36 Prolactin (PRL)

Lothar Thomas

The primary source of PRL (also called lactogenic hormone) production is the lactotrophs of the anterior pituitary gland. However, there are a number of other organs, cells, and tissues in which PRL is expressed and secreted (extrapituitary PRL).

PRL is very versantile, with many biological functions in different species that fall into the following general categories: reproduction, pregnancy and lactation, growth and development, metabolism, immune modulation, electrolyte transport, regulation of the integument, behavior, and carcinogenesis. The main role, of pituitary PRL is to stimulate the production of milk in women after childbirth. The specific function of pituitary PRL in men is not well known, however, it is most commonly measured in individuals with reproductive disorders. The regulation of extrapituitary PRL is dissimilar to that of pituitary PRL and is typically cell- or tissue specifc. The biologically active form of PRL is the monomeric 23 kDa peptide /1/.

Circulating prolactin exists as 23 kDa bioactive PRL and in a number of post-translational modifications of inactivated forms (macroprolactins), which are measured in varying quantities depending on the PRL immunoassay used /2/:

• Big PRL, with a molecular mass of 48–56 kDa, which is a covalently bound dimer of 23 kDa PRL.
• Big-big PRL (macro PRL), with a molecular mass of 150–205 kDa, which consists of antigen-antibody complexes of 23 kDa PRL with IgG, IgM and IgA or with other proteins, or is a hyperglycosylated form of PRL.

All prolactin immunoassays currently available are affected with 5–25% results indícating hyperprolactinemia falsely elevated due to macroprolactin with normal concentrations of bioactive monomeric prolactin /30/. Big PRL and macro PRL are clinically irrelevant as they do not bind to the PRL receptors. They can, however, cause apparent hyperprolactinemia (pseudo-hyperprolactinemia), which can lead to misdiagnosis and mismanagement of patients. Therefore, in cases of elevated serum PRL levels, laboratories are advised to remove the big PRL and macro PRL components by precipitation with polyethylene glycol (PEG), and to report the results for total PRL and the hormonally active 23 kDa PRL. The clinical evaluation should be based on the concentration of 23 kDa PRL. Only increased PRL secretion (i.e., hyperprolactinemia) is of clinical significance.

Pituitary PRL and extrapituitary PRL proteins are identical in terms of their primary, secondary, or tertiary structure, and both bind to the same receptor.

36.1 Indication

Both sexes

• Suspected pituitary adenoma and pituitary insufficiency
• Treatment with anti-psychotics.

Females

• Amenorrhea, oligomenorrhea, anovulatory cycles, corpus luteum insufficiency
• Galactorrhea, mastodynia, mastopathy, osteopenia
• Acne, virilization symptoms
• Workup of infertility.

Males

• Hypogonadism, sometimes combined with headache and impaired vision (macro prolactinoma)
• Reduced libido
• Erectile dysfunction
• Gynecomastia, in rare cases with galactorrhea.

36.2 Method of determination

Enzyme immunoassay, immunometric assay. Assays are calibrated against the WHO’s Third International Standard IS 84/500. Prolactin units can be converted from mIU/L to μg/L by dividing by 21.2.

Determination of 23 kDa prolactin

250 μL of serum is mixed with an equal volume of polyethylene glycol (250 g/L, dissolved in 137 mmol/L of NaCl, 10 mmol/L of sodium phosphate) and incubated at room temperature for 10 minutes. The clear supernatant is removed from the suspension by centrifugation at 1,800 × g for 30 minutes. PRL is measured in the serum and in the clear supernatant. The serum contains the total PRL, the clear supernatant the post-PEG PRL (23 kDa prolactin) /3/.

The reference method of separation 23 kDa prolactin from macro prolactin is gel filtration chromatography.

36.3 Specimen

Serum (blood collection 8–10 a.m.): 1 mL

36.4 Reference interval

Reference intervals vary between diagnostics manufacturers and are age- and sex dependent /4, 5/. Refer to:

• Tab. 36-1 – Prolactin reference intervals determined by PEG precipitation (23 kDa prolactin)
• Tab. 36-2 – Reference intervals for total prolactin in children.

36.5 Clinical assessment

In normal sera, monomeric (23 kDa) PRL accounts for 85–95%, big PRL for less than 10%, and macro PRL (big-big PRL) for less than 5% of the serum PRL. Depending on the study, macro PRL is identified as the sole cause of hyperprolactinemia in 4% of cases /7/, while other studies report a prevalence of macro prolactinemia of about 10% in patients with hyperprolactinemia /8/. It is therefore recommended to perform PEG precipitation on all hyperprolactinemic sera and to report the concentrations of total PRL and post-PEG PRL (bioactive PRL) /5/.

36.5.1 Macroprolactin

Macro PRL is a large antigen-antibody complex of molecular weight greater than 100 kDa and constitutes less than 1% of circulating PRL. The antigen-antibody complex consists of monomeric PRL and anti-PRL autoantibodies, these being IgG antibodies with low receptor affinity. Macro-PRL is cleared more slowly than monomeric (23 kDa) PRL /9/.

Many laboratories perform the determination of post-PEG PRL if prolactin levels are at or above 600 mIU/L (30 ug/L) in females or above 400 mIU/L (20 ug/L) in males. The findings should be commented as follows /5/:

• Elevated post-PEG PRL: all sera with elevated total PRL concentrations are routinely screened for the presence of macro PRL. This abnormal form of PRL cross reacts with the biologically active PRL in the immunoassay but is biologically inactive. When corrected for macro PRL, the bioactive PRL concentration in this serum is increased.
• Normal post-PEG PRL: all sera with elevated total PRL concentrations are routinely screened for the presence of macro PRL. This abnormal form of PRL cross reacts with the biologically active PRL in the immunoassay but is biologically inactive. When corrected for macro PRL, the bioactive PRL concentration in this serum is normal.

The prevalence of hyperprolactinemia ranges from 0.4%, as found in Japanese factory workers, to as high as 9% and 17% in women with infertility or polycystic ovarian syndrome /9/. There is a clear gender predilection for hyperprolactinemia, with women being affected six times more often than men /9/. If hyperprolactinemia is present, confirmatory testing of a second blood sample must be carried out. Prior to this, pregnancy should be ruled out in women of reproductive age.

If PRL levels are only mildly elevated or within the upper reference interval and a consistent clinical picture is observed, it is recommended to measure the basal prolactin concentration with an intravenous cannula in situ three times at 20 minute intervals to rule out stress induced hyperprolactinemia, or to take a blood sample in the morning on two different days.

Hyperprolactinemia can have physiological, pathological or pharmacological causes (Tab. 36-3 – Causes of hyperprolactinemia). The most common causes of pathological hyperprolactinemia are prolactinomas, which account for 25–30% of clinically diagnosed pituitary tumors. Prolactinomas are treated with dopamine agonists or surgically.

36.5.2 Clinical symptoms

Hyperprolactinemia presents predominantly with hypogonadism, galactorrhea, and symptoms of the mass effect of the pituitary tumor on the surrounding structures (Tab. 36-4 – Clinical symptoms of hyperprolactinemia) /10/.

36.5.3 Evaluation of PRL levels

Hyperprolactinemia can be diagnosed based on the test results from two blood samples. While PRL levels above 250 μg/L (5,000 mIU/L) are virtually always diagnostic of a prolactinoma, less elevated levels may also be due to a prolactinoma /9/. Causes of hyperprolactinemia are listed in Tab. 36-5 – Conditions and diseases with hyperprolactinemia.

Other possible causes are pregnancy, drugs, and central hypothyroidism. Once these causes have been ruled out by anamnesis, determination of TSH levels, and pregnancy testing, the next step is to image the base of the skull /10/.

The behavior of PRL in the presence of different conditions and diseases is outlined in Tab. 36-3 – Causes of hyperprolactinemia.

36.5.3.1 Hyperprolactinemia in females

Hyperprolactinemia is the reported cause of amenorrhea in 10–40% of patients. Approximately 70% of women with hyperprolactinemia have galactorrhea, which in most cases is expressible rather than spontaneous. The most common form of hyperprolactinemia in women are micro prolactinomas with normal sella turcica /10/.

Pituitary adenoma comprises 10–15% of all diagnosed intracranial tumors. Prolactinoma is the most common type of functional pituitary adenoma, with a prevalence of 100 per 1 million people. According to the 2016 WHO classification of tumors, prolactinomas are grade I/II, which are considered to be benign tumors. In addition, prolactinoma is one of the functional pituitary tumors, which are classified into adrenocoticotropic hormone-secreting pituitary adenoma, growth hormone-secreting pituitary adenoma and prolactinoma /11/. In approximately 20% of hyperprolactinemic women, a large pituitary tumor (macroprolactinoma) is found with diagnostic imaging. If no adenoma is diagnosed and other possible causes of hyperprolactinemia, such as drugs and hypothyroidism, have been excluded, the hyperprolactinemia is of functional origin.

36.5.3.2 Hyperprolactinemia in males

Men with hyperprolactinemia often present with large (> 2 cm) pituitary adenomas with markedly elevated PRL levels. The clinical picture is characterized by loss of libido, impotence, symptoms of hypogonadism with gynecomastia, and occasionally galactorrhea. Since the adenoma mass often leads to compression of the anterior pituitary, further symptoms resulting from anterior pituitary insufficiency are not uncommon. In addition, visual field defects (chiasm syndrome) with suprasellar expansion may develop. Very large pituitary adenomas, in particular in young men, are usually prolactinomas, and the endocrine axes are inactive, and prolactin levels are high.

36.6 Comments and problems

Blood sampling

Blood sampling should not be performed after a gynecological examination (stress) or test for galactorrhea. It is important to note that stress alone (e.g., anxiety over blood drawing) can cause mildly elevated PRL levels and that manipulation of the nipple (e.g., provocation of galactorrhea) can cause PRL to rise to hyperprolactinemic levels /12/. The blood draw should be performed 3 to 4 hours later i.e., between 8 a.m. and 10 a.m. /12/.

Method of determination

All immunoassays for prolactin are affected by interference from macroprolactin which renders their overall clinical performance unsatisfactory. This problem would be resolved if manifacturers complied with the requirements of the in vitro diagnostic medical devices (IVDR).

The determination of monomeric endocrinologically active PRL (post-PEG PRL) is still a rarely performed procedure. One drawback of PEG precipitation is that it is not quantitative and up to 25% of the monomeric PRL may be co precipitated with the macro PRL. However, PEG precipitation has the advantage that, if monomeric PRL levels after PEG treatment are higher than the reference interval, biologically active hyperprolactinemia is confirmed /4/. One of the main causes of the co precipitation are matrix effects of the sample /3/.

Reference interval

PRL levels follow a circadian rhythm. During the course of the day, they fall to approximately half the morning levels, then rise continuously during sleep to reach a peak in the early morning hours. Parametric total and monomeric gender-specific reference intervals were determined for six immunoassay methods using female and male sera /13/.

Half life

The half life in plasma is 40–50 minutes.

Stability in serum

Stable for 1 day at 20° and 4 °C; for long term storage samples should be frozen.

36.7 Pathophysiology

The lactotroph cells account for 20–50% of pituitary cells. They are located in the inner region of the organ and respond to dopamine. The PRL produced by these cells belongs to the same family as GH and human placental lactogen. PRL is a single chain peptide with 199 amino acids which contains six cysteine residues and three disulfide bonds and has a molecular mass of 23 kDa. PRL binds to a receptor of the class 1 cytokine family and is also expressed in organs such as the liver, pancreas, prostate and uterus.

Refer to Section 20.1 – Definition, classification, structure and function of cytokines.

PRL is released by the anterior pituitary by pulsatile secretion with approximately 10 pulsations per day in young people. PRL levels follow a circadian rhythm, with the highest levels occurring during sleep and the lowest between 8 a.m. and 10 a.m.

PRL secretion is controlled and inhibited by dopamine. Dopamine is released by hypothalamic, dopaminergic neurons and transported to the anterior pituitary via the hypophysial portal vessels. The lactotroph cells have a high basal PRL secretion which is adapted to the relevant situation through appropriate inhibition by dopamine. The released PRL in turn regulates the release of dopamine via feedback inhibition.

While PRL secretion is mainly regulated by dopamine, it is also influenced by other hormones such as TSH or vasoactive polypeptide. In contrast to the inhibitory effect of dopamine, these hormones stimulate PRL secretion.

PRL stimulates lactogenesis and galactopoiesis (i.e., the onset and maintenance of milk secretion after delivery). Furthermore, suckling induced PRL secretion maintains postpartum anovulation. Approximately 50% of women with non physiological galactorrhea have hyperprolactinemia.

In hyperprolactinemia, dopamine and opiate concentrations are increased in the basal hypothalamic regions without having any significant influence on the autonomous pituitary PRL secretion. However, the pulsatility of the GNRH producing neurons is inhibited, leading to suppression of the pulsatile LH secretion. Since proper gonadal function relies on pulsatile LH secretion, functional hypothalamic normogonadotropic hypogonadism develops. Approximately 25% of women with secondary amenorrhea have hyperprolactinemia. Absence of ovarian cyclicity leads to a lack of estrogen with atrophy of the vaginal mucosa, and to osteoporosis. Replacement therapy with pulsatile application of GnRH leads to normalization of gonadal function despite persistently elevated PRL /14/.

Dopamine and L-dopa, the direct precursor of dopamine which crosses the blood-brain barrier, inhibit PRL secretion. This is also the case with dopaminergic agonists (bromocriptine, cabergoline, lisuride, metergoline, quinagolide) /14/. They are used in the treatment of hyperprolactinemia. Following medication with these drugs, PRL falls within 2.5 to 5 hours and remains low for a good day. Dopamine agonists not only inhibit PRL secretion but in prolactinomas also lead to shrinkage of the tumor in up to 85% of cases /15/. This can be achieved even more effectively with second generation dopamine agonists such as cabergoline or the non-ergot preparation quinagolide /16/. After normalization of PRL secretion, hypogonadism is reversible and fertility can be restored.

The dopamin agonists are the best medical treatment of prolactinoma and can reduce the secretion of PRL. Approximately 70–90% of patients with microadenomas have normalized prolactin concentrations after dopamin agonist treatment, with menstruation resuming, lactation ceasing, fertility restored and shrinkage of the tumor. However 20% of patients are resistant to medications and below 4% of properly regulated patients with prolactinoma can develop acromegaly. Although prolactinomas can be removed by a frontal or a butterfly pathway, it is often difficult to completely excise and postoperative PRL levels are difficult to recover. Approximately 40–80% of surgeries will result in temporary improvement and half of them will relapse /17/.

Prolactinoma expression of specific ErbB receptors is associated with tumor invasion, symptoms, and response to doamin agonists /18/.

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