SEX-HORMONE-BINDING GLOBULIN

Transcription

1 Clinical Endocrinology (1974) 3, RE VE W A RT CLE SEX-HORMONE-BNDNG GLOBULN DAVD C. ANDERSON The Medical Professorial Unit, St Bartholomew's Hospital, London A. NTRODUCTON Since the discovery of thyroxine-binding globulin by Gordon and co-workers in 1952, a whole class of related plasma hormone-binding glycoproteins has become recognized. For glucocorticoids, higher vertebrates have been shown to possess corticosteroid-binding globulins (CBG), that vary somewhat in structure and properties from species to species (Daughaday, 1956, 1958; Seal & Doe, 1965, 1966; Westphal, 1970). For human CBG cortisol and progesterone bind with high affinity and testosterone binds less well. The existence of a separate human sex-hormone-binding globulin (SH BG) was suggested by the experiments of Daughaday (1958) on testosterone-binding in plasma and was first clearly demonstrated by Mercier et al. (1 966). Recently, many other steroid-binding plasma proteins have been demonstrated including ones for progesterone in the pregnant guinea-pig and oestradiol in the rat, and a sex-hormone-binding globulin similar to the human one in the rabbit (Westphal, 1971 ; Raynaud et al., 1971 ; Mahoudeau & Corvol, 1973). Similar sex-hormone-binding proteins probably exist in most primates (McCormack, 971 ;Anderson & Fisher, 1973). n this paper current knowledge of the properties, measurement and role in health and disease of the human sex-hormone-binding globulin (SHBG)* is reviewed. This protein binds reversibly and with high affinity the main biologically-active circulating androgen testosterone (T), and somewhat less well the active oestrogen, oestradiol (E2). Recent work suggests that it may be important both physiologically and in a number of pathological states including hirsutism and gynaecomastia. n order to understand how a plasma binding protein might influence sex-hormone action in target tissues we must first consider current knowledge of the mechanism of steroid action at the cellular level. * Other names used for this protein include testosterone-binding globulin; oestradiol-binding globulin ; testosterone-oestradiol-binding globulin (TeBG); sex-steroid (SBBG); sex-steroid binding protein (SBP); and 17j3-hydroxysteroid binding globulin. Correspondence: Dr D. C. Anderson, The Medical Professorial Unit, St Bartholomew's Hospital, London EClA 7BE. 69

2 Synlnesis Secretion \ \ / Specific plasma (U), (high affinity but ropidly reversible) \ / \, blnding protein,aq & a a i,/ + Steroid circulates mainly bound / / 0 -; membrane 1 Sleroid c y toplosmic Cytoplasmic rdu.lew L tu A receptor receptsr receptor + orotein + repressor steroid 1 -L --i Opened repressor bindinq Site Cei membrane Nuclear membrane (bl DNA de-repressed (cl $ / Steroid released Nuclear membrane Protein synthesis FG.. A model for steroid hormone action in target tissues. (a) Secretion and circulation; unbound steroid diffuses into cells. (b) Steroid binds to cytoplasmic receptor protein. S-P complex passes into nucleus. (c) S-P complex binds to chromatin, and allows transcription of DNA. (Binding of repressor to S-P complex, illustrated here, is speculative.) RNA and protein synthesis follows.

3 Sex- hormone- binding globulin 71 B. MECHANSM OF ACTON OF SEX HORMONES 1. Target-hormone action of androgens and oestrogens n the past decade knowledge of the mechanism of steroid hormone action has increased greatly. Fig. 1 depicts a model which is applicable to a number of different steroid hormones. n the circulation steroids are bound predominantly to plasma proteins, but only the unbound steroid enters the cell. Target tissues respond specifically to physiological levels of the steroid and differ from non-target tissues in being able to synthesize a receptor protein or proteins which are responsible for specific uptake of that steroid (Jensen & Jacobson, 1962; Gorski et al., 1968; Jensen et al., 1969; Wilson & Gloyna, 1970; James & Fotherby, 1970; O Malley, 197 1). The steroid-protein (S-P) complex is then transferred into the nucleus, a process which involves some alteration in the structure of the complex. nside the nucleus the S-P complex binds to nuclear chromatin at specific acceptor sites, which are probably of an acidic protein nature (Steggles et al., 1971), and this in some way allows ribonucleic acid (RNA) polymerase to transcribe specific sections of deoxyribonucleic acid (DNA). ncreased synthesis of ribosomal and messenger RNA, with subsequent increased protein synthesis on the ribosomes, follows rapidly. The mechanisms suggested in Fig. (c), in which binding of a repressor-molecule by the S-P complex is depicted, may prove to be incorrect in detail. However, there is no doubt of the existence and central importance of the receptor proteins in steroid hormone action. They appear to differ from the plasma steroid-binding proteins in that the binding is generally of somewhat higher specificity for one or a few closely related steroids, the association-constant is higher, and the binding is less rapidly reversible (Korenman, 1968). n oestrogen target-tissues it is E2 itself that is bound to the receptor protein. n the case of androgens it is now known that while T is the main circulating steroid it is converted to 5crdihydrotestosterone (DHT) in target tissues and this appears to be the active metabolite (Bruchovsky & Wilson, 1968; Liao & Fang, 1969). The enzyme responsible for the conversion, 5%-reductase, is present in many tissues and does not appear to be specific to target cells; however, only target tissues have been shown to possess a specific DHT-binding protein. The presence of DHT receptor protein(s) has been shown, for instance, in rat and human prostate and hamster sebaceous gland (Tveter el a[., 1970; Baulieu et al., 1971; Adachi & Kano, 1972). Baulieu s group have also produced evidence that another metabolite, Sr-androstane-3P, 17P-dio1, which is not specifically bound to a receptor-protein but does bind to SHBG may be responsible for some of the effects of testosterone on the prostate (Baulieu et al., 1969; Robe1 et al., 1971). Uptake of active steroids by these target-tissue binding proteins can be readily saturated; it can also be prevented by other active steroids competing for the same binding sites. Thus target-organ uptake of E, is inhibited by the hormonally-active non-steroidal oestrogen stilboestrol which competes for the protein binding-site. t is also known that steroidhormone antagonists (e.g. clomiphene for E,, cyproterone for DHT) act by binding onto the receptor protein and thus blocking the action of the true hormone (Jensen et al., 1966; Liao & Fang, 1969; Neumann et al., 1970). t now seems likely that the defect in the syndrome of testicular feminization, and its two animal counterparts (the pseudohermaphrodite rat and mouse), is in the structure of the DHT receptor-protein, which eads to targetorgan androgen insensitivity (Sherrins & Bardin, 1971 ; Gehring et al., 1971 ; RitzCn er al., 1972).

4 72 David C. Anderson 2. Circulation of testosterone and oestradiol Only very small fractions of the biologically-active steroid hormones in the plasma exist in the unbound state. n the case of cortisol, it is about 5-10% (Burke & Roulet, 1970) whereas for T and Ez it is between 1 and 3% (Burke & Anderson, 1972; Galviio-Teles et al., 1973). T is also bound partly to albumin (Eik-Nes et al., 1954; Schellman et al., 1954) which has a low binding affinity (K, approximately 6 x lo4 M-') but is important because of its very high capacity. A little of the T is bound to CBG; however, the main fraction of T in plasma is bound to SHBG (Mercier et al., 1966; Pearlman & CrCpy, 1967). This binding is of high affinity (K. approximately lo9 M-'), readily reversible at 37 C and, in men, is nearly saturated since the molar concentration of SHBG binding-sites in adult male plasma is only marginally greater than the molar concentration of T. n female plasma the SHBG concentration is twofold higher and the T concentration tenfold lower than in men and therefore most of the binding sites are unoccupied. E, binds less well than T to SHBG, better than T to albumin, and does not bind significantly to CBG. 3. Evidence that only the unbound fraction is biologically active Despite the small size of the unbound fraction of steroid hormones, it appears that it is this and not the bound fraction which is biologically active. Being freely interchangeable between the plasma and extravascular and intracellular compartments it is presumed to determine the intracellular concentration of the steroid. Much of the early evidence for the inactivity of the bound fraction came from studies with other steroids such as cortisol. Thus, oestrogens increase total cortisol levels in vivo by elevating CBG, but this reduces the metabolic effect (Robertson et al., 1959; Peterson et al., 1960; Mills et al., 1960). Prior injection of CBG nullified the effect of cortisol in vivo in the mouse liver glycogen assay (Slaunwhite et a/., 1962), and cortisol antagonism of insulin in mouse ear preparations was much reduced by CBG (Matsui & Plager, 1966). Other studies with albumin, a,-acidic glycoprotein and CBG have shown that protein-bound progesterone is biologically inactive (see Burton & Westphal, 1972, for review). Recently several groups of workers have also presented evidence for the inactivity of protein-bound T and E,. Mowszowicz et al. (1970) found that the in vitro aromatization of T to E, by isolated rat liver microsomes is markedly slowed by the addition of small amounts of partially-purified human SHBG, or by large amounts of albumin. Lasnitzki & Franklin (1972) studied the uptake of tritium-labelled T and its metabolism to DHT, and the degree of maintenence of androgen-induced histological changes in rat prostate explants in organ culture. They found that increasing amounts of human or animal serum inhibited T uptake and action; human pregnancy serum with its high SHBG level was more inhibitory than male serum. Further strong evidence for the inactivity of bound steroid comes from Raynaud (1973) who compared the relative biological potencies of two oestrogens in the immature rat at different ages. The rat possesses a plasma oestradiol-binding protein (EBP) whose concentration is high at birth and falls off progressively to reach undetectable levels in the adult. E, binds strongly to the protein, whereas the synthetic oestrogen R 2858 does not. Large doses of E2 produced no effect on uterine weight in the young rat, when EBP levels were still high, but the steroid was extremely potent in the older animals when EBP levels had fallen. n contrast, the synthetic oestrogen was also active in the immature animals. Finally, circumstantial evidence of the inactivity of bound T comes from the clinical

5 Sex-hormone-binding globulin 73 observations in situations, associated with increased or decreased levels of SHBG, to be discussed later. Keller et al. (1969), while accepting that in most tissues only the free steroid exerts biological activity, have suggested that the liver is an exception. They found that corticosterone readily stimulated production of the enzyme alanine aminotransferase in the liver, regardless of CBG level, while in the pancreas its induction occurred only if the CBG level was low. They interpret this as evidence for pinocytosis of CBG-bound corticosterone (possible in the liver because plasma may directly bathe the cells) with subsequent intracellular release of CBG-bound steroid. t could equally be that this close proximity ofpfasma and hepatic cells permits more rapid dissociation of steroid off CBG than can occur in other tissues, allowing a greater flux of cortisol into liver cells at any given cortisol or CBG concentration. An additional factor which would make for more rapid flux of cortisol into the liver might be a high intracellular CBG level, since CBG is synthesized in the liver. 4. Functions of plasma steroid-binding proteins t seems therefore that the concept of a steroid-transport function for proteins such as SHBG and CBG (Sandberg et al., 1957) analogous to the transport of iron by transferrin, is incorrect. Similarly, the proteins are not needed to solubilize the small amounts of steroids in plasma (De Moor el al., 1968). Daughaday (1959) suggested a buffering role for proteinbinding in stabilizing the unbound concentration, a view favoured by Burton & Westphal (1972). Thus, it might be of importance to have a relatively slow rate of change in unbound plasma steroid concentration for accurate monitoring by the hypothalamus. Alternatively, since protein-binding reduces the metabolic clearance rate of steroids it might have economic function in lowering the production rate required to achieve a given unbound level. Raynaud s work (Raynaud, 1973), discussed above, on the rat EBP suggests that here the protein may have a quite different function, namely to inactivate E, in the fetal rat. Burke & Anderson (1972) have recently presented evidence for a special role of human SHBG in oestrogen-androgen balance and this is discussed in detail below. This function does not exclude the possibility that the protein also has other important effects. C. DSCOVERY AND PHYSCO-CHEMCAL PROPERTES OF SEX- HORMONE-BNDNG GLOBULN The first clear evidence for a specific testosterone-binding globulin came from the studies of Mercier et al. (1966), who separated a testosterone-binding B-globulin from albumin and CBG by eectrophoresis. Rosenbaum et al. (1966) produced evidence for an oestradiolbinding protein in human plasma, and it was soon shown by competition experiments that the two steroids were bound to the same site on the same protein (Van Baelen et al., 1968; Murphy, 1968). Mercier-Bodard et al. (1970) have recently reviewed the early work characterizing the protein and have described its partial purification. Unfortunately, much of its binding-activity was destroyed in the process, and others have found similar difficulty in achieving its purification (Rosner et al., 1969). Mowsowicz er al. (1970) have purified SHBG 220-fold by means of precipitation, gel filtration and ion-exchange chromatography, with retention of its binding properties, Cautrecasas & Anfinson (1971) have purified SHBG by means of affinity chromatography, but the method used and results obtained were not described in detail.

6 74 David C. Anderson SHBG is a 1-globulin, and its molecular weight has been variously estimated at 52,000 (Mercier-Bodard et al., 1970) and 95, ,000 (Van Baelen et al., 1968; Rosner et al., 1969; Corvol et al., 1971; Hansson et al., 1972); this suggests that it may exist in plasma as a dimer, which dissociates to form a monomer on extensive purification (Hansson et af., 1972). The lower estimated molecular weight of 52,000 is virtually identical with that of CBG (Westphal, 1970). SHBG is rapidly denatured by heating to 60 C (Pearlman & CrCpy, 1967; Steeno et al., 1968). Like other specific plasma steroid binding-proteins, its affinity is 0 _OO % Relative bioassav potency (rat seminal vesicle weignt ) FG. 2. Relation between binding to SHBG (as ability to displace T off SHBG) and bioassay potency of androgens (testosterone = 100%) in rat seminal vesicle assay, as given by Dorfman & Shipley (1956). Key: ( ) testosterone; (2) Sa-dihydrotestosterone; (3) 5a-androstane-3a-17/3- diol; (4) A4 androstenedione; (5) androsterone; (6) dehydroepiandrosterone; (7) A5 androstenediol. higher at low temperatures. This is principally due to a marked decrease in the dissociation rate constant with falling temperature which is more marked than the decrease in association rate constant (Heyns & de Moor, 1971a). Studies using isoelectric focusing indicate considerable micro-heterogeneity of SHBG, probably due to variable amounts of sialic acid in the molecule (Van Baelen et al., 1969; Hansson et al., 1972). A number of studies of its binding specificity in dilute plasma or with partially purified SHBG have been reported (Kato & Horton, 1968; Steeno et af., 1968; Vermeulen & Verconck, 1968; Mayes & Nugent, 1968; Murphy, 1968, 1970; Anderson, 1970). n dilute plasma, at low temperatures, SHBG has about three times higher affinity for T than it has for E2. Changes in structure around both the 'A' and 'D' rings affect the binding of steroids to SHBG; in particular a planar 'A' ring and a 178 hydroxyl group in the 'D' ring are necessary. Among the androgens there is an apparent relationship between in vivo biological activity of steroids and their ability to bind to SHBG; the more potent the androgen the

7 ~~ Sex-hormone- binding globulin 75 greater its binding to SHBG (Fig. 2). Thus DHT binds almost three times as well as T and in Some systems isa more potent androgen than T. By contrast A4-androstenedione is a much weaker androgen (about 20% as potent as T, and part of this is due to conversion to T), and it binds with only 4% of the affinity. E,, the biologically active oestrogen, binds to SHBG whereas oestrone and oestriol, which are much weaker oestrogens, do not bind. The approximate normal mean plasma concentrations of steroids that bind significantly to SHBG, taken from the literature, are shown in Table 1. Several androgens that bind to SHBG, in addition to T, are present in plasma. n men clearly T is the main circulating TABLE 1. Approximate normal mean plasma concentration (ng/ml) of steroids that bind significantly to SHBG Steroid Adult men Adult women Testosterone a-Dihydrotestosterone * A5-androstene-3b, 17b-diol a-androstene-3a, 17b-dio Oestradi0l-7j3~*~ * References: to & Horton (1970); Tremblay et al. (1970); Rosenfield & Otto (1972); Strickland er a/. (1973); Baird & Guevara(1969); Chopra era/. (1972). * Depending on the stage of the menstrual cycle. androgen that binds to SHBG while in women levels are much lower, and the other 17phydroxy-androgens are relatively more important. E, is present at very much lower concentrations than T, even in female plasma. The specificity of binding to SHBG can be used as the basis for a competitive proteinbinding assay of plasma 17P-hydroxy-androgens, which is of considerable clinical value in both sexes (Horton et al., 1967; Murphy, 1969; Anderson, 1970; Rosenfield, 1971 ; Anderson ef al., 1972a). Although many specific testosterone assays have been developed using SHBG as binding protein, these have been largely superseded by specific radioimmunoassays which require less prior purification (Furuyama et al., 1970; smail et al., 1972). D. METHODS OF MEASUREMENT 1. SHBG Many of the earlier studies on changes in SHBG in normal and abnormal conditions used indirect methods to give a binding index or apparent binding capacity. The techniques included Sephadex equilibration, equilibrium dialysis of diluted plasma, paper electrophoresis, competitive absorption and polyacrylamide gel electrophoresis (Pearlman & Crtpy, 1967; Rosner & Deakins, 1968; Vermeulen & Verdonck, 1968; Vermeulen et al., 1969; August et al., 1969). Whilst much of the information derived was qualitatively correct, these methods did not provide direct quantitative data on either the changes in SHBG concentration or the free hormone concentration in the plasma. Several recent methods give more nearly quantitative results (Corvol et al., 1971 ; Heyns & de Moor, 1971b; Rosner, 1972; Anderson et al., 1972b). This is achieved in part by using tritiated DHT as ligand,

8 76 David C. Anderson as this is preferable to tritiated T because of its higher affinity for SHBG and the fact that it does not bind to CBG. n Rosner s method, saturating amounts of tritiated DHT are added to dilute plasma, and SHBG is selectively precipitated with ammonium sulphate; the amount of DHT precipitated gives a direct measure of SHBG binding-site concentration. Our own method is based on the principle of competitive adsorption (Heyns et al., 1968), using florisil as adsorbent.. The effect of albumin is minimized by using low concentrations of DHT, and by adding a constant amount of exogenous albumin to each tube (Anderson, 1973); for each plasma sample serial dilutions are incubated with constant amount of tracer tritiated DHT, and relative concentrations are derived from the relative positions of plasma dilution curves. The molar SHBG binding-site concentration in the standard reference plasma which is run with each batch was determined independently by steady-state gel filtration and Rosenthal s graphical analysis (Rosenthal, 1967). We find close agreement between these two methods except at very high concentrations, when Rosner s method gives higher figures. 2. Unbound testosterone and oestradiol Several methods have been used and all have their disadvantages. The three principal P a C 3 4 z 3 2 MkSO MtSO SH9G cancentrotion (x O-~M) FG. 3. n vitro experiment showing differential effect of SHBG on the percentage unbound of tracer tritiated testosterone (0) and oestradiol (0). Normal male and female concentrations indicated by the horizontal bars. Reproduced from Burke & Anderson (1972) with permission of the Editor.

9 Sex- hormone-binding globulin 77 techniques are equilibrium dialysis, ultrafiltration (see Baulieu et al., 1970) and steady-state gel filtration (Burke, 1969; Marshall et al., 1972; Burke &Anderson, 1972). t is not possible to extrapolate to the in vivo situation unless observations are made on undiluted plasma, at physiological temperature and ph; for this purpose steady-state gel filtration has considerable advantages, although quite large volumes (8 ml of plasma) are required. t is usually necessary to measure the percentage unbound of added tritium-labelled steroid, and the total concentration independently, to arrive at the actual unbound level. Such methods require special apparatus and are quite time-consuming; for clinical purposes a binding index may give useful semi-quantitative information much more readily. E. PHYSOLOGCAL ROLE OF SHBG 1. Effect of changes in SHBG concentration on the relative unbound fractions of testosterone and oestradiol: servo-amplijier function Burke & Anderson (1972) have recently carried out an in vitro study of the effect of changing the SHBG concentration in plasma on the relative binding of T and E,. A range of SHBG concentrations was achieved by mixing pools of heat-inactivated plasma, with male, female and late pregnancy plasma in various proportions. Endogenous steroids were previously removed by charcoal adsorption. Fig. 3 shows that changes in SHBG concentration produce a much greater alteration of percentage unbound T than E,. This is partly because E2 binds less well than T to SHBG, and partly because E, binds better than T to albumin (Fig. 4). n the same study it was found that increasing the T concentration over the physiological range did not significantly increase the unbound E,; similarly a marked increase in cortisol did not significantly increase the unbound T by displacement off CBG. 2 i SHBG A bumin u D X v, F 0 FG. 4. Equilibrium constants at 37 C (K.1 of testosterone (0) and oestradiol(0) for SHBG (left) and albumin (right) (assuming single binding site). These were derived using steady-state gel filtration and Roesenthal s (1967) graphical method. Results shown for two male and female pools. Data from Burke & Anderson (1972).

10 78 David C. Anderson These findings, taken with the known fact that oestrogens stimulate and androgens inhibit SHBG production (Pearlman et a[., 1967; Vermeulen et al., 1969) suggested a new role for SHBG. This is diagramatically illustrated in Fig. 5. Here, the unbound T and E, levels are denoted by the boxes, at either end of a see-saw. SHBG is the fulcrum, whose position moves in response to oestrogens and androgens. f E, production increases (A), the unbound E, rises, and SHBG production is stimulated; this causes a differential fall in unbound T, tipping the sex-hormone balance further in favour of oestrogens. Conversely, a rise in T production (C) will amplify its own effect by causing a fall in SHBG and a differential rise in unbound T relative to Ez. Furthermore, we predict from this that any other factor altering SHBG concentration may tip the balance in favour of either T or E,, depending on the direction of the change in SHBG concentration. 2. Efect of protein-binding on the metabolism of steroids There is considerable evidence that one of the effects of SHBG-binding is to cause a reduction in the rate of hepatic degradation of steroids it binds. The metabolic clearance rate (MCR) of androstenedione, which is poorly bound to SHBG, exceeds the hepatic blood flow. For E,, T and DHT it is considerably lower, bearing an inverse relationship to the affinity of binding to SHBG (Southren et al., 1967; Rivarola et al., 1967; Longcope et al., 1968; Mahoudeau el al., 1971; Saez ef af., 1972). The MCR for T and DHT is about twice as great in men as in women; this does not appear to be due to the higher plasma T level in men than women (Vermeulen et al., 1969) and is probably due principally to the differences in SHBG concentration. n women the clearance rate of testosterone rises with chronic androgen administration when the SHBG level falls (Vermeulen et al., 1969). Conversely, the MCR is reduced in states associated with raised SHBG levels, for instance due to increased oestrogens or thyroid hormones (Mahoudeau et al., 1971 ; Saez et a/., 1972). There appears to be a relationship between plasma binding to SHBG and 5a-reduction of testosterone (Mauvais-Jarvis et al., 1970a, b). t is not clear, however, to what extent the greater production of Sa-reduced metabolites of testosterone in men and hirsute women compared with normal women is due to lower SHBG levels, and to what extent it is a consequence of the stimulation by androgens of enzymes present in target cells. Conversely, the reduction in the production of these metabolites caused by oestrogens may be due to direct inhibition of the enzyme 5a-reductase, rather than to the rise in SHBG. This is supported by the greater degree of Sa-reduction of testosterone in thyrotoxicosis than in pregnancy, while SHBG levels are comparably raised in the two states (Saez et al., 1972). Thyroid hormones stimulate 5x-reductase while oestrogens inhibit it (Mauvais-Jarvis & Bercovici, 1967). 3. Hormonal injluences on SHBG There have been several studies showing that the administration of androgens in both sexes causes a reduction in SHBG level, and that oestrogens cause a rise in SHBG level (Pearlman et al., 1967; Migeon et al., 1968; De Moor et al., 1969; Vermeulen et al., 1969; Tochimoto et al., 1970; Ruder et a/., 1971; Murray ef al., 1973). The level of SHBG appears to be remarkably sensitive to a change in relative production of androgens and oestrogens, Thus, the administration of only 20 pg daily of ethinyl oestradiol for 5 weeks to male criminal sex-offenders produced a 150% increase in SHBG, which was accompanied

11 Sex-hormone- binding globulin 79 by a 50% increase in plasma T and lutehizing hormone (LH) (Murray et al., 1973). The rise in SHBG with low doses of oestrogen occurs slowly, and the inference from these data was that the resulting fall in unbound T caused a reduction in negative feedback suppression, and a rise in serum LH despite the presence of increased oestrogen levels. This appears to be clear evidence of separate feedback control of LH by androgens independent of that of oestrogens in man. Unbound testosterone Unbound oestradiol A. Oestrogen servo - Oestradiol ncreases SHBG 8. Balance C. Androgen servo Testosterone - Decreases SHBG FG. 5. Diagram illustrating proposed role of SHBG in regulating relative unbound T and Ez concentrations. SHBG is denoted by the fulcrum, and unbound T and EZ by the boxes on each end of the see-saw. (A) ncreased E, production; unbound E2 rises, and by stimulating SHBG production causes a differential fall in unbound T. Here SHBG change acts as an oestrogen servo, (B) Balance. (C) ncreased androgen production. Unbound T rises, and effect is then amplified by a fall in SHBG, which tips the balance further in favour of T (Androgen servo). Normal adult female situation = A; normal adult male situation = C. Any third factor (such as thyroid hormones) which alters the SHBG concentration will also be expected to alter the ratio of unbound T to Ez. (From Burke & Anderson, 1972, by permission of the Editor of Nuture.) The studies of Forest et al. (1968) and Forest & Bertrand (1972) suggest that the progestational agent medroxyprogesterone acetate may lower SHBG in both sexes before and after puberty. Chlormadinone and lynestranol, however, have no effect (Vermeulen et al., 1969). The synthetic anti-oestrogen clomiphene citrate produces a moderate rise in SHBG and CBG, presumably because of the weakly oestrogenic nature of this substance (Dray er al., 1970; Marshall et a[., 1972). The unbound T level nevertheless rises in men treated with clomiphene, indicating that the T rise is not entirely due to the rise in SHBG and is in part due to increased T production (Marshall et al., 1972). Anti-androgens, curiously, have F*

12 80 David C. Anderson no effect (Murray et al., 1973). Growth hormone has been found to lower SHBG levels in normal children (De Moor et al., 1972), a fact of relevance to the findings in acromegaly, discussed below. Glucocorticoids do not produce any fall in the already low SHBG levels in hirsute women (Anderson et al., 1972b), but Vermeulen et al. (1969) have produced evidence for a possible inhibitory effect of large doses of glucocorticoids on SHBG in men with chronic asthmatic bronchitis. LH.20 t n!k- (subject J.McN.) 0 C.M.H. Subec J.kcN. SHBC? Doys of cycle FG. 6. Serial measurements of SHBG throughout the menstrual cycle in two normal women. No consistent change was related to times of cycle. The LH peak is also shown in one of the subjects. One final hormonal influence, which is so marked that it may well be of physiological importance, is that of thyroid hormones. These produce an elevation of SHBG in normal subjects of both sexes and in hirsute women, when given in slightly more than replacement doses (Dray et al., 1969; Ruder et al., 1971 ; Anderson et al., 1972b). 4. Physiological changes in SHBG concentration The concentration of SHBG is higher in children than in adults, and with puberty falls slightly in the female and markedly in the male. As indicated in Table 2, and Fig. 8, levels in adult men are half those in women of comparable age (Vermeulen et al., 1969 ; August et al., 1969; Corvol et al., 1971 ; Anderson et al., 1972b). The reason for this difference lies in the opposing effects of androgens and oestrogens on SHBG synthesis by the liver. n the prepubertal child production of both T and E2 is very low and SHBG production is probably uninfluenced by sex hormones. t may well depend on adequate levels of thyroid hormones

13 Sex- hormone- binding globulin 81 for its maintenance before puberty, since thyroid hormones exert a marked effect in adults (see below). At the time of puberty in the female there is an increase in E, and in ovarian and adrenal androgen production. t is possible that at least one factor responsible for increased adrenal androgen production is an effect of oestrogens on adrenal steroid biosynthesis (Bongiovanni et al., 1967; Cathro, 1969; Sobrinho et af., 1970). t appears that the balanced increase in oestrogen and androgen production produces only a small fall in SHBG in the female. n males, in contrast, the increase in T production is much greater and that of E2 much less than in the female, and the combined effect is to produce a considerably greater fall in circulating levels of SHBG. 'Or S x W m ;*x 0 0 v) 4' e Age (years) FG. 7. ncrease in SHBG concentration in normal men after 50 years. Several workers have studied SHBG by indirect methods during the menstrual cycle, and have found no consistent changes (Pearlman & CrCpy, 1967; Vermeulen et al., 1969). This is borne out by our own findings in two complete menstrual cycles (Fig. 6). This failure to demonstrate any pre-ovulatory rise in SHBG is perhaps surprising in view of the marked rise in plasma E2 at this time. t may be that the time-course of response of SHBG is too slow to show a consistent change. There is a definite increase in SHBG levels in adult men after the age of 50, associated with a fall in total testosterone level (Vermeulen et al., 1972). This is demonstrated in Fig. 7. SHBG levels rise markedly in pregnancy to some 5-10 times those in the normal nonpregnant female; precise estimates vary according to the methods used. The protein appears to behave as 'normal' SHBG (Corvol et al., 1971). Our own findings confirm that levels remain high until delivery, and thereafter fall to normal with a half-life of about 5 days. The maternal plasma levels of T rise two- to four-fold during pregnancy (Rivaroa et al.,

14 82 David C. Anderson 1968; August et al., 1969) and other 178-hydroxy-androgens even more (Murphy, 1971); because of the increased binding, maternal virilization is rare. t is of considerable interest that the levels of SHBG in the fetal plasma at term are in the adult male range, and rise soon after birth (Rivarola et al., 1968, Vermeulen et al., 1969; August et al., 1969; Forest et al., 1971). Our own estimates indicate a twenty-fold gradient in SHBG concentration across the placental bed, but with no difference in fetal levels between the sexes. t seems possible that fetal adrenal androgens inhibit SHBG production in the face of high oestrogen levels. We would predict that a marked concentration gradient across the placenta will serve to reduce the unbound androgen level in the fetus. SHBG may thus act as a magnet drawing androgens from the fetal into the maternal circulation, for degradation in the maternal liver; this might be important in preventing virilization of the female fetus. F. ROLE OF SHBG N DSEASE Reducedplasma levels 1. Hirsutism andpolycystic ovaries One of the most consistent abnormalities in hirsute women regardless of their menstrual history, is a reduction in plasma levels of SHBG (Dray et al., 1969; Southren et al., 1969; Vermeulen et al., 1969; Rosenfield, 1971 ; Anderson et al., 1972b). This is well illustrated by our findings in thirty-five hirsute women (Fig. 8; Table 2) in whom the SHBG concentration was reduced in over 80%. This finding is independent of the presence or absence of polycystic ovaries (PCO). This fall appears to account for the equally consistent finding of an elevated MCR of T (Bardin & Lipsett, 1967; Southren et al., 1969; Kirschner et al., 1970; Kirschner & Bardin, 1972). n many cases it is associated with a rise in plasma T and other 17P-hydroxy-androgens (170HA); for instance the mean plasma 170HA level, measured by competitive protein binding (Anderson, 1970), is increased two-fold, with considerable overlap with normal (Fig. 8, bottom half). ndirect measurements of free T in plasma demonstrate a rise in unbound androgen level in most hirsute women (Rosenfield, 1971 ; Vermeulen et al., 1971). The simplest index of this type, which is roughly related to the unbound 170HA, is the ratio of the molar concentrations of 170HA to SHBG. This ratio separates hirsute and normal women almost completely, as shown in Fig. 9, and provides a clear indicator of the degree of the androgen abnormality, of potential value in following treatment. Recently Shuster (1972) has focused attention on the skin, suggesting that it is abnormally sensitive to androgens in hirsute women. But these findings of a marked rise in 170HA : SHBG ratio are strong evidence that the primary abnormality is hormonal, although no doubt individuals vary considerably in their cutaneous response to the raised unbound androgen levels. We still do not know, however, what the primary abnormalities in hirsutism are or why the SHBG is low. Many of these women have menstrual disturbances, associated with polycystic ovaries (PCO). A common assumption is that here the ovaries are primarily at fault, and this gains some support from the return of periods, and the finding of a fall in plasma T and its production rate, in most women with PCO who are submitted to ovarian wedge resection (Lloyd et al., 1966; New et al., 1968; Southren et al., 1969). However, Yen et al. (1970) showed high fluctuating LH levels in these patients and this suggests that the ovaries may be abnormal as a secondary result of abnormal gonadotrophic stimulation. Other evidence suggests that the adrenals are at fault, since plasma levels of T and related 17/?-hydroxy-androgens are usually suppressed markedly by glucocorticoids (Bardin et al.,

15 3 Sex-hormone- binding globulin ; Rosenfield et al., 1972; Anderson et al., 1972b). Furthermore, data from adrenal vein catheterization has indicated increased adrenal production of dehydroepiandrosterone and A4 androstenedione in hirsute women (Weinheimer et al., 1966). n contrast, the catheterization data of Kirschner & Jacobs (1971) suggests a major ovarian source for T and androstenedione, although this conclusion rests on some questionable assumptions regarding their data. 15 r Normal 0 Hirsure? Normal cf ~? z m E! n FG. 8. Plasma SHBG and 178 hydroxyandrogen (170HA) levels in hirsute women. SHBG levels subnormal in 80% of subjects. Mean 170HA raised two-fold, but marked overlap with normal. (Note: log scale used because of log-normal distribution of 170HA; right-hand scale for 170HA concentration in molar terms, for comparison with SHBG.) The main possible sites for the primary abnormality in hirsutism, and the likely sequence of secondary effects, are as follows: (1) The primary abnormality might be in the hypothalamus or pituitary, resulting in failure of the normal mid-cycle surge of LH. This would be analogous to the androgenized female rat, whose hypothalamus has been masculinized by testosterone at birth (Matsutama et al., 1966). These animals do not ovulate, and after puberty they develop polycystic ovaries. (2) Excess production of ACTH may cause increased adrenal androgen production ; this is the primary cause of the hirsutism in Cushing s disease.

16 84 David C. Anderson (3) The defect may be in the adrenal itself or in some factor other than ACTH that affects adrenal androgen production. (4) Similarly, it may be in the ovary or some factor other than gonadotrophins controlling ovarian androgen production. (5) Production of precursors such as androstenedione might be normal, but their conversion to T excessive. Lesions in these five sites could cause the observed effects, namely increased T production; a rise in unbound T, a fall in SHBG level, and an increase in MCR for T. 2.c 2 0.o (201 (351 (171 FG. 9. Ratio of molar concentrations of 170HA: SHBG in normal women, hirsute women and normal men; virtually complete separation of the three groups. 0 0' (6) A sixth possible site of primary abnormality is in the structure or production of SHBG itself. Levels may also be low through inadequate stimulation by oestrogens. Raised unbound androgen levels, by disturbing feedback mechanisms controlling gonadotrophins, might lead secondarily to increased LH production, ovarian stimulation, and further androgen production. Therapeutic implications in hirsutism. The therapeutic implication of the consistent findings of a low SHBG and a raised 170HA : SHBG ratio in hirsutism is quite simple. n such women it seems reasonable that measures should be directed towards producing a maximum fall in this ratio, and therefore in the unbound level of testosterone and other biologically-active 17b-hydroxy-androgens (170HA). We find a consistent fall in 170HA on prednisolone in moderate dosage ( mg/day), but the SHBG does not rise on this therapy. A rise in SHBG without any parallel rise in 170HA is produced either by a low

17 Months FG. 10. llustration of the value of SHBG measurements in assessing therapy in a patient with polycystic ovaries and hirsutism. 17OHA level suppresses with prednisolone alone, but SHBG remains low; addition of triiodothyronine produces a brisk rise in SHBG to normal, without a corresponding rise in 170HA. Overall 170HA : SHBG ratio fall to about 10% of control values. The hirsutism is improving clinically. TABLE 2. Sex hormone binding globulin levels (x lo-" M)* Diagnosis NO. Meant 95% Confidence limitst Normal men Normal women Men with endocrine disease Thyro toxicosist Myxoedema Acromegaly Women with endocrine disease Thyrotoxicosis Untreated? Treated medically for 3 months Myxoedemat Hirsutismt ' '22-11' * Method of Anderson et af. (1972b) slightly modified by inclusion of 2 mg/ml albumin in final incubate. t P<O.005 for significance of difference from normal. $ Calculated as loglo and back-transformed to arithmetic values.

18 David C. Anderson dose oestrogen-progestogen contraceptive pill, or by triiodothyronine (40-80 pg/day). t remains to be seen which of these two is better; on theoretical grounds oestrogens seem preferable, since in addition to their effect on SHBG they inhibit 5a-reductase, the enzyme that converts T to DHT, while thyroid hormones stimulate it (Mauvais-Jarvis & Bercovici, 1967). We find that by combining prednisolone with an oestrogen-progestogen containing contraceptive pill, or moderate doses of triiodothyronine the 170HA : SHBG ratio can be dramatically reduced in most hirsute women. Fig. 10 shows the results in one such subject, where prednisolone was given first and produced a marked fall in 170HA without affecting the SHBG. The effect of triiodothyronine on the SHBG is obvious, and the 170HA : SHBG ratio has fallen to a tenth of control levels on combined therapy. There is considerable variation in these responses, making it important to make these measurements serially on each patient. t remains to be seen just how effective a marked reduction in unbound androgens is in different types and at different stages of severity of hirsutism. 2. Other endocrine disorders SHBG levels are low in myxoedema (see Table 2) and return to normal with thyroid hormone replacement (Olivo et al., 1970). Levels also appear to be low in acromegaly (De Moor et al., 1971, 1972; Anderson, 1973), possibly due to a direct effect of growth hormone. This may be an important factor in causing hirsuties in acromegalic women, a not uncommon presenting symptom. SHBG levels are certainly very low and the 170HA : SHBG ratio raised ig Cushing s syndrome (De Moor et al., 1968; Anderson, 1973); this may be in part the result of increased adrenal androgen production and in part due to increased glucocorticoid production inhibiting SHBG synthesis. Levels rise to normal after adrenalectomy. Elevated plasma levels 3. Hypogonadism and testicular feminization SHBG levels are in the female range in men with hypogonadism (Vermeulen et al., 1969) and in subjects with testicular feminization, where there is end-organ androgen resistance (Mauvais-Jarvis ef al., 1970b; Rosenfield el al., 1971). t has been suggested that SHBG may be abnormal in structure in testicular feminization. f this was so, we would predict a difference in plasma binding for T and E2 in this syndrome. However, Fig. 11, showing results of competitive adsorption, demonstrates that SHBG is qualitatively normal with respect to T and E2 binding in this syndrome. 4. Hepatic cirrhosis in men Several workers have shown that SHBG levels are higher in plasma from cirrhotic subjects (Rosenbaum et al., 1966; Vermeulen et al., 1969; Rosner, 1972). We have recently studied the levels of unbound 178 hydroxyandrogens (170HA), unbound E2, SHBG and LH in twenty-five men with chronic liver disease due to a variety of causes (Galviio-Teles et al., 1973). The main findings were a modest reduction in unbound 170HA due to a twofold rise in SHBG concentration. Unbound E, levels were normal, probably because here the effect of a fall in albumin cancels out the opposite effect of a rise in SHBG. The changes were most marked in eight alcoholic cirrhotics, in whom the mean unbound 170HA was

19 Sex-hormone-binding globulin pg/ml, compared with 160 pg/ml in control subjects. t appears likely from these studies that in chronic liver disease the SHBG level rises first, as a result of an increase in hepatic SHBG production. As a consequence, the unbound T level falls and the hypothalamus/ pituitary responds by increasing LH production. However, apparently this does not produce sufficient Leydig-cell stimulation to return the unbound T to normal. t appears probable that the changes of hypogonadism and gynaecomastia, which are seen especially in alcoholic cirrhosis, result more from a fall in unbound T than from any rise in unbound E,. *\* Subject N.W. 28 years (normal male).* '*/* *\ Plnsrno dllution FG. 11. Demonstration of qualitatively normal SHBG in a patient with testicular feminization. Competitive adsorption of 3H-T and 'H-E2 (separately) from increasingly dilute plasma by florisil. Similar relative positions of dilution curves in testicular feminization (left) and normal male plasma (right) make it unlikely that SHBG is structurally abnormal. The cause of the raised SHBG levels in cirrhosis is uncertain. One possibility is that it results from impaired conversion of E, to oestriol, known to occur in chronic liver disease (Zumoff et al., 1968; Adlercreutz, 1970). This might result in a local intrahepatic elevation of E, concentration. t is of interest that in two hirsute women with cirrhosis of liver the SHBG levels were in the normal female range, and the plasma 170HA levels unusually high for hirsutism (Anderson, 1973). 5. Gynaecomastia The majority of non-cirrhotic men with gynaecomastia do not have abnormal SHBG levels. However, we have found one subject (Fig. 12) with gynaecomastia and impotence in whom SHBG levels were raised two- to three-fold (Anderson et al., 1972~). He had high total 170HA and T levels, high LH, high SHBG, and normal unbound 170HA. Levels of E, were only marginally raised. We were able to demonstrate by stimulation and suppression tests that the hypothalamus-pituitary-leydig cell axis was functioning normally. Thyrotoxicosis

20 88 David C. Anderson was excluded, and the primary abnormality was probably hepatic since the gynaecomastia developed after an attack of infectious hepatitis; liver function at the time of study, however, appeared normal. This interesting case demonstrates that SHBG should be measured in men with gynaecomastia and impotence, especially if the total T levels are raised, as it may be elevated. n such cases treatment with androgens might be expected to lower the SHBG although in this case it was not successful. The biochemical findings in this patient were strikingly similar to those seen in men given low doses of oestrogen, referred to above. 7 LH HA SHBG Unbound Unbound Oestrodiol FG. 12. Basal measurements in S.W., a man presenting with gynaecomastia and impotence. High LH, 170HA (and T) and marked rise in SHBG. Per cent unbound T reduced, and unbound 170HA (total 170HA x % unbound T) low normal. E2 marginally raised. See text for discussion. (From Proceedings of the Royal Society of Medicine, by permission of the Editor,) 6. Thyrotoxicosis in women As shown in Table 2, the SHBG levels in thyrotoxicosis are very high, and they fall to normal on treatment with anti-thyroid drugs (CrCpy et a[., 1967; Gordon et a/., 1969; Olivo et al., 1970). Thyrotoxic women often have amenorrhoea, and this, too, responds to anti-thyroid drugs. Recently, Akande & Hockaday (1972a, b) have shown that the pattern of oestrogen and LH levels in plasma is abnormal. Levels of E2 in the follicular phase are higher than normal, as are LH levels in both follicular and luteal phases; however, the midcycle LH peak is reduced. t seems quite possible that some or all of these findings may result from the marked increase in SHBG levels, impairing by increased binding both the positive and the negative feed-back effects of E2 on the hypothalamus and pituitary. 7. Thyrotoxicosis in men The elevation of SHBG in thyrotoxic men has been noted by several workers (CrCpy et al., 1967; Dray et al., 1969; Olivo el al., 1970; Ruder et al., 1971; Chopra et af., 1972). t is accompanied by a moderate rise in total T, 170HA, and LH levels. t is therefore important to consider thyrotoxicosis as a possible diagnosis in any man with high plasma T levels. Gynaecomastia and reduced libido often occur in men with thyrotoxicosis (Starr, 1935 ;

21 Sex-hormone- binding globulin 89 Becker et ai., 1968; Chopra et a)., 1972). t is pertinent to ask whether SHBG might be responsible for this, by tipping the sex-hormone balance (unbound T : unbound E,) in plasma in favour of unbound E,. From the measurements of gonadotrophins, SHBG, T and E, in two recent studies (Ruder et al., 1971 ; Chopra et a/., 1972), we can construct a plausible hypothesis to account for the gynaecomastia of thyrotoxicosis, as follows. SHBG rises as a consequence of primary hepatic stimulation by thyroid hormones. The unbound T therefore falls, and the pituitary responds by increasing LH production. This in turn stimulates the Leydig cells, which increase their production of both T and E,, until the unbound T is returned to normal. This, however, can only be done at the expense of an abnormally high i Morrh A m May SeDf FG. 13. Parallel fall in 170HA, SHBG and free thyroxine index with treatment in a man with thyrotoxicosis and gynaecomastia. unbound E,, because of the weaker binding of E, to SHBG. Therefore, gynaecomastia may result from an imbalance between unbound T to E, in plasma. The gynaecomastia, as well as the sex-hormone changes, rapidly resolves once the thyrotoxicosis is treated. The parallel fall in SHBG, 170HA, and free thyroxine index in one man with thyrotoxicosis and gynaecomastia is shown in Fig. 13. G. CONCLUSONS These recent developments in understanding the properties and changes in concentration of SHBG introduce a new dimension to our understanding of the endocrinology of sex hormones. Because SHBG was found to bind both T and E2 it was not immediately obvious that changes in SHBG concentration would significantly affect the ratio of unbound (and therefore biologically-active) T to E, in plasma. Evidence reviewed above, however,

22 90 David C. Anderson indicates that changes in its concentration will exert a significant effect on this ratio. An increase in oestrogen production, by raising the SHBG level, will simultaneously damp down T action, while androgens by inhibiting SHBG production will augment T action. We therefore have a self-servo mechanism whereby a rise in one of these two groups of sex hormones tips the balance further in favour of its own kind. t appears likely that this represents an important mechanism for the maintenance of differentiation of secondary sexual characteristics in the human adult. The evidence reviewed also suggests that disturbances of this mechanism may lead to states of abnormal adult sex differentiation-notably hirsutism and some forms of gynaecomastia such as that seen in thyrotoxicosis and in chronic liver disease. The development of simple competitive protein-binding methods for measuring SHBG concentration opens the way for a more critical appraisal of the hormonal response to glucocorticoid, or oestrogen administration in various forms of hirsutism. Furthermore, the marked effect of thyroid hormones on SHBG may yet prove to have therapeutic implications in these disorders. t is to be hoped that these and other aspects of the role of SHBG in endocrine disease will soon be clarified, and treatment thereby improved. ACKNO W L ED G MENTS am grateful to Dr C. W. Burke, Mr R. Fisher, Dr A. GalvZo-Teles, Mr R. Peppiatt, Mrs J. Woodham and many others who have collaborated in the studies discussed; and to Dr G. M. Besser for his comments on the manuscript. This work has been supported by a grant from the Medical Research Council. REFERENCES ADACH, K. & KANO, M. (1972) The role of receptor proteins in controlling androgen action in the sebaceous glands of hamsters. Steroids, 19, 567. ADLERCREUTZ, H. (1970) Oestrogen metabolism in liver disease. Journal of Endocrinology, 46, 129. AKANDE, E.O. & HOCKADAY, T.D.R. (1972a) Plasma luteinizing hormone levels in women with thyrotoxicosis. Journal of Endocrinology, 53, 173. AKANDE, E.O. & HOCKADAY, T.D.R. (1972b) Plasma oestrogen and luteinizing hormone concentrations in thyrotoxic menstrual disturbance. Proceedings of the Royal Society of Medicine, 65,789. ANDERSON, D.C. (1970) A simple and clinically useful method for plasma testosterone-like substances by competitive protein-binding. Cinica Chimicu Acta, 29, 513. ANDERSON, D.C. (1973) Studies on plasma androgens and sex-hormone-binding globulin in health and disease. Thesis for Doctorate in Medicine, submitted to University of Dundee. ANDERSON, D.C. & FSHER, R. (1973) Unpublished data. ANDERSON, D.C., MARSHALL, J.C., YOUNG, J.L. & FRASER, T.R. (1972a) Stimulation tests of pituitary- Leydig cell function in normal male subjects and hypogonadal men. Clinical Endocrinology, 1, 140. ANDERSON, D.C., PEPPAT, R., SCHUSTER, L. & FSHER, R. (1972b) A new method for measurement of sexhormone-binding globulin in plasma, and its clinical application. Journal of Endocrinology, 55, xi (proceedings). ANDERSON, D.C., MARSHALL, J.C., GALV~O-TELES, A. & CORKER, C.S. (1972~) Gynaecomastia and irnpotence associated with abnormal testosterone binding. Proceedings of the Royal Society of Medicine, 65, 787. AUGUST, G.P., TKACHUK, M. & GRUMBACH, M.M. (1969) Plasma testosterone-binding affinity and testosterone in umbilical cord plasma, late pregnancy, prepubertal children, and adults. Journal of Clinical Endocrinology and Merabolism, 29,89 1. BARD, D.T. & GUEVARA, A. (1969) Concentration of unconjugated estrone and estradiol in peripheral

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