Cortisol levels in saliva. "Free cortisol (saliva)
If you suspect you have low thyroid or adrenal function and want to confirm this with laboratory tests, I recommend the list below. However, you should always remember rule N1 when diagnosing thyroid gland - do not let “good” results blind you to the standard symptoms of hypothyroidism + low temperature. Unfortunately, with hypothyroidism, tests can look “healthy,” especially if you have little understanding of what they mean or how to interpret them.
1) FREE CORTISOL IN DAILY URINE OR SALIVA.
to assess adrenal function.
I put this test in first place for the reason that it is extremely informative, unlike thyroid tests. If it is optimal, then this is amazing news and means that you do not have a problem with cortisol! If cortisol is low, then you also have hypothyroidism. Adequate cortisol levels are very important for the full conversion of thyroid hormones, as well as the proper functioning of cell receptors. Your basal temperature will be lowered, and your daytime temperature will fluctuate greatly from day to day.
UNDER NO CIRCUMSTANCES SHOULD YOU TAKE A MORNING TOTAL BLOOD CORTISOL TEST unless you want to confuse yourself or your doctor.
This test shows the level of total (bound + free) cortisol and says absolutely nothing about the level of free, i.e. biologically active. In most cases of adrenal insufficiency (cortisol “deficiency”), the problem is not in the adrenal glands themselves (they cope with the production of their hormones), but outside them. And in response to this problem, the body often binds almost all free cortisol to coticoid-binding globulin. You might have high levels of total cortisol but
At the same time, these are near-zero levels of free!!! And accordingly, all the symptoms of adrenal insufficiency. The above example was my own case. Sending for general cortisol in the blood is a gross, unforgivable mistake, which leaves a large number of patients undiagnosed. If your endocrinologist sends you for a general blood cortisol test, you are looking at a lazy, brainless ignoramus and it is extremely unlikely that he will be able to cure you.
Optimal free cortisol according to Terry Hertog (President of the European Hormonal Association) is 40-70 mcg/day (ug/24). Or 110-192 nmol
/day. But according to my observations in patients, 40 mcg/day (110 nmol/day) for a man already corresponds to chronic fatigue and brain fog.
2) TSH (Thyroid Stimulating Hormone) is informative ONLY if it is above 2, and then this means hypothyroidism. TSH less than 2 means nothing. Most Russian doctors still use outdated TSH standards of 0.5 -4.5. And many diagnose
hypothyroidism only when TSH > 10.
TSH more than 4 and less than 10 they call subclinical hypothyroidism, i.e. meaning an increase in TSH with “healthy” levels of free T4 and free T3, and therefore not requiring treatment.
Run away from these "doctors".
TSH more than 2 = hypothyroidism, according to top thyroid experts! And subclinical hypothyroidism DOES NOT EXIST! There are lazy, undeveloped endocrinologists!
The obsession of most doctors with TSH testing is one of the main reasons for undiagnosed hypothyroidism.
And the attempt to select a dose of hormones by adjusting TSH to one (another brainless strategy of doctors) is the reason for the huge number of untreated hypothyroidism. On top of that, TSH is significantly lower in smokers.
3) FREE T4. It is optimal when it is in the middle of the reference range. If it falls to the lower 30% of the reference range, this is already a problem and already hypothyroidism. If fT4 is in the middle and you have hypothyroidism, it means that the gland itself is coping with the production of hormones, but there is a problem outside of it (low cortisol / low iron / low zinc / low B12 / chronic inflammation, etc. .) A fairly reliable test and does not contain pitfalls.
4) FREE T3 Unfortunately, the standard analysis for free T3 shows “free T3 + reverse T3” and it is impossible to understand how much T3 is there (accelerates metabolism, gives energy), and how much reverse T3 (inhibits metabolism) without an additional test for reverse T3. As far as I know, at the moment, reverse T3 is not being tested anywhere in Russia.
Provided that your reverse T3 is no more than 15, a healthy T3 should be in the upper quarter of the reference range, and ideally at the upper limit of normal.
If it is below the upper quarter, it is already a problem and is usually accompanied by symptoms of hypo.
5) FERRITIN Iron is toxic to the cells of the body, therefore, during evolution, a special transport protein ferritin was developed, which “preserves” iron in a form that is safe for long-term storage. Ferritin is produced by the liver in response to iron intake and, in the absence of inflammation, infection or liver disease, is the best marker for diagnosing iron deficiency anemia. because reflects the body's iron reserves. In addition, ferritin is a marker of chronic inflammation, since it is a rectant of the acute phase - its production soars in the presence of inflammation.
What does ferritin have to do with diagnosing thyroid disease?
The fact is that low iron reserves create hypothyroidism, regardless of the amount of hormones produced by the thyroid gland, since they lead to excessive conversion of T4 to reverse T3, and also make the body's cells resistant to T3. Hypothyroidism itself leads to a decrease in iron stores due to underproduction of hydrochloric acid in the stomach, which is extremely important for adequate absorption of iron, zinc, selenium, B12, vitamin D, calcium, magnesium, copper, and vitamin K from food.
Low ferritin will help diagnose hypothyroidism even if TSH, fT4 and fT3 appear normal, but there is a low temperature and symptoms of hypothyroidism. It will also help to identify hidden inflammation, which is one of the worst enemies of the thyroid gland, disrupts the conversion of T4 to T3, makes cells resistant to T3 and drives the body into an “economy” mode.
Optimal ferritin, according to
Janie Bowthourpe (author of the best site on hypothyroidism Stop Thyroid
Free cortisol (saliva)
Cortisol is a steroid hormone synthesized in the zona fasciculata of the adrenal cortex from the common precursor of cholesterol under the control of ACTH. Interaction in the hypothalamus-pituitary-adrenal system is carried out according to the classical principle of negative feedback. ACTH increases cortisol secretion, and elevated cortisol levels block ACTH secretion. The adrenal cortex secretes cortisol in free form, entering the bloodstream, cortisol binds to blood plasma proteins, more than 90% of cortisol binds to a specific transport protein - transcortin (CSG) and in a small amount - to albumin. In a bound state, cortisol loses its biological activity, and only free cortisol provides its effect on target tissues. Metabolized in the liver, filtered in the glomeruli, and excreted in the urine in free form. The half-life is 80-100 minutes. Cortisol plays an important role in glucose metabolism, maintaining vascular tone, regulating the immune response, and in the body's response to stress.
The concentration of ACTH and cortisol in plasma has a clear circadian rhythm: the peak concentration of ACTH is observed between 4 and 6 a.m., the maximum level of cortisol is at 8 a.m., and the minimum is in the evening (11 p.m.).
In healthy people, the difference between cortisol concentrations in the morning and afternoon reaches 50%, and between morning and evening - 350%.
Cortisol secretion changes little with age. The daily rhythm of ACTH and cortisol secretion is formed in children by 6-12 months of life and is clearly established only after 3 years of life. In older children and adults, the individual daily rhythm of ACTH and cortisol secretion is quite stable and changes with a change in time zone only after 15-20 days.
Determination of free biologically active cortisol in saliva
The cell membrane (barrier) of the salivary glands does not allow biological molecules with a MV > 400 Da to pass into the salivary ducts. Molecular weight of all steroids< 400 Да, и в результате только свободные формы стероидов проникают в слюнной проток (кортизол, тестостерон, эстрадиол и др.), а гормоны, связанные с альбумином или глобулинами, транспортными белками стероидов, не проникают.
The level of cortisol in saliva does not depend on the volume and speed of its secretion, which is of fundamental importance when interpreting the results.
In endogenous hypercortisolism syndrome, the circadian rhythm of cortisol secretion is disrupted and patients with Cushing's syndrome have high plasma levels of the hormone in the evening hours, which correlates with the level of the hormone in saliva. Therefore, determination of salivary cortisol at 23:00 is a convenient and effective screening test in patients with suspected endogenous hypercortisolism. Sensitivity - 92-100%, specificity - 93-100%. A double determination is recommended. Normal salivary cortisol levels at 23-24 hours do not exceed 145 ng/dL (4 nmol/L).
Monitoring of therapy for adrenal insufficiency with hydrocortisone derivatives is possible.
| Indicator | Characteristic |
|---|---|
| Analyzer and test- system | EUROIMMUN (Germany) |
| Reference values, ng/ml Children: Adults: 0,2-4,4 |
|
| Interfering factors. Medicines | |
| Smoking, disruption of the physiological rhythm of sleep and wakefulness, crossing time zones | |
| Raise | Reduce |
|
|
| Indications for use | |
Marker of the functional state (glucocorticoid activity) of the adrenal glands
|
|
| Interpretation of results | |
| Level Up | Level reduction |
|
|
Indicator
Characteristic
Analyzer and test- system
EUROIMMUN (Germany)
Reference values, ng/ml
Children: up to 4 days of life: up to 0.95; 4-6 days of life: 1.3-3.02; 6-8 days of life: 2.07-5.05; 8-10 days of life: 1.04-4.78; 10-12 days of life: 0.49-1.83; 12-14 days of life: 0.46-1.26; 14-16 days of life: 0.55-1.23; 16-18 days of life: 0.13-1.15; 18-20 days of life: 0.03-1.15; 20-22 days of life: 0.18-0.72; 22-24 days of life: 0.06-0.96.
Adults: 0,2-4,4
Cortisol (saliva)
1. Full name of the study:
Cortisol (saliva)
Cortisol (saliva)
A marker of disorders of the regulatory function of the pituitary gland, an indicator of adrenal function.
2. Section name:
Hormonal studies
3. Research code in the price list :
1200
Price - see
4. Research method, analyzer:
Enzyme immunoassay:
- Automatic enzyme immunoassay analyzer Lazurite “DynexTechnologiesLTD”,, (USA) - analytical sensitivity: 0.035 - 45 ng/ml
5. Units of measurement and conversion factors:
- ng/ml
6. Type of biomaterial:
Saliva
7. Type of test tube/container for biomaterial:
Special container "Sarstedt Cortisol-Salivette"
8. Description of the study:
Cortisol is a steroid hormone, the main representative of glucocorticoid hormones, synthesized in the zona fasciculata of the adrenal cortex from the common precursor of cholesterol under the control of pituitary ACTH and corticotropin-releasing hormone of the hypothalamus (indirectly through ACTH). Interaction in the hypothalamus-pituitary-adrenal system is carried out according to the classical principle of negative feedback. In the bloodstream, cortisol binds to blood plasma proteins: more than 75% of cortisol binds to a specific transport protein, transcortin (CSG), synthesized in the liver, 15% to albumin. About 10% of circulating cortisol is in free form and it is this that provides the effect on target tissues.
Glucocorticoids directly or indirectly regulate almost all physiological and biochemical processes; receptors for them are found in all tissues of the body. They play a key role in the body’s reactions to stressful situations, maintaining vascular tone, and regulating the immune response. Cortisol affects carbohydrate metabolism - it increases the concentration of glucose in the blood by increasing its synthesis in the liver and reducing its utilization in the periphery (it is an insulin antagonist). The effect of the hormone on lipid metabolism leads to inhibition of synthesis and increased breakdown of fats with the release of glycerol and fatty acids, an increase in cholesterol levels in the blood, and the accumulation of ketone bodies. There is also a catabolic effect of cortisol on protein metabolism. Cortisol has little mineralocorticoid activity. Cortisol has a powerful anti-inflammatory effect. It inhibits almost all stages of the inflammatory process, suppresses the migration of phagocytes to damaged tissues and the release of inflammatory cytokines, reduces the synthesis of prostaglandins, and stabilizes lysosome membranes.
Cortisol is metabolized in the liver, filtered in the glomeruli, and excreted in the urine in free form. The half-life is 80-100 minutes. Saliva contains free, stable cortisol, the level of which does not depend on the content of cortisol-binding globulin. The level of cortisol in saliva is not affected by the volume and speed of saliva secretion, which is of fundamental importance when interpreting the results.
Like the content of cortisol in the blood, cortisol in saliva is also characterized by a daily rhythm of secretion. In healthy people, the cortisol content is highest in the morning (6.00-8.00) and 2-5 times lower in the evening (22.00-24.00). The daily rhythm of cortisol secretion is disrupted in patients with Cushing's syndrome, during pregnancy, and under the influence of stress - physical or psychological. Thus, patients with Cushing's syndrome have high levels of the plasma hormone in the evening hours, which correlates with the level of the hormone in saliva. Therefore, determination of salivary cortisol at 23:00 is a convenient and effective screening test in patients with suspected endogenous hypercortisolism.
9. Reference values of the norm :
- Night: 0.035 - 1.0 ng/ml
- Morning peak (1.5 hours after waking up): 0.9 - 11.5 ng/ml
- 3.5-4.5 hours after the morning peak: 0.28 - 5.8 ng/ml
- 7.5-8.5 hours after the morning peak: 0.18 - 3.0 ng/ml
10. Indications for use:
- diagnosis of Itsenko-Cushing's syndrome/disease (obesity, arterial hypertension, hyperglycemia, myopathy, hirsutism, plethora, osteoporosis, pathological fractures, red stretch marks, increased trauma to the skin and soft tissues, menstrual irregularities)
- Diagnosis of Addison's disease
- atypical clinical symptoms for the patient’s age (osteoporosis, arterial hypertension)
- children with growth retardation, overweight, premature sexual development
- differential diagnosis of primary and secondary adrenal insufficiency when combined with ACTH
11. Interpretation of results:
Level Up:
Cushing's disease/syndrome, nodular adrenal hyperplasia, ectopic CRH syndrome (corticotropin-releasing hormone), ectopic ACTH syndrome, polycystic ovary syndrome (PCOS), hyperthyroid state, hypoglycemia, obesity, depression, liver cirrhosis (decreased catabolism), uncompensated diabetes mellitus, alcoholism, pregnancy.
Downgrade:
hypopituitarism, Addison's disease, condition after taking glucocorticoids, adrenogenital syndrome, hypothyroid state (decreased secretion), hepatocellular failure - decreased secretion (liver cirrhosis, hepatitis), sudden weight loss
12. Level up factors:
Microbleeding from the gums, chewing tobacco, licorice, synthetic glucocorticosteroids (oral, parenteral, inhalation, topical), anticonvulsants, aspirin, atropine, benzodiazepines, clomipramine, oral contraceptives, furosemide, insulin, interferon (gamma), methoxamine, metoclopramide, naloxone, ranitidine, spironolactone, opiates, vasopressin, ACTH, alcohol
13. Level reduction factors:
Barbiturates, dexamethasone, ephedrine, indomethacin, ketoconazole, labetalol, levodopa, morphine, nifedipine, oxazepam, pravastatin, rifampicin, synthetic corticosteroids (prednisolone, methylprednisolone)
14. Deadline:
3 working days
15. Preparation rules:
cm. « »
Features: on the eve of the study, it is necessary to exclude physical activity (sports training), alcohol intake, licorice, and not smoking; before collecting biomaterial, the patient needs complete physical and emotional rest for an hour.
in "Research Handbook"
The active steroid hormone produced by the adrenal cortex is cortisol. It plays an important role in the human body, is responsible for stress, regulates the immune system and is involved in reactions with glucose. It is present in the body in several forms:
- Together with red blood cells.
- In the form of conjugates with proteins.
- And also in a free form, where its action is expressed in an active form.
The main functions of cortisol include its participation in energy metabolism, the metabolism of fats, proteins and carbohydrates, as well as the regulation of water-salt metabolism. Thanks to its active participation, the body develops protective properties of the body to external stimuli, that is, stressful conditions.
Cortisol affects the immune system, blocking allergies. Artificial preparations based on this hubbub are able to relieve hypersensitive reactions, block the manifestations of anaphylactic shock, and help reduce inflammatory processes, which is important in the treatment of autoimmune diseases. A normal amount of cortisol in a healthy person has a positive effect on biorhythms.
Since cortisol plays a significant role in the body’s vital functions, an analysis of the patient’s saliva is taken to confirm the authenticity of the disease. An increase in cortisol levels may be a consequence of the development of such diseases:
- Formation of tumors in the adrenal glands.
- Development of Itsenko-Cushing syndrome, Addison's disease.
- Insufficient production of adrenocorticotropic hormone.
- A reduced level of cortol quite often becomes the cause of the development of osteoprosis.
To test salivary cortisol, first of all, little preparation is required. The procedure for taking an analysis is quite simple for the patient and the doctor, and does not present any particular difficulties. However, approximately three to four weeks before the procedure, you need to notify the doctor about drug therapy, preferably stopping it.
One day before, eliminate fatty and spicy foods from your menu, as well as alcoholic beverages. Several hours before collecting saliva, brushing your teeth, eating, smoking and drinking is prohibited, and you should not use refreshing lozenges for the oral cavity. Failure to follow these recommendations will lead to inaccurate indicators of the true picture of the disease.
As a rule, saliva collection is carried out during the period of lowest cortisol concentration, this is from 11 to 12 pm. To do this, a tampon and a special container are used in which the biomaterial is placed, and the exact time is indicated. Usually the procedure is done within two days, this allows you to get the most accurate results.
Saliva collection for cortisol analysis.
The plasma half-life of cortisol (60-90 minutes) depends on the degree of its protein binding and metabolic rate.
Plasma binding proteins
When released into the blood, cortisol and adrenal androgens bind to plasma proteins. Cortisol interacts primarily with corticosteroid binding globulin (CBG, or transcortin) and to a lesser extent with albumin, while androgens interact primarily with albumin. Bound steroids are biologically inactive. Only the unbound, or free, faction of them is active. Plasma proteins protect cortisol from decay and smooth out sharp fluctuations in its level during episodic releases from the adrenal glands. There are no binding proteins in saliva, and therefore the level of cortisol in it reflects the concentration of its free form in the blood.
Free and bound cortisol
Under basal conditions, free cortisol in plasma accounts for about 10%, about 75% bound to DSG, and the rest bound to albumin. The concentration of free cortisol in plasma is approximately 1 mcg%, and the level of this biologically active hormone is regulated by ACTH.
Corticosteroid binding globulin (CBG)
CSH (molecular weight approximately 50,000) is produced in the liver and has a high affinity for cortisol. Contained in 100 ml of plasma, CSG binds approximately 25 μg of cortisol. When the total plasma cortisol concentration exceeds this level, the concentration of its free form increases rapidly. Other endogenous steroids generally do not affect the binding of cortisol to DRGs. The exception is progesterone in late pregnancy, when it can occupy up to 25% of the DRG binding sites. Synthetic steroids (with the exception of prednisolone) practically do not bind to CSG. Estrogens increase plasma DRG levels (as observed during pregnancy or oral contraceptive use). Its concentration also increases with hyperthyroidism, diabetes mellitus, certain blood diseases or due to genetic reasons. A decrease in the level of CSH is characteristic of its familial deficiency, hypothyroidism and protein deficiency (severe liver disease or nephrotic syndrome).
Albumen
The capacity of albumin for cortisol is much higher, but the affinity is lower. Normally, albumin binds about 15% of the cortisol present in the blood, but in cases where the total cortisol concentration exceeds the binding capacity of the DRG, the proportion of cortisol bound to albumin increases. Albumin actively binds synthetic glucocorticoids (for example, approximately 75% of plasma dexamethosone is bound to albumin).
Androgen binding
Androstenedione, DHEA and DHEA sulfate circulate in the blood, loosely bound to albumin. Testosterone interacts with high affinity with other sex hormone binding globulin (SHBG).
Laboratory indicators
There are specific methods for determining cortisol and adrenal androgens in plasma. The activity of the adrenal cortex can also be judged by the level of steroids in the urine (in particular, by the daily excretion of cortisol). Determining plasma ACTH concentration provides useful information. The results of determining the total level of steroid hormones in plasma depend on the concentration of binding proteins. In addition, since plasma concentrations of ACTH and adrenal hormones fluctuate significantly throughout the day, the results of single determinations are highly unreliable. To obtain more reliable diagnostic information, dynamic tests (stimulation or suppressive), as well as indicators of the rate of cortisol secretion, are usually used.
Plasma ACTH concentration
Determination methods
Plasma ACTH concentration is a very accurate indicator of the state of the pituitary-adrenal axis. When determined by a sensitive immunometric method, it normally ranges from 9-52 pg/ml (2-11.1 pmol/l).
Interpretation
Plasma ACTH levels are most often determined to determine the location of the primary cause of adrenal dysfunction.
- With primary adrenal insufficiency, the ACTH level is elevated, and with pathology of the pituitary gland and secondary adrenal insufficiency, its level is “normal” or does not reach 10 pg/ml (2.2 pmol/l).
- In Cushing's syndrome, caused by a cortisol-producing adrenal tumor, a drop in plasma ACTH level below 5 pg/ml (1.1 pmol/l) is diagnostic, whereas in Cushing's disease (hypersecretion of ACTH), its plasma level is normal or elevated.
- Plasma ACTH levels are usually sharply elevated in ectopic ACTH secretion syndrome, although equally significant increases occur in Cushing's disease. In addition, when using the immunoradiometric method with double antibodies (IRMA), the concentration of ACTH in rare cases of this syndrome is increased to a lesser extent, since high molecular weight ACTH precursors are not determined by this method.
- Plasma ACTH levels are also sharply elevated in patients with common forms of congenital adrenal hyperplasia, which is used to diagnose these disorders and monitor their treatment.
Plasma cortisol concentration
Determination methods
Plasma cortisol levels are most often determined by radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC). These methods determine total cortisol (bound and free) in plasma. Saliva contains only free cortisol, and saliva testing allows a simple and reliable assessment of the concentration of the free form of this hormone.
The principle of RIA is to inhibit the binding of labeled cortisol to antibodies by cortisol present in the plasma sample. The sensitivity of modern RIA methods allows the use of small volumes of plasma. In addition, currently used antisera have little or no interaction with other endogenous steroids, and therefore RIA results accurately reflect plasma total cortisol levels. The antibodies used may cross-react with some synthetic glucocorticoids (eg, prednisone), but other commonly used drugs do not interfere with these determinations.
Interpretation
Single determinations of plasma cortisol concentration have no diagnostic meaning, since cortisol is secreted episodically and, in addition, its level increases with any stress. More reliable information is obtained through dynamic testing of the state of the HPA system.
- Normal indicators. Normal plasma cortisol levels depend on the method of determination. According to RIA data, the cortisol content at 8 o'clock in the morning ranges from 3 to 20 μg% (80-550 nmol/l), averaging 10-12 μg% (275.9-331.1 nmol/l). In later hours these values decrease, and at 4 o'clock in the afternoon they are approximately half as low as in the morning. Between 10 pm and 2 am, cortisol concentrations are usually less than 3 mcg% (80 nmol/L). Salivary cortisol levels at midnight are below 0.15 μg% (4 nmol/L).
- Levels during stress. The secretion of cortisol increases during various diseases, during surgery and during injuries. Under these conditions, plasma cortisol concentrations can reach 40-60 μg% (1100-1655 nmol/l).
- Increased concentration of estrogen. The level of total plasma cortisol increases with increasing DRG binding capacity, which most often occurs under conditions of high estrogen levels in the blood (for example, during pregnancy or the use of exogenous estrogens or oral contraceptives). The concentration of cortisol in such cases can be 2-3 times higher than normal.
- Other conditions. The content of DRGs may also change in other conditions. The concentration of total cortisol increases with anxiety and depression, with fasting, anorexia nervosa, alcoholism and chronic renal failure.
Cortisol in saliva during late night hours
In most patients with Cushing's syndrome, the daily rhythm of cortisol secretion is disrupted, and its level (unlike the norm) does not decrease by 11-12 o'clock at night. This can disrupt sleep and cause psychological problems. Elevated plasma cortisol levels at midnight (in sleeping people) distinguish Cushing's syndrome not only from the norm, but also from conditions such as depression or alcoholism. One study found that a midnight serum cortisol concentration greater than 5.2 μg% (140 nmol/L) diagnosed Cushing's syndrome with 100% sensitivity and 77% specificity. However, such definitions are difficult, and many clinicians prefer to diagnose Cushing's syndrome by the level of cortisol in saliva at night. The content of cortisol in saliva is in equilibrium with the level of free cortisol in the blood, does not depend on the amount of saliva and remains stable for several days.
With the help of various devices, saliva can be easily collected at home. Recent studies have shown that elevated salivary cortisol levels at night are a reliable sign of Cushing's syndrome. The sensitivity and specificity of this indicator exceed 90%, and such determinations may be the simplest and most effective way to screen patients with suspected hypercortisolemia. Reference values for cortisol concentrations in saliva depend on the determination method.
(module direct4)
Corticosteroids in urine
Free cortisol
- Determination methods. Determination of urinary free cortisol excretion is a reliable way to diagnose Cushing's syndrome. Normally, free cortisol in urine accounts for less than 1% of the secreted hormone. However, when it is secreted excessively, the concentration of cortisol exceeds the binding capacity of DSG, and the level of free hormone in plasma and its excretion in urine increase. The content of free cortisol is determined in 24-hour urine samples using HPLC, RIA, and more recently using gas chromatography/mass spectroscopy.
- Normal indicators. Currently, the most specific method for determining cortisol in urine is HPLC. The normal level of free cortisol determined by this method ranges from 5 to 50 mcg (14-135 nmol) per day. When using RIA methods, the normal content of cortisol in urine is 20-90 mcg (50-250 nmol) per day.
- Diagnostic significance. Urinalysis makes it possible to distinguish simple obesity from Cushing's syndrome, since the content of free cortisol in the urine (unlike 17-hydroxycorticosteroids) does not increase with obesity. The level of cortisol in urine increases under the same conditions as its content in plasma, and may increase slightly during pregnancy. In case of adrenal insufficiency, this method of assessing cortisol secretion is not used, both due to the low sensitivity of the methods and because low cortisol excretion is typical for many healthy people.
17-Hydroxycorticosteroids
Currently, the determination of the excretion of these compounds in urine has lost its importance, since it has become possible to determine the levels of cortisol in plasma and free cortisol in urine.
Suppressive tests with dexamethasone
Low dose test
Using this test, you can reject or confirm the diagnosis of Cushing's syndrome, regardless of its cause. Normally, dexamethasone, being an active glucocorticoid, suppresses the pituitary secretion of ACTH, which leads to a drop in the level of glucocorticoids in plasma and urine. This checks the integrity of the feedback mechanism in the HPA system. In Cushing's syndrome, this mechanism is disrupted, and steroid secretion is not reduced when dexamethasone is administered. Dexamethasone in the doses used does not interfere with the determination of cortisol in plasma and urine.
To screen patients with Cushing's syndrome, an overnight test with 1 mg of dexamethasone is usually performed. Dexamethasone is administered orally at 11 p.m., and plasma cortisol levels are determined the next morning. If its level is below 1.8 mg% (50 nmol/l), then Cushing's syndrome is most likely excluded. If the cortisol level exceeds 10 mg% (276 nmol/l), then (in the absence of reasons for false positive test results) this indicates the presence of this syndrome. In these cases, the diagnosis should be confirmed by other means. Cortisol levels between 1.8 and 10 mcg% are not informative.
Impaired response to dexamethasone is found in 80-99% of patients with Cushing's syndrome. False-negative results of this test are more common in mild hypercortisolemia, as well as in cases of slow metabolism of dexamethasone, when its plasma level is higher than expected. In such cases, the plasma levels of dexamethasone and cortisol are simultaneously determined.
False-positive results occur in hospitalized patients and people with chronic or acute illnesses, as well as in depressive and anxiety states, alcoholism, high estrogen levels and uremia. In patients taking phenytoin, barbiturates and other drugs that induce the synthesis of liver microsomal enzymes, dexamethasone may be destroyed more quickly and may not reach the concentration necessary to reduce ACTH levels.
High dose tests
Such tests are carried out in order to distinguish Cushing's disease (hypersecretion of ACTH by the pituitary gland) from the syndrome of ectopic ACTH secretion and adrenal tumors, since in the first case, supraphysiological doses of dexamethasone can still suppress the secretion of ACTH and cortisol, whereas in adrenal tumors and, in most cases, ectopic ACTH secretion does not. Unfortunately, exceptions to this rule are very often observed, which requires great caution when interpreting the results of such samples.
- A short trial with a high dose of dexamethasone. The initial morning plasma cortisol concentration is preliminarily determined. Oral dexamethasone 8 mg is given at 11 p.m., and plasma cortisol levels are determined at 8 a.m. the next morning. In Cushing's disease, the plasma cortisol concentration is usually half the initial value, and in ectopic ACTH secretion syndrome it either does not decrease or decreases to a much lesser extent. Plasma cortisol concentrations do not decrease in adrenal tumors that secrete cortisol autonomously. In these cases, ACTH secretion is already initially suppressed by high levels of endogenous cortisol, and the effect of dexamethasone does not appear.
- Long trial with a high dose of dexamethasone. Dexamethasone is taken orally 2 mg every 6 hours for 2 days. Before and on the second day after starting dexamethasone, daily urine is collected. In Cushing's disease, urinary cortisol excretion is reduced by more than 50%, while in adrenal tumors or ectopic ACTH secretion it remains almost at the original level. However, in some patients with ectopic ACTH secretion syndrome, high doses of dexamethasone still reduce cortisol excretion, and in some cases of Cushing's disease, the excretion of this hormone is reduced to a much lesser extent. The diagnostic sensitivity, specificity and accuracy of this test are only about 80%. Its specificity and accuracy can be increased by focusing not on cortisol excretion, but on other criteria. However, it is increasingly clear that the high-dose dexamethasone test does not reliably distinguish pituitary from non-pituitary ACTH hypersecretion, regardless of the criteria for assessing its results.
Pituitary-adrenal reserves
Determination of pituitary and adrenal reserves is used to assess the ability of the HPA axis to respond to stress. This is done by administering ACTH (which directly stimulates the secretion of corticosteroids), metyrapone (which inhibits the synthesis of cortisol and thereby stimulates the secretion of ACTH) or insulin (which, by causing hypoglycemia, stimulates the secretion of CRH and therefore ACTH). Later, CRH was used to directly stimulate ACTH secretion by pituitary corticotrophs.
ACTH tests
- Methodology and normal indicators. A short test with ACTH, which allows assessing the acute response of the adrenal glands to stimulation, is carried out to diagnose both primary and secondary adrenal insufficiency. Synthetic human α 1-24 ACTH (tetracosactide, or cosyntropin) is used. Fasting before the test is not necessary, and it can be done at any time of the day. After determining the initial cortisol level, 0.25 mg of tetracosactide is administered intramuscularly or intravenously and the plasma cortisol level is determined 30 or 60 minutes after injection. Since the maximum concentration of ACTH during the test exceeds 10,000 pg/ml, the results of this test reflect the maximum secretory capacity of the adrenal cortex. The maximum rise in cortisol levels after 30-60 minutes should exceed 18-20 mcg% (497-552 nmol/l). This rise at 30 minutes is constant and does not depend on the basal concentration of cortisol. Moreover, it does not depend on whether ACTH is administered at a dose of 250, 5 or even 1 mcg. The rise in cortisol levels in response to the administration of 1 mcg of ACTH is a more sensitive indicator of adrenal function and makes it possible to identify among those receiving long-term corticosteroid therapy those patients whose response to 250 mcg of ACTH is normal, but to 1 mcg is weakened. The 1 mcg ACTH test is better tolerated and appears to be the test of choice when adrenal insufficiency is suspected. No less informative may be the determination of cortisol in saliva after administration of ACTH.
- Decreased response. Poor adrenal response to acute stimulation supports the diagnosis of adrenal insufficiency. In primary adrenal insufficiency, cortisol secretion is reduced and ACTH secretion is increased. Therefore, the adrenal glands are initially stimulated to the maximum extent, and the administration of exogenous ACTH does not increase cortisol secretion. In other words, in this case there is a decrease in the secretory reserves of the adrenal glands. With secondary adrenal insufficiency (caused by ACTH deficiency), the zona fasciculata and reticularis of the cortex atrophy and practically do not respond to acute ACTH stimulation. In both primary and secondary adrenal insufficiency, their weakened response to acute ACTH stimulation predicts a weakened response to insulin hypoglycemia, metyrapone, and surgical stress.
- Normal reaction. A normal response to acute ACTH stimulation excludes both primary adrenal insufficiency (since it indicates the preservation of the secretory reserve of these glands) and obvious secondary adrenal insufficiency (due to their atrophy). However, a normal reaction does not exclude partial ACTH deficiency (reduced pituitary reserve), since the basal secretion of this tropic hormone may be sufficient to prevent adrenal atrophy, but will not increase under stress. In such cases, tests with metyrapone, insulin hypoglycemia or CRH are indicated.
Test with metyrapone
The metyrapone test is used to diagnose adrenal insufficiency and to assess pituitary-adrenal reserves. Metyrapone blocks cortisol synthesis by inhibiting llp-hydroxylase, which converts 11-deoxycortisol to cortisol. This stimulates the secretion of ACTH, which in turn increases the secretion of 11-deoxycortisol and its plasma level. If pituitary ACTH deficiency is suspected, a short test with dexamethasone is usually used. Suspicion of primary adrenal insufficiency is best checked with a short ACTH test. Normally, during an overnight test with metyrapone, the plasma 11-deoxycortisol level should exceed 7 ng% (0.2 nmol/l), and the ACTH concentration should exceed 100 pg/ml (22 pmol/l). A reduced response indicates adrenal insufficiency. A normal response to metyrapone predicts a normal response to stress and insulin hypoglycemia.
Test with insulin hypoglycemia
Stress caused by hypoglycemia is accompanied by the release of CRH, which leads to increased secretion of ACTH and cortisol. Thus, this test assesses the functional integrity of the HPA axis and its ability to respond to stress. Normally, the increase in plasma cortisol levels should exceed 8 mcg% (220 nmol/l), and its maximum concentration should be 18-20 mcg% (497-552 nmol/l). Plasma ACTH levels typically increase by more than 100 pg/mL (22 pmol/L) in response to hypoglycemia. A normal cortisol response rules out adrenal insufficiency and decreased pituitary ACTH reserves. This means that people with a normal response to hypoglycemia do not need additional amounts of cortisol during illness or surgery.
Test with CRH
The ACTH response to CRH is enhanced in primary adrenal insufficiency and is absent in hypopituitarism. With pathological processes in the hypothalamus, this reaction may be delayed. A CRH test is also performed to determine the cause of Cushing's syndrome.
Androgens
Androgen secretion is usually assessed by its basal level, since suppressive and stimulation tests in this case are less informative than for disorders of glucocorticoid secretion. There are methods for determining DHEA, DHEA sulfate, androstenedione, testosterone and dihydrotestosterone. The level of free testosterone in plasma directly reflects its biological activity, but commercial kits for its determination do not always provide sufficiently accurate results.
