Clinical relevance of macroprolactin

The anterior pituitary hormone PRL was identified in animal species as early as 19331 but only purified in humans in 1972.2 Since then, the clinical syndrome of hyperprolactinaemia has been characterized extensively, the predominant symptoms being galactorrhoea, oligomenorrhoea or amenorrhoea and infertility in women and reduced libido, impotence and galactorrhoea in men.3–8 Hyperprolactinaemia has an estimated prevalence of 15% in women with secondary amenorrhoea,9,10 a condition that affects at least 3% of women of reproductive age.11 Pathological hyperprolactinaemia results from a lactotroph adenoma, conditions that increase TRH, such as hypothyroidism, conditions that decrease dopamine action at the lactotroph cell, such as hypothalamic or pituitary tumours, drugs such as dopamine D2 receptor antagonists, or conditions in which reduced clearance of PRL occurs, such as renal failure. By contrast, macroprolactinaemia, the presence of elevated levels of PRL of high molecular mass with little, if any, bioactivity, remains a largely under-recognized phenomenon and is not considered in the differential diagnosis of hyperprolactinaemia in current comprehensive endocrinology texts.3,4,6 Recent studies have indicated that macroprolactinaemia accounts for up to 26% of biochemical hyperprolactinaemia depending on the immunoassay in use,12–18 and thus macroprolactinaemia represents a common diagnostic pitfall, which is responsible for frequent misdiagnosis and mismanagement of hyperprolactinaemic patients.19–23 Prolactin, a globular protein consisting of 199 amino acids with three intramolecular disulfide bonds, is synthesized as a prehormone with a molecular weight of 26 kDa.24 When the prehormone is proteolytically cleaved, the resulting mature polypeptide has a molecular weight of 23 kDa, and this monomeric form accounts for the majority of total PRL in the serum of normal subjects and most patients with hyperprolactinaemia. Prolactin is secreted episodically by the anterior pituitary and is primarily under tonic inhibitory control of the hypothalamus.25 Physiological levels of PRL are higher during pregnancy and lactation than otherwise and mean serum levels are higher in women than in men.26,27 In addition to monomeric PRL, which accounts for approximately 85% of the total circulating PRL in the majority of normal subjects and in those patients with hyperprolactinaemia, other molecular weight variants of PRL can be demonstrated in serum.28,29 Big PRL, which has a molecular mass in the 50 kDa range and is thought to be a covalently bound dimer of PRL, accounts for approximately 10–15%. Big big PRL, or macroprolactin, which has a molecular mass of more than 150 kDa, usually contributes a small, though variable amount to circulating levels.28,30 Moreover, post-translational modification of pituitary PRL generates a variety of additional species, including glycosylated and phosphorylated variants, together with 14, 16 and 22 kDa proteolysed forms.25 In 1974 Rogol and Rosen31 reported for the first time that big PRL may be the predominant form of PRL in the serum of some patients with hyperprolactinaemia, while in 1981 Whittaker et al. observed that fertility was maintained even when circulating levels of big big PRL were significantly elevated. Numerous subsequent case reports and case series have demonstrated that high levels of PRL, predominantly composed of macroprolactin, are compatible with preserved menstrual cyclicity and ovulation.15,32–37 Following the simultaneous demonstrations by Hattori et al.36,37 and Leite et al.38 that the sera of a significant number of patients with idiopathic hyperprolactinaemia contained an anti-PRL antibody, it has become clear that in most cases macroprolactin is a macromolecular complex of 23 kDa monomeric PRL and an immunoglobulin. This high molecular mass form of PRL, despite being present at supraphysiological levels, does not elicit the common signs and symptoms of the hyperprolactinaemic syndrome.34,39,40 Less commonly, other forms of macroprolactin have been described, often in patients with prolactinomas. Such forms are heterogeneous with molecular mass ranging up to approximately 500 kDa. They are incompletely characterized but are often composed of either covalent or noncovalent polymers of monomeric PRL, although some hyperglysolated variants have also been described.24,41–46 Although direct biochemical or structural evidence on the nature of macroprolactin is lacking, there is a considerable amount of indirect evidence that is consistent with the concept that macroprolactin is a PRL autoantibody complex. Such evidence includes the results of immunoadsorption and precipitation experiments using anti-human immunoglobulin G (IgG), protein A and protein G,38,47–49 together with Western blot anlysis.47,50 The molecular mass of macroprolactin, 150–200 kDa on gel filtration chromatography (GFC), is also consistent with the concept of a PRL–immunoglobulin complex. Scatchard analysis by Hattori et al.37 demonstrated that a low affinity (10−6 l/mol) but high capacity (2·1 mg/l) antibody of the IgG subclass directed against PRL was present in the sera of macroprolactinaemic patients. However, evidence by Bonhoff et al.47 suggests that the PRL autoantibody complex is of high affinity. Antibody titres in such individuals were shown to remain relatively constant over a number of years. Recent clinical experience has revealed that macroprolactin in the form of a PRL–autoantibody complex is much more frequently encountered in hyperprolactinaemic sera than any of the other high molecular mass forms.51,52 Consequently, this review focuses primarily on that form of macroprolactin that behaves as a PRL–autoantibody complex. Experiments that examined the disappearance of human macroprolactin injected into rats suggested that reduced clearance of the high molecular mass complex was likely to account for the persistent hyperprolactinaemia observed in patients harbouring this PRL–autoantibody complex.39 Ahlquist and Fahie-Wilson53 examined the compartmental distribution of macroprolactin in a patient with hyperprolactinaemia and a coexisting pituitary tumour. They observed that while monomeric PRL was identified by GFC in serum, cerebrospinal fluid (CSF) and a pituitary extract, macroprolactin was present only in serum. These results indicate that macroprolactin is formed subsequent to the release of 23 kDa PRL from the pituitary. Furthermore, they demonstrated that the macroprolactin complex is confined to the intravascular space and is not present in the CSF, presumably because of its high molecular weight. Intravenous administration of TRH or the dopamine antagonist metoclopramide (MCP) to normal subjects is followed by an increase of approximately 1000–2000 mU/l in serum PRL levels, which peak within half an hour of administration.26 A blunted response is characteristic of patients with true hyperprolactinaemia.26 By contrast, in most macroprolactinaemic subjects the maximal response to these stimuli appears to be normal.38 The largest study to address this was carried out by Vallette-Kasic et al.,54 who reported a normal response of PRL to TRH in 63% and a normal response to MCP in 88% in a series of more than 100 macroprolactinaemic patients. Studies that have examined in greater detail the response to TRH or MCP have identified different kinetic profiles in macroprolactinaemics compared to normal subjects. In macroprolactinaemic subjects, an initial normal peak response, probably caused by release of pituitary monomeric PRL, is followed by a more delayed increase in macroprolactin levels, which has been explained by the binding of monomeric PRL to circulating anti-PRL autoantibodies together with continued PRL secretion.35 Bjøro et al.55 demonstrated that following TRH stimulation, an increase in macroprolactin to levels above baseline could still be demonstrated 24 h following TRH administration. These observations provide convincing evidence of delayed clearance from the circulation of macroprolactin. Similar conclusions are supported by the different rates of decline of monomeric PRL and macroprolactin following administration of bromocriptine. While bromocriptine rapidly suppressed monomeric PRL by approximately 80% after 6 h, a fall of only 20% occurred in macroprolactin during the same time,35 although suppressed values are eventually achieved. These findings in humans are supported by animal studies by Carlson et al.44 and Hattori and Inagaki,39 who observed delayed clearance rates for macroprolactin injected into rats relative to monomeric PRL. It is not surprising that PRL immunoassays detect macroprolactin. However, what is surprising and of ongoing concern to both clinicians and laboratory scientists is the significant variability in the detection of macroprolactin in hyperprolactinaemic sera by different PRL immunoassays in routine use.56–59 In a comprehensive study, Smith et al.18 examined the ability of nine of the most commonly used immunoassay platforms to measure PRL in 10 sera containing predominantly macroprolactin. The results demonstrated gross variability in the detection of PRL across the nine assay systems with 2·3–7·8-fold differences in measured PRL levels in the 10 sera (Fig. 1). Moreover, comparison of monomeric PRL levels in the 10 sera measured following GFC with the reported PRL levels following immunoassay revealed that all of the automated immunoassay systems detected macroprolactin to some extent. Although the absolute PRL levels varied in the 10 sera examined, there was consistent stratification so that the hierarchy of results obtained was reproduced in each given assay. For example, the Roche Elecsys and Wallac Delfia assays exhibited high reactivity towards macroprolactin while the Bayer Centaur and systems demonstrated low with other assays in these serum PRL levels reported by nine different in from 10 macroprolactinaemic subjects. For the PRL in each following of macroprolactin by gel filtration chromatography is reproduced from of and The The relatively consistent hierarchy in immunoassay reactivity observed the 10 examined in the study by Smith et al.18 suggests a common anti-PRL autoantibody directed against a on PRL. this is the the variability of PRL immunoassays to detect macroprolactin probably in the or detection immunoassay used by the diagnostic It is likely that the of the to which the immunoassay or detection are relative to the to which the autoantibody is may the differences in reactivity of the A variety of have been used to the of macroprolactin in serum. GFC has been used to the of PRL including macroprolactin in profiles obtained on chromatography of normal and macroprolactinaemic sera are in have been as of PRL present in the high molecular weight or macroprolactin For example, macroprolactin accounts for 85% of PRL in the macroprolactinaemic serum in However, it is also to provide the absolute of macroprolactin a diagnosis of macroprolactinaemia has been to hyperprolactinaemic patients when more than of PRL was in the macroprolactin filtration profiles obtained following chromatography of serum from a macroprolactinaemic patient on and a true hyperprolactinaemic patient for The of PRL macroprolactin and monomeric PRL are indicate the of molecular weight human immunoglobulin G 150 serum monomeric 23 kDa. Although the of GFC is and and is often considered the it does from with a low affinity antibody complex as reported by Hattori et there the for of PRL from the autoantibody during the gel filtration to an of the macroprolactin in serum. In to any does not to that macroprolactin is probably a high affinity complex. there is considerable with the levels of PRL and macroprolactin in to an of the of macroprolactin of PRL or during the gel filtration if that is a of either PRL or macroprolactin, to either or of the the nature and of GFC usually its in all but at a of has the ability to and this has been used to for the presence of macroprolactin in hyperprolactinaemic serum by Hattori et al. in The has been against GFC by a number of and PRL of following of sera with have been for the detection of While there is a relatively GFC and macroprolactin levels Following precipitation of sera with macroprolactin levels are significantly higher than values following et demonstrated that sera contained and macroprolactin as by while the same sera with an macroprolactin of and of a significant amount of monomeric PRL, together with the amount of macroprolactin present in normal is likely to account for the The precipitation is and is the of for most However, using relative than absolute PRL the to For example, of may be consistent with true hyperprolactinaemia, that is the simultaneous presence of an amount of macroprolactin and of supraphysiological levels of monomeric and reported three patients to have macroprolactinaemia on the of PRL of although monomeric PRL levels from to Such patients have an of both macroprolactin and monomeric PRL. a clinical of the presence of monomeric PRL is of concern and a diagnosis of macroprolactinaemia in this is and have a more for the diagnosis of This that monomeric PRL levels in hyperprolactinaemic sera fall to within a normal range following of macroprolactin. PRL were using sera from Prolactin levels from to mU/l in sera to mU/l in Consequently, for a diagnosis of macroprolactinaemia to be it is that PRL levels in hyperprolactinaemic sera with fall to those levels obtained when sera was that is to than of an range for the of monomeric PRL when serum is with of the used and it is on each laboratory to an the precipitation is not to all PRL immunoassays systems for some immunoassay such as is with the immunoassay In other to Elecsys and or variable as by of the PRL of PRL immunoassays have been to or for to macroprolactin using in assay this to be with the of for macroprolactin that have it is on serum PRL levels to clinicians of assay and In demonstrated that both anti-human immunoglobulin and protein A the ability to macroprolactin, evidence that macroprolactin a a of has the ability to the of immunoglobulin with the protein A has been used to for the presence of macroprolactin by from hyperprolactinaemic sera to of protein G which is more for human has findings to those obtained with protein Smith et have also examined the ability of both protein A and protein G to macroprolactin from sera containing predominantly macroprolactin to indicated that both of these are in macroprolactin from serum, although monomeric PRL levels obtained in sera were approximately higher than those obtained by both protein A and protein G of and when compared to on the molecular mass of for this to sera containing macroprolactin, and et and et the to be a to However, when et compared macroprolactin levels by and they observed that the were in a significant number of Similar findings have been reported by this both a clinical and a biochemical the concern be to those patients with true hyperprolactinaemia in the first so as to subsequent and the only of hyperprolactinaemic sera is to such sera for PRL after macroprolactin the that macroprolactin from serum to with is the most commonly used or for macroprolactin is only hyperprolactinaemia is detected in the first immunoassays that with macroprolactin, for each 100 hyperprolactinaemic for macroprolactin approximately be a low assay of the 100 hyperprolactinaemic as detected in a high assay only be When these are than be for macroprolactin, than The clinical of evidence is of significantly reduced or and of macroprolactin in When over 10 subjects were macroprolactinaemia was in of all of were of However, of the macroprolactinaemia and clinical of hyperprolactinaemia is by the in which macroprolactin has been In all of the series of macroprolactinaemic patients symptoms characteristic of hyperprolactinaemia, such as oligomenorrhoea or galactorrhoea or have been observed in a of However, as of PRL is by symptoms of hyperprolactinaemia and these symptoms are not it is not surprising that the of the symptoms and macroprolactinaemia are but not studies also reported on hyperprolactinaemic patients in for macroprolactin was carried out because of clinical Furthermore, the that some macroprolactinaemic patients to dopamine does not that the patient been to supraphysiological levels of PRL because of oligomenorrhoea may It is that galactorrhoea to to PRL or the for a of PRL for galactorrhoea to However, the of the of macroprolactinaemia in patients may be greater than by an in by et the of to is have examined the of macroprolactin. of such studies are with normal or reduced PRL in patients with The of such studies is that macroprolactin has been reported to be to some in of the for the above findings may be to differences in the PRL with some sera from macroprolactinaemic patients while have purified or purified macroprolactin by or that macroprolactin is a molecular mass complex of monomeric PRL and an IgG that appears to be confined to the PRL is likely to be in than In a series of 10 and hyperprolactinaemia. A of these hyperprolactinaemic subjects were of were of these 10 subjects symptoms or signs of other study from consisting of individuals identified only patient with These findings that macroprolactinaemia probably in the at a prevalence of in but only in The of hyperprolactinaemic sera by macroprolactinaemia depending the assay used to measure PRL and on the of patients the reported prevalence of macroprolactinaemia in studies in which all hyperprolactinaemic were for macroprolactin. While a of was reported by et al.,54 of the subjects in that study were not as they were to have true hyperprolactinaemia on characteristic clinical and thus it is likely that that represents a significant By contrast, the reported of macroprolactinaemia is of patients by et The that the high of macroprolactin in study probably because of the nature of the study which from other when the diagnosis of macroprolactinaemia was In a of macroprolactinaemic patients by Vallette-Kasic et al.,54 mean PRL levels constant during of although were Leite et a high of macroprolactin in three of of macroprolactinaemic patients. In of the low prevalence of macroprolactinaemia in the these findings are of a but When sera from with macroprolactinaemia were examined at frequent serum PRL was primarily in the macroprolactin Furthermore, obtained at was shown to of macroprolactin. Ahlquist and Fahie-Wilson53 have reported findings in a and that of the complex from the to the may be responsible for the Similar to the macromolecular forms of PRL from the more common autoantibody complex have been reported to in et the presence of anti-PRL in the sera of women at different of precipitation revealed macroprolactinaemia in of women and anti-PRL were in of the there is some evidence that macroprolactinaemia may be more during pregnancy than in the Furthermore, to that macroprolactinaemia during pregnancy to high levels of PRL has to diagnostic least case has including in a who was to have Hyperprolactinaemia in may be with reduced libido, galactorrhoea and These symptoms are at least to of the although a direct of PRL may also reports macroprolactinaemia in subjects, but those that reduced in et reported subjects who were for and to have elevated PRL levels but normal levels and evidence of any of the pituitary on When to serum PRL from these subjects to be predominantly macroprolactin. A surprising of this study was that subjects with macroprolactinaemia were identified from a total of patients with impotence that were This represents a prevalence of or approximately 100 that for in the The same reported on with pituitary in the majority of circulating PRL was These normal and normal of and However, both high levels of circulating PRL 10 and there was evidence that both secreted PRL. These findings can be explained by the concept that directed against PRL may have reduced the of PRL and thus the clinical of hyperprolactinaemia. Hyperprolactinaemia has been in which may the common of PRL in However, there has been a series of with macroprolactinaemia of the three or each in of pituitary were et reported patients and with hyperprolactinaemia who over a of to years. In all of these cases levels of either macroprolactin or big PRL were In to the consistent clinical findings in hyperprolactinaemia with elevated levels of monomeric PRL as in the case of a the reported clinical of macroprolactinaemia have varied in different the that the of PRL reports and series in which macroprolactin was measured because clinical were not of hyperprolactinaemia have not that clinical of macroprolactinaemia from those of true hyperprolactinaemia. This was first by Whittaker et who reported maintained fertility in a with hyperprolactinaemia predominantly for by macroprolactin. et women with hyperprolactinaemia and reported that in all of these patients macroprolactin was the form of PRL in Since then, reports have macroprolactinaemia in patients who biochemical hyperprolactinaemia but the clinical of However, it is not surprising that symptoms and signs of hyperprolactinaemia have frequently been to with macroprolactinaemia as it is these symptoms that initial of PRL Leite et al.38 identified macroprolactinaemia in hyperprolactinaemic subjects, of with galactorrhoea, menstrual or Vallette-Kasic et macroprolactinaemic of were In this of for macroprolactin was frequently by of hyperprolactinaemia, in some cases of a probably However, galactorrhoea was observed in of subjects and menstrual in Following of women who were or of syndrome a total of menstrual et observed that while symptoms were more likely to in subjects with true they were also frequently in macroprolactinaemic subjects In to the findings of these studies have suggested that of hyperprolactinaemia are relatively in macroprolactinaemic subjects. and 16 macroprolactinaemic patients and cases of menstrual that could not be to coexisting and of et macroprolactinaemic and symptoms of hyperprolactinaemia in only a number of subjects. The low of clinical of hyperprolactinaemia in the study probably with a low for PRL. The reported for of PRL symptoms and symptoms of oligomenorrhoea or galactorrhoea were the for PRL in only 20% of these subjects. In to into the of to macroprolactinaemia in hyperprolactinaemic it is to analysis of the of hyperprolactinaemic patients who were to have either true hyperprolactinaemia or macroprolactinaemia following of the studies these While the patients by et were the study was and patients were with the that they macroprolactinaemia or true hyperprolactinaemia. the studies of et and et reported findings in hyperprolactinaemic patients when clinical the of and and et examined hyperprolactinaemic sera to patients with true hyperprolactinaemia or macroprolactinaemia The of these of patients were also the findings of et that while oligomenorrhoea or amenorrhoea and galactorrhoea occurred more frequently in patients with true hyperprolactinaemia, and these symptoms also occurred in and of macroprolactinaemic patients. These though are not to the of patients on clinical with these and symptoms to hyperprolactinaemia in all of patients who were identified to have In of this frequently of symptoms and biochemical of hyperprolactinaemia, it is not surprising that and been that may to be but only with the that these patients with this is the that at least patient has pituitary This reported that a was to have hyperprolactinaemia during for revealed a pituitary that was there was in the symptoms and hyperprolactinaemia was to have was for of the identified macroprolactinaemic patients by et and for of macroprolactinaemic patients reported by and or amenorrhoea in the of true hyperprolactinaemia is characterized by low levels of and low or normal of and the hypothalamic of the In the study of et levels of and significantly higher in macroprolactinaemic compared with true hyperprolactinaemic subjects, consistent with of macroprolactin The prevalence of or from to in the macroprolactinaemic in which it was However, in the for other than pituitary indicate that of such are consistent with the presence of a pituitary findings are the prevalence of findings consistent with a pituitary in macroprolactinaemic subjects is to that in the that macroprolactinaemia can account for up to 26% of hyperprolactinaemia and up to of a may an adenoma, it is to be that some patients be identified who have both macroprolactinaemia and findings consistent with a pituitary identified macroprolactinaemic subjects symptoms been to hyperprolactinaemia the that macroprolactin for hyperprolactinaemia, may be for some patients. For example, in the series of patients examined by et patient with a diagnosis of infertility secondary to was with and patient with was with In be and the patient et in a study of that most patients with macroprolactinaemia and galactorrhoea, but not those with macroprolactinaemia and menstrual exhibited during This is consistent with the of on galactorrhoea in subjects and can be explained by the of on circulating levels of PRL for lactation to The fall in PRL levels to within the normal range observed in macroprolactinaemic patients on with dopamine may also convincing evidence of the of out macroprolactinaemia in hyperprolactinaemic subjects, routine has not been It is that of the series of patients with macroprolactinaemia have from the 1). It has been that approximately of hyperprolactinaemic sera reported in the may be for by This is on the reactivity of PRL immunoassays and the of in the of the of have that macroprolactin levels only be in biochemical and clinical are or a diagnosis of idiopathic hyperprolactinaemia has been have suggested that macroprolactin be measured in patients in the response to PRL is However, the findings in the study of et indicate that of macroprolactinaemic patients from patients with true hyperprolactinaemia on clinical is to diagnosis and a or delayed to the Such subjects are likely to studies and be for macroprolactin following pituitary or is of and in the case of the patient to In with routine of all hyperprolactinaemic sera for the presence of macroprolactin. In the majority of clinical macroprolactin results from the binding of monomeric PRL to an anti-PRL In this high molecular mass PRL is and appears in of reduced macroprolactin in serum to hyperprolactinaemia, which is identified by all PRL immunoassays diagnosis of true hyperprolactinaemia is of clinical as it is with significant which is to By contrast, misdiagnosis of hyperprolactinaemia to the presence of macroprolactin to patient This may and either or Furthermore, of the true of symptoms at least be delayed and may to to for those patients with It is estimated that of biochemical hyperprolactinaemia reported is because of the presence of The true hyperprolactinaemia and macroprolactinaemia be on the of clinical a for macroprolactin be in the routine of all hyperprolactinaemic patients. be to PRL assays of and serum monomeric PRL than total PRL, as is the case at the studies are to address the of macroprolactin and the clinical of the in patients. for