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The Endocrine Function of the Human Testis
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The Endocrine Function of the Human Testis, Volume 1, contains papers that comprise the series of lectures given in a short course on the "Endocrine Function of the Human Testis" which was organized by the Post-Graduate School of Endocrinology in the University of Florence. The course was held in two parts, the first on April 25-27 and the second on October 24-26, 1972. The topics which were covered were: methods for the assay of androgens; binding of androgens in plasma; metabolism of testosterone; the chemistry, biology, and radioimmunoassay of hypophiseal gonadotrophins; the mechanism of control of the secretion of pituitary gonadotrophins; and the biosynthesis of androgens in the human testis.
Azioni libro
Inizia a leggereInformazioni sul libro
The Endocrine Function of the Human Testis
Descrizione
The Endocrine Function of the Human Testis, Volume 1, contains papers that comprise the series of lectures given in a short course on the "Endocrine Function of the Human Testis" which was organized by the Post-Graduate School of Endocrinology in the University of Florence. The course was held in two parts, the first on April 25-27 and the second on October 24-26, 1972. The topics which were covered were: methods for the assay of androgens; binding of androgens in plasma; metabolism of testosterone; the chemistry, biology, and radioimmunoassay of hypophiseal gonadotrophins; the mechanism of control of the secretion of pituitary gonadotrophins; and the biosynthesis of androgens in the human testis.
Anteprima del libro
The Endocrine Function of the Human Testis
Martini
Determination of Androgens in Human Plasma
V.H.T. James and A.E. Rippon, Steroid Research Unit, Department of Chemical Pathology, St. Mary’s Hospital, London, W.2
Publisher Summary
This chapter illustrates the methods of determination of androgens in human plasma. Luteinizing hormone (LH) can cause increased secretion of testosterone when administered to subjects with normal testicular function; however in human subjects, there is no direct relationship between the plasma LH levels and the plasma testosterone levels. Testosterone is not the only steroid present in the male plasma which is androgenic. It is well recognized that a major metabolite, 5α-dihydrotestosterone, is of fundamental importance in the mode of action of testosterone at the cellular level, and it is likely that certain aspects of testosterone action are expressed through this metabolic product. Testosterone is not the only potentially active C19 steroid that is present in human plasma in a free form, and these other steroids share with testosterone the property of binding to the testosterone-estradiol binding globulin (TEBG) which is present in plasma. Although testosterone accounts for a major proportion of the 17-hydroxysteroids, this proportion is varied, even in a series of plasma samples from one subject taken over a few hours. In some samples, it accounts for almost all the total 17-hydroxysteroids, while in others less than 50%. Under basal conditions, the assay of 17-hydroxysteroids reveals virtually the same pattern as does the testosterone assay. The problem of deciding upon an appropriate methodology for plasma androgen assay therefore depends upon the particular requirement to be met.
For many years, virtually the only means of studying androgen metabolism in experimental animals or in human subjects was by examination of the urinary steroid metabolites which were presumed to originate from gonadal and adrenocortical androgens. Nevertheless, even though the techniques of urinary assays were refined extensively to enable the quantitative measurement of groups of steroids (such as 17–oxosteroids) or individual steroids, it was only rarely possible to deduce information of physiological or clinical importance from those measurements. It awaited the availability of a remarkably versatile biochemical tool before any real progress could be made, that tool being the radio–labelled chemical. In the steroid field, radio–isotopically labelled hormones have entirely revolutionised a situation that had progressed little over several years, and have made possible extensive and definitive studies of hormone metabolism. Although used initially as metabolic tracers, the use of high specific activity labelled steroids to develop extremely sensitive assay methods has at last made it possible to examine reasonably small quantities of blood quantitatively for the presence of various hormones. In the androgen field, this was particularly gratifying since these are virtually the only techniques which are capable of achieving the high sensitivity needed, particularly for studies of female plasma.
However, since this symposium is devoted to the endocrine function of the testes, it is appropriate for us to concentrate more upon the problems of androgen levels in male subjects, although even here, certain situations still require a degree of sensitivity in the assay which is normally appropriate to female plasma levels.
The techniques which have been successfully employed for the assay of testosterone have been the double isotope dilution derivative assay, gas liquid chromatography (usually with electron capture detectors), and saturation analysis (in which term I include competitive protein binding assays and radioimmunoassay). Unlike the situation with female plasma, the improved assay methods of the last few years have not greatly altered out concept of the normal range of plasma testosterone levels in normal male adult subjects. Thus some representative levels from different investigators are shown in Table 1.
Table 1
Peripheral Plasma Testosterone Levels in Normal Male Subjects
DIDA Double isotope derivative assay
GLC Gas liquid chromatography
CPB Competitive protein binding
RIA Radioimmunoassay
As the sensitivity of the methods has increased, so it has become possible to make more extensive studies and to investigate the extent to which plasma testosterone levels fluctuate through the day and night. There is a well–known pattern of plasma cortisol change through the 24 hours and it has been shown that corticotrophin release occurs sporadically and thus plasma cortisol levels follow a similar pattern of intermittent sharp increases. Evans et al. (1) reported finding changes of considerable magnitude in plasma testosterone levels, in man during sleep, and found a relationship with REM sleep. Our experience confirms the existence of these variations in plasma testosterone levels and in four normal subjects studied by sampling at frequent intervals, plasma testosterone levels fluctuated through 24 hours from 400 to 825 ng/100 ml; 300 to 640 ng/100 ml; 300 to 580 ng/100 ml and 340 to 1060 ng/100 ml. A part of the total 24 hour study in one subject is shown in Fig.1. The variations which are found are well in excess of the error of the method – in this case, a radioimmunoassay method was used which has a coefficient of variation at the levels involved of 7%. In the same figure, the changes in plasma cortisol are shown, peaking characteristically at approximately 0800 hours. We have not been able to discover any obvious relationship between plasma cortisol levels and plasma testosterone levels and whatever mechanism is responsible for changes in the latter, it appears not to be related to the release of corticotrophin.
Fig. 1 Changes in plasma cortisol, 17β–hydroxysteroids
and testosterone in a normal male subject from 0200 hours to 1000 hours.
We have not found any clear indication of a nyctohemeral rhythm in plasma testosterone levels, although in general, plasma levels tended in all our subjects to be lower at night. In most of the studies reported in the literature, there has been only inconclusive evidence for a nyctohemeral rhythm, and since sampling has usually been at intervals of hours, rather than minutes, and bearing in mind the considerable fluctuations in plasma testosterone levels which can occur, it is not surprising that no clear conclusions have been drawn. Boon et al. (2), reviewing the existing literature, concluded that if any rhythm exists at all, it must be one which is easily over–ridden by other stimuli.
The variations which occur in plasma testosterone levels pose a problem in defining what is a ‘normal’ plasma testosterone level, apart from the interesting but unsolved question of what mechanism is involved in causing these changes in steroid levels. Luteinising hormone (LH) can cause increased secretion of testosterone when administered to subjects with normal testicular function, but in human subjects, unlike other species, there is no direct relationship between plasma LH levels and plasma testosterone levels (3).
Testosterone is not the only steroid present in male plasma which is androgenic. It is now well recognised that a major metabolite, 5a–dihydrotestosterone, is of fundamental importance in the mode of action of testosterone at the cellular level, and it is likely that certain aspects of testosterone action are expressed through this metabolic product. Attempts have therefore been made to determine this steroid in human plasma, and it has been shown (4) that there is a progressive increase through puberty, reaching the adult male levels of approximately 50 ng/100 ml (5, 6). How far the assay of this steroid will produce useful clinical or physiological information remains to be seen.
More recently, methods have been developed for the determination of other C19 steroids, which may also have some physiological or clinical significance, since they have been demonstrated to have androgenic activity. These are androstanediol (5a–androstane–3β, 17β–diol) and androstenediol (androst-5-ene–3β, 17β–diol). These compounds are present in relatively low concentrations in peripheral plasma and as yet, insufficient information is available to indicate what changes, if any, might occur in pathological conditions, or how far they might play any physiological role.
It is clear therefore, that testosterone is not the only potentially active C19 steroid which is present in human plasma in a free form, and all these compounds share with testosterone the property of binding to the testosterone–estradiol binding globulin (TEBG) which is present in plasma. This is a useful property since it provides a means of assaying these steroids by the competitive protein binding technique. This principle has been exploited by several workers who were seeking to assay testosterone (7, 8) or androstenediol (9) and who described methods involving initial chromatographic purification. Murphy, however, who has pioneered much of the work on competitive binding assay techniques, put forward a different philosophy in describing an assay (10) in which minimal purification was used, and the specificity of the binding protein for 17β–hydroxysteroids was invoked. On this basis, the assay would be regarded as a group analysis for steroids which would include testosterone and other 17β–hydroxysteroids which might be potentially androgenic. Anderson, following the same reasoning, described in detail his method (11) for testosterone–like substances and demonstrated its considerable clinical value, and Horton, Kato and Sherins (12) have also described a simple assay of this type.
The virtue of these methods, is that they are relatively inexpensive in terms of reagents, and are less demanding technically and can be performed far more rapidly than methods which are more specific for testosterone. These qualities are valuable when there is a clinical demand for androgen assays, and as Anderson has shown, (11), used in conjunction with dynamic endocrine function tests, they offer clinical information which is probably as useful as the specific assays. The possibility that, as suggested by Murphy, more relevant information might be available from this group assay approach is also worth bearing in mind. We have therefore looked in more detail at this technique, in terms of its specificity and also in relation to changes in plasma testosterone levels. We wished also to improve the technique so as to make possible the use of relatively small volumes of plasma.
In our hands, ammonium sulphate precipitation has proved a more reliable method for the separation of bound and unbound steroid in saturation analyses than have adsorbents such as charcoal or florisil, and so our efforts were directed towards developing an assay along these lines. Initially though, crude plasma extracts caused considerable problems, since the ammonium sulphate invariably caused precipitation of lipid with subsequent poor separation. After various experimental approaches, it was found that hexane – ether produced a very satisfactory extract from plasma and the method ultimately adopted was as follows.
Plasma samples are stored deep frozen prior to analysis. After thawing overnight at 4°C the samples are mixed vigorously on a vortex mixer and 0.1 ml pipetted into glass test tubes of approximate capacity 5 ml (Quickfit type MF 24/0/4) followed by the addition of 0.2 ml of 0. IN sodium hydroxide solution. The contents of the tubes are again mixed thoroughly on a vortex mixer and extracted twice with 2 mls of n–hexane:ether (8:2) by shaking horizontally for ten minutes. The extracts are transferred into a second set of Quickfit tubes, using a pasteur pipette, where the pooled extracts are washed twice with 1 ml of distilled water by shaking horizontally for five minutes. The first water wash is removed carefully with a pasteur pipette. After the second water wash the tubes are centrifuged at 1, 000 g for ten minutes at 4°C. The extracts are then transferred, in two stages, to 10 × 50 mm glass specimen tubes, care being taken not to transfer any of the aqueous phase. The n–hexane:ether is evaporated to dryness in a vacuum oven at a temperature not greater than 40°C and evacuated by means of a filter pump.
Standards in duplicate of 0, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 ng of testosterone are taken through the extraction procedure together with the plasma samples.
A 2% solution of third-trimester pregnancy plasma is prepared by adding the appropriate volume of plasma to borate buffer pH 8.0 containing 2% methanol and approximately 50, 000 dpm/ml of (1, 2−³H) testosterone. 0.25 ml of this solution is added to each of the standard curve and test samples and also to two counting vials containing 0.05 ml of water. The specimen tubes are shaken gently but thoroughly on a vortex mixer and returned to their rack which is placed in a transparent plastic box with a tight fitting lid. The tubes are not stoppered individually.
After overnight incubation at 4°C the tubes are immersed in an ice bath and 0.25 ml of cold saturated ammonium sulphate added. The contents of each tube are mixed for approximately ten seconds on a vortex mixer immediately after the addition of the ammonium sulphate. When the addition of ammonium sulphate to all the tubes is complete each tube is again mixed for two more periods of ten seconds, returning them to the ice bath between each mix. The tubes are left for a further five minutes in the ice bath before centrifugation at 1,000 g for ten minutes at 4°C. The tubes are returned to the ice bath and 0.3 ml of the supernatant transferred to a counting vial. To all the counting vials 10 ml of scintillator is added. The scintillator is prepared by dissolving 6 gm of p–terphenyl and 0.08 gm of dimethyl-P.O.P.O.P. in toluene, adding 40 ml of methanol, and making up to two litres with toluene. All the counting vials are capped securely and shaken mechanically for ten minutes before counting in a liquid scintillation spectrometer to an error of 2%. The activity of the standard curve and test samples is calculated as a percentage of the activity in 0.25 ml of the pregnancy plasma solution multiplied by 0.6. A curve is plotted of percentage free radioactivity against the mass of testosterone contained in the standard curve samples. From this curve the mass of ‘17β–hydroxy steroids’ in ng per 0.1 ml of plasma is read directly and the final results expressed as ng per 100 ml plasma.
The number of plasma samples analysed in one particular batch will of course depend upon the facilities available in each laboratory. Due to the fact that it is necessary to process the standard curve alongside the plasma samples fourteen places in each batch will be taken up with standards. The plasma extracts will however store, at least, overnight at 4°C with no apparent damage provided that they are not allowed to evaporate to dryness. This means that one can extract plasmas on two consecutive days and then incubate them all with one standard curve. Using this approach one technician can easily process 45 plasma samples plus the standard curve in two and a half days.
The recovery of (1, 2−³H) testosterone from plasma when taken through the extraction and washing procedure was 102% with a range of 91 – 108, N = 9. The recovery of (³H)–androstanediol from three separate plasmas when taken through the extraction procedure was 89%, 91% and 89%. The recovery of 0.25 ng, 0.5 ng and 0. 75 ng of testosterone added to plasma when taken through the method was 104%, 105% and 106% respectively. Precision data were obtained from the assay of aliquots of a plasma pool in eleven separate batches. This gave a mean of 967 ng/100 ml with a coefficient of variation of ± 6.7%. The analysis of within batch duplicates with a mean of 1075 ng/100 ml gave a coefficient of variation of ± 5%.
Although it is possible to store plasma deep frozen without a great deal of difficulty, it is important to realise that the practise of centrifuging such plasma samples after they have been unfrozen (to remove precipitated protein) carries the risk of removing some part of the endogenous testosterone, which is presumably bound to the precipitate. This phenomenon is particularly marked if this happens repeatedly (e.g. with a frozen pool which is unfrozen on several occasions). Fig. 3 illustrates the progressive fall in steroid concentration (as shown by the radio–active tracer) when this is done, and shows that when the precipitate is resuspended, the plasma testosterone level returns towards the original value.
Fig. 3 The effect of storage on plasma testosterone concentrations. (1−2³H) testosterone was added to a plasma sample which was deep frozen at weekly intervals up to 9 weeks. The plasma was un–frozen, centrifuged and the radioactivity in the plasma measured. The progressive fall in radioactive concentrations shows that the precipitate formed on each occasion is adsorbed by the radio–labelled steroid.
A group of 49 male control subjects was used to obtain normal data; their ages ranged from 15 to 59 years and the mean level of plasma 17–hydroxysteroids found was 614 ng/100 ml with a range of 270 - 1200. It is interesting to compare these data with those in Table 1. The values obtained are shown in Figure 2 in relation to age.
Fig. 2 17β–hydroxysteroid
concentrations in normal male subjects in relation to age.
The question of specificity, or what is being measured in this assay is more difficult to answer. Figure 1 shows the relationship between the 17–hydroxysteroids measured by the method described above and testosterone as measured by radioimmunoassay. Although testosterone accounts for a major proportion of the 17–hydroxysteroids, this proportion is very variable, even in a series of plasma samples from one subject taken over a few hours. In some samples it accounts for almost all the total 17–hydroxysteroids; in others less than 50%. It is not likely that this is in any major part due to assay errors, since the precision is, as described above, quite good for both assays. This variability in the proportion of the 17–hydroxysteroids which is made up of testosterone is further illustrated by Figure 4, comparing testosterone measured by immunoassay and the 17–hydroxysteroid content of a number of plasma samples. The difference between the two assay results arises, at least in part, from the contribution of the 17–hydroxysteroids in plasma mentioned above, i.e. dihydrotestosterone, androstanediol and androstenediol. So far, we have no knowledge of how the plasma levels of these other steroids alters through the day, but it would not be surprising if they varied to some extent independently of testosterone, since some may originate from the adrenal cortex. Thus Wieland et al. (13) from their studies of adrenal venous blood, have found evidence for adrenocortical secretion of androstenediol in human subjects. Under basal conditions though, it is not easy to assess the significance of the small fluctuations in the levels of 17–hydroxysteroids although there is no obvious relationship to plasma cortisol levels and by implication, to corticotrophin secretion.
Fig. 4 Comparison of 17β–hydroxysteroids
and testosterone concentrations in a series of samples taken from a normal male subject. Testosterone was measured by radioimmunoassay (RIA).
Under conditions in which changes of plasma testosterone are being studied, as in gonadal stimulation tests, the assay of 17–hydroxysteroids reveals virtually the same pattern as does the testosterone assay. To illustrate this point, figure 5 shows a comparison of the results of the two assays in two patients being treated by the administration of clomiphene.
Fig. 5 
) in a normal male subject treated with clomiphene.
The problem of deciding upon an appropriate methodology for plasma androgen assay must therefore depend very much upon the particular requirement to be met. Studies in which testosterone levels are primarily of interest still require fairly complicated assay methods to provide the necessary specificity. The double isotope and gas chromatographic methods, although excellent in highly skilled and experienced hands, are technically too demanding to attract unqualified recommendation, and most investigators would now prefer to employ radio–immunoassay after some initial purification stage. Separative techniques are also essential if dihydrotestosterone, androstenediol or androstanediol are to be measured. Inevitably, these methods prove relatively more difficult to employ in a busy diagnostic laboratory, and where the main need is for a method which rapidly reveals changes in testosterone levels, particularly as in the investigation of male gonadal dysfunction, then simpler assays, either as described by Anderson (11) or Horton (12) or the method used here, may prove entirely adequate.
Acknowledgement
We are glad to acknowledge Mrs. J. E. Prescott’s excellent technical assistance.
References
1. Evans, J.I., MacLean, A.W., Ismail, A.A.A., Love, D. Nature. 1971; 229:261.
2. Boon, D.A., Keenan, R.E., Slaunwhite, W.R. Steroids. 1972; 20:269.
3. Murray, M.A.F., Corker, C.S. J. Endocr. 1973; 56:157.
4. Gupta, D., McCafferty, E., Rager, K. Steroids. 1972; 19:411.
5. Tremblay, R.R., Beitins, I.Z., Kowarski, A., Migeon, C.J. Steroids. 1970; 16:29.
6. Ito, T., Horton, R. J. clin. Endocr. 1970; 31:362.
7. Kato, T., Horton, R. Steroids. 1968; 12:631.
8. Mayes, D., Nugent, C.A. J. clin. Endocr. 1968; 28:1169.
9. R.L. Rosenfield, Programme 53rd Meeting of the Endocrine Society, Abstract 201(1971).
10. Murphy, B.E.P. Recent Prog. in Hormone Res. 1969; 25:563.
11. Anderson, D.C. Clin. Chim. Acta. 1970; 29:513.
12. Horton, R., Kato, T., Sherins, R. Steroids. 1967; 10:245.
13. Wieland, R.G., de Courcy, C., Levy, R.P., Zala, A.P., Hirschmann, H. J. clin. Invest. 1965; 44:159.
14. Bardin, C.W., Lipsett, M.B. Steroids. 1967; 9:71.
15. Vermeulen, A.Scholler R., Jayle M.F., eds. Gas Chromatography of Hormonal Steroids
. Gordon and Breach: New York, 1968; 375.
16. Maeda, R., Okamoto, M., Wegienka, L.C., Forsham, P.F. Steroids. 1969; 13:83.
17. Furuyama, S., Mayes, D.M., Nugent, C.A. Steroids. 1970; 16:415.
METHODOLOGIC CONSIDERATIONS ON THE MEASUREMENT OF PLASMA ANDROGENS BY COMPETITIVE PROTEIN BINDING METHODS AND RADIO-ASSAYS
Mario Serio, Gianni Forti, Gianna Fiorelli and Mario Pazzagli, Endocrinology Unit, University of Florence, Italy
Abstract
The principal methodologic problems which arise in the measurement of plasma androgens by means of competitive protein binding and radio–assay are discussed. Particular examination is made of the properties of plasma proteins and antibodies, the characteristics of the labeled steroid, the conditions of assay, the methods of separation of bound and unbound fractions, the factors which cause high blank values and the practicability of the various chromatography systems (thin layer, paper, sephadex LH 20, column).
Symbols
P = binding protein
S = steroid
Sr = labeled steroid
CPB = competitive protein binding
SHBG = sex–hormone binding globulin
Introduction
Competitive protein binding analysis can be defined as a form of saturation assay in which the specificity is essentially dependent upon the particular binding a protein. The binding protein can be a plasma protein, a tissue protein, an enzyme or an antibody.
The competitive binding methods are based on the following principle: if a protein P (antibody, tissue protein etc.) is mixed with a steroid S for which it has specific binding sites, a PS complex forms when equilibrium is reached. The same labeled steroid will behave similarly, forming a PSr complex. If the concentration of S and Sr is superior to the number of binding sites available, they will compete with each other for the binding sites according to their concentration.
If the quantity of P and Sr is constant, while the quantity of S is variable, it is evident that the quantity of Sr which results as bound by the protein will be inversely proportional to the quantity of S present in the system. On the basis of this principle, it is possible to construct a calibration curve plotting the percentage of Sr bound to the protein (or free) as a function of the concentrations of S.
Consequently, on the basis of the percentage of bound Sr which is found in the protein–steroid system following the addition of an unknown quantity Xs (from plasma, urine or other biological fluid), it is easy to obtain the concentrations of the hormone which is to be measured.
Various steroids have been measured by this method and among them some plasma androgens (1). So far plasma proteins and antibodies have been used for the assay of the plasma androgens, as reported in table 1.
Table 1
Competitive protein–binding analysis for androgens
Reliability Criteria of Competitive Binding Analysis.
A competitive binding method, like any other chemical assay method, must possess certain reliability criteria.
Accuracy: this is without doubt the most important of the reliability criteria and that to which all the others are related. Accuracy can be defined as the probability an assay method has of furnishing results truly corresponding to the quantity of the substance present in the sample which is to be measured. Accuracy of a measurement depends, however, on the good quality of the system used, which, in the case of the competitive binding, depends on sensitivity, precision, the slope of the calibration curve, the blank value and specificity.
Sensitivity, usually defined as the smallest amount of a substance which can be measured with accuracy by the assay method used, really corresponds to the ability of the assay method to distinguish between slightly different steroid concentrations; thus the sensitivity is identified in the resolving power. Two aspects of sensitivity which contribute to defining the resolving power of a competitive binding method are its precision and the slope of its calibration curve.
Precision corresponds in practice to the repeatability of the measurement and is related to various factors depending on the system used. The slope of the curve is an important factor for competitive binding methods.
The reason is easily understandable from fig. 1, where it is seen that different fiducial limits in relation to the different slope of the curve correspond to the same standard error in the measurements.
Fig. 1 Effect of the slope of calibration curves on the concentration confidence limits.
The concept of accuracy embraces not only sensitivity and precision, but also the idea of the deviation from the reference level. In this regard the blank value is very important. In fact, if the precision of two methods is the same, but the blank values are different, the method with the smaller blank has more probability of obtaining a high accuracy. Since the blank value varies according to the type of interference and does not have a constant influence on all the concentrations of the calibration curve (fig. 2), it cannot be subtracted without making errors of accuracy.
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