infection: Diagnostic Approach and Treatment
• Canine Respiratory Virus: Diagnostic and Management Considerations

References and Further Reading

• Agreement in histologic assessments of the pituitary pars intermedia in aged horses.. 2005.
• BEVA primary care clinical guidelines: Diagnosis and management of equine pituitary pars intermedia dysfunction.. 2024.
• The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.. 2021.
• Diagnosis of canine hypothyroidism. Perspectives from a testing laboratory.. 2001.
• Health risk assessment procedures for endocrine disrupting compounds within different regulatory frameworks in the European Union.. 2009.
• Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?. 2023.
• ACVIM Consensus Statements. Journal of Veterinary Internal Medicine.
• MSD Veterinary Manual, Professional Edition. MSD Veterinary Manual.
• American Veterinary Medical Association Practice Resources. American Veterinary Medical Association.

Related Articles

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• Canine Hypoadrenocorticism: Diagnostic and Therapeutic Monitoring
• Canine Proteinuria: Diagnostic Approach and Management
• Canine Vomiting: Diagnostic Approach for Pancreatitis
• Canine Autoimmune Disease Overview: Diagnostic Considerations

This article is educational professional reference material for veterinary audiences. It is not a substitute for veterinary diagnosis, individual clinical judgment, current product labeling, or applicable regulatory requirements.