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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 7 2062-2069
Copyright © 1997 by The Endocrine Society

Clinical Studies

Disparate Serum Free Testosterone Concentrations and Degrees of Hypothalamo-Pituitary-Luteinizing Hormone Suppression Are Achieved by Continuous Versus Pulsatile Intravenous Androgen Replacement in Men: A Clinical Experimental Model of Ketoconazole-Induced Reversible Hypoandrogenemia with Controlled Testosterone Add-Back1

Alexander D. Zwart2, Ali Iranmanesh and Johannes D. Veldhuis

Endocrine Section, Medical Service, Veterans Affairs Medical Center (A.I.), Salem, Virginia 24153; and the Division of Endocrinology, Department of Internal Medicine, University of Virginia Health Sciences Center, National Science Foundation Center for Biological Timing (A.D.Z., J.D.V.), Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology, Department of Internal Medicine, Box 202, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: JDV{at}Virginia.Edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the neuroendocrine mechanisms underlying the negative feedback actions of testosterone on both the pulsatile mode of LH release and the entropy or disorderliness of the LH release process, we blocked testicular androgen biosynthesis using oral high dose ketoconazole treatment with concomitant low dose glucocorticoid replacement for 48 h in six healthy young men. Volunteers were then infused iv with saline or a total of 8.0 mg testosterone base over the second 24 h via either a continuous or a pulsatile (90-min boluses) delivery pattern. Discrete peak detection (Cluster analysis) was applied to obtain a model-independent estimate of the frequency of serum LH concentration peaks, maximal and incremental LH peak amplitudes, peak area, and interpeak nadir serum LH concentrations. Approximate entropy was used to quantify the relative orderliness/disorderliness of the LH release process over 24 h. Ketoconazole treatment markedly lowered 24-h mean serum total and free testosterone concentrations (by 17- and 9-fold respectively), and significantly increased LH pulse frequency, maximal LH peak height, and interpeak nadir serum LH concentrations. Continuous iv testosterone add-back increased 24-h pooled serum free testosterone concentrations 3-fold more and concomitantly reduced mean (24-h) serum LH concentrations by at least 2-fold more than pulsatile delivery of the same total daily amount of androgen. Both modes of testosterone infusion suppressed pulsatile LH release, but the effects were distinguishable; namely, treatment with continuous vs. intermittent androgen add-back, respectively, decreased LH pulse frequency and incremental LH pulse amplitude. Ketoconazole treatment alone also significantly increased approximate entropy values, indicating greater disorderliness of LH release during androgen removal. Approximate entropy/orderliness was restored to baseline by continuous, but not pulsatile, iv testosterone replacement.

In conclusion, the present novel testosterone add-back clinical experimental paradigm indicates that 1) remarkably different 24-h mean serum free testosterone concentrations can result from continuous vs. pulsatile testosterone delivery into the bloodstream; 2) androgen negative feedback can exert frequency- as well as amplitude-dependent suppression of pulsatile LH release; and 3) testosterone is required to maintain an orderly 24-h LH release process in young men.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADAL HORMONES participate in regulating the pulsatile output of the hypothalamo-pituitary-gonadotroph unit in men and women. In particular, both steroidal and nonsteroidal feedback effectors are produced by various gonadal compartments. Feedback from the testis is important, because rodents and men with bilateral orchidectomy and/or primary testicular failure exhibit increased serum LH (and FSH) concentrations, defined as a castration response (1, 2, 3, 4, 5). Conversely, the administration of synthetic androgens or testosterone will suppress blood gonadotropin concentrations (6, 7, 8, 9, 10, 11, 12, 13). However, to date virtually all androgen replacement experiments have employed continuous testosterone delivery via injections of long acting testosterone esters in lipophilic vehicles (e.g. testosterone enanthate in oil im) or steady state iv infusions of testosterone or other androgens, such as testosterone’s 5{alpha}-reduced product (14). Such experiments do not mimic the physiological time structure of episodic testosterone release in vivo. Indeed, direct catheterization of the spermatic vein in the human has revealed a prominently pulsatile mode of testosterone (and estradiol and inhibin) secretion, occurring approximately circhorally (15, 16). Furthermore, simultaneous measurements of serum LH and testosterone concentrations in the peripheral blood of the ram, bull, rodent, and young men have revealed a significant temporal correlation between serum LH and testosterone concentrations (17, 18, 19, 20, 21, 22, 23, 24). Such observations suggest a dynamic feed-forward model of normal male hypothalamo-pituitary-testicular physiology, in which bursts of hypothalamic GnRH secretion trigger corresponding pulses of pituitary LH release, which, in turn, drive intermittent production of testosterone by responsive Leydig cells. However, in this so-called servocontrol model, virtually nothing is known about the impact of the physiologically pulsatile testosterone signal produced by the LH-responsive Leydig cells as a feedback regulator of the hypothalamo-pituitary-LH unit.

To evaluate the impact of pulsatile testosterone feedback on the secretory activity of the hypothalamo-pituitary-gonadotroph unit, we have applied two new investigative strategies, namely 1) short term (48-h) oral administration of ketoconazole to inhibit adrenal and Leydig cell steroidogenesis in healthy young men, who are concurrently replaced with physiological amounts of glucocorticoid; and 2) during the resulting profound hypoandrogenemia, infusion of saline vs. testosterone iv in either of two temporal modes: 90-min bolus injections or continuous delivery of the same total daily testosterone dose. This reversible Leydig cell chemical castration model in healthy young men allowed us to test the hypotheses that blood concentrations and/or the feedback actions of testosterone on the hypothalamo-pituitary-LH axis depend on the pattern of testosterone’s entry into the bloodstream.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical paradigm

Six healthy young men (aged 18–31 yr) participated, after providing written informed consent approved by the human investigation committee. After a compete history and physical examination and screening tests of hepatic, renal, metabolic, and hematological function, volunteers were admitted to the General Clinical Research Center four times in randomly assigned order: 1) dexamethasone replacement only (0.75 mg, orally, twice daily throughout), 2) dexamethasone (as above) and ketoconazole (1000 mg, orally, at midnight followed by 400 mg every 6 h for 56 h); and 3 and 4) dexamethasone and ketoconazole combined with bolus iv testosterone injections as 0.5 mg crystalline testosterone (dissolved in 0.1 mL absolute alcohol diluted in 5% dextrose in water for a 2-mL final volume delivered over 1 min) every 90 min or continuous ivinfusion of testosterone (8 mg delivered continuously over the last 24 h), as described previously (6). The 24-h testosterone infusions and concurrent contralateral blood sampling every 10 min were begun 32 h after the loading dose of ketoconazole at midnight (thus encompassing day 2 from 0800 h through 0800 h on the next day). All 145 blood samples obtained during each session were later assayed for serum LH and testosterone concentrations (below).

Validation of Cluster analysis for serum LH pulse detection

We used a combination of in vivo biological validation and computer-assisted biophysical modeling to determine the approximate sensitivity and specificity (or positive accuracy) of detecting serum LH concentration pulses by Cluster analysis (25, 26, 27). Cluster analysis is a largely model-free discrete peak detection method (28), since a priori assumptions about hormone half-life, secretory burst waveform, basal secretion, etc. are not required (29).

Parameters of Cluster analysis

For LH pulse detection, we used two points in the test nadir cluster and one in the test peak, with pooled t statistics for significant upstrokes and downstrokes of 2.0 in each case (27). For testosterone peak detection, the same t statistic was used, but with a 2 x 2 test cluster nadir and peak. The following pulse attributes were determined: frequency (number of pulses per 24 h), maximal peak height (highest absolute serum hormone concentration attained within the peak), incremental peak amplitude (algebraic difference between maximal peak height and prepeak nadir), area under the peak, and interpulse nadir serum LH concentrations.

Approximate entropy (ApEn)

The regularity or orderliness of LH release over 24 h was quantified by an approximate entropy statistic, ApEn (30). ApEn provides a relative measure of the pattern repetition within the hormone profile by assigning a single nonnegative number whose value increases with greater disorder or more irregularity. This statistic exhibits high sensitivity (>90%) and specificity (>90%) in distinguishing the relative orderliness of GH, aldosterone, and ACTH release in normal vs. tumoral secretory profiles and, in the case of GH, in healthy men compared to women (31, 32, 33, 34). For LH time series each comprising 145 observations, we used ApEn (1, 20%) as a scale- and model-independent statistic calculated for a window length (m) of 1 and a tolerance (r) of 20% of the overall SD of the individual subject’s 24-h serum LH concentration profile (31, 32, 34). Adjusting the tolerance to each subject’s series SD normalizes ApEn to otherwise unequal mean hormone levels.

Hormone assays

LH was assayed in a duplicate in a two-site immunoradiometric assay (IRMA) that correlates well with the rat Leydig cell in vitro bioassay (35, 36) (Nichols Laboratories, San Juan Capistrano, CA; First International Reference Preparation of human menopausal gonadotropin). The assay sensitivity was 0.5 IU/L, and the mean within-assay coefficient of variation (CV) ranged from 3.5–8.7%, with an interassay CV of less than 10%. Serum total testosterone was measured with a solid phase RIA (Diagnostic Products Corp., Los Angeles, CA), with a sensitivity of 20 ng/dL (0.68 nmol/L), and intra- and interassay CVs from 6–8%. Serum free testosterone in 24-h pools was measured with similar within- and between-assay CVs and a sensitivity of 1 pg/mL (3.4 pmol/L) via Coat-A-Count (Diagnostic Products Corp.), an analog-based nonequilibrium RIA (36).

Statistical analysis

Differences among specific measures of pulsatile LH or testosterone release (e.g. number of peaks, maximal peak amplitude, interpeak nadir, etc.) were assessed by ANOVA after logarithmic transformation, in view of the nonnormality of these measures (29). Data are given as the mean \ SEM. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
One volunteer experienced nausea after treatment with ketoconazole and was discharged after 24 h. He subsequently returned and completed the same treatment session without symptoms. No other adverse symptoms or signs were observed.

Figure 1Go illustrates the observed 24-h serum LH concentration profiles in one individual for all four treatment conditions. The mean (24-h) serum LH concentration at baseline was 3.8 \ 0.25 IU/L and increased after ketoconazole treatment to 10.5 \ 1.1 IU/L (P < 0.01). During bolus testosterone infusions, serum LH concentrations remained significantly elevated over baseline at 9.2 \ 0.84 IU/L. This mean was also significantly higher than that observed during continuous testosterone infusion, namely 7.0 \ 0.52 IU/L.



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  		Figure 1. Twenty-four-hour serum LH concentration profiles illustrated for one of six young men who underwent blood sampling at 10-min intervals under baseline conditions, during ketoconazole administration alone (KTCZ), or during ketoconazole treatment accompanied by bolus or continuous iv testosterone replacement (see Materials and Methods). Serum LH concentrations were measured by IRMA in blood sampled every 10 min during the last 24 h of 56 h of treatment with placebo or the steroidogenic enzyme inhibitor without or with testosterone add-back (see Materials and Methods). Vertical error bars denote within-assay SDs assessed by a dose-dependent variance function for all 145 samples measured in that assay in that subject.

 
The mean (\SEM) serum total testosterone concentration measured in the 10-min (24-h) profiles was markedly suppressed from 524 \ 24 ng/dL (18 \ 0.84 nmol/L) basally to 32 \ 6 ng/dL (1.1 \ 0.21 nmol/L) in response to ketoconazole treatment alone (P < 0.001), with an absolute range of 20–71 ng/dL (0.70 to 1.4 nmol/L). During bolus and continuous testosterone add-back, the mean serum total testosterone concentrations rose to 415 \ 260 ng/dL (14 \ 8.8 nmol/L) and 1100 \ 94 ng/dL (38 \ 3.2 nmol/L), respectively. The former was statistically indistinguishable from that during either baseline or ketoconazole treatment, and the latter was significantly higher than that during ketoconazole treatment alone or ketoconazole with pulsatile testosterone add-back (P < 0.01). A large variance was introduced by one of six subjects receiving bolus testosterone add-back, who had occasional peak serum total testosterone concentrations 3- to 10-fold in excess of 524 ng/dL (18 nmol/L) immediately after the bolus injections. If these data are omitted, the mean total serum testosterone concentration during pulsatile testosterone add-back was 164 \ 30 ng/dL (5.4 \ 1.0 nmol/L; P < 0.01 vs. ketoconazole treatment alone, baseline, or ketoconazole combined with continuous testosterone infusion). All individual 24-h serum total testosterone profiles are shown in Fig. 2Go (including patient 6, above).



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  		Figure 2. Serum total testosterone (RIA) concentration profiles in all six individuals sampled at 10-min intervals over 24 h. The experimental paradigm is described in Fig. 1Go. To convert nanograms per dL testosterone to nanomoles per L, multiply by 0.034. Note that two subjects (no. 5 and 6) during bolus testosterone add-back had occasional serum total testosterone concentrations in excess of 500 ng/dL (17 nmol/L), when some blood samples were withdrawn immediately after steroid injection. The y-axes for these profiles (bottom two, pulsatile infusion column) have been rescaled.

 
Serum free testosterone concentrations were reduced 9-fold by ketoconazole treatment and were restored by pulsatile and continuous iv infusions of testosterone to the mean baseline (pulsatile injections) or to approximately 2.5 times the mean baseline value (continuous androgen infusion; see Table 1Go). Continuous infusions achieved significantly (3.0-fold) higher serum free testosterone concentrations over 24 h than pulsatile delivery of the same total testosterone dose (P < 0.01). If the data from the one subject (no. 6, above) with occasional blood samples withdrawn immediately after bolus iv testosterone injections were omitted, the mean serum free testosterone concentration was 9-fold [absolute value, 6.0 \ 1.2 pg/mL (20 \ 4.1 pmol/L)] lower during pulsatile than during continuous testosterone replacement.


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  		Table 1. Serum hormone concentrations in normal men treated with dexamethasone and placebo or ketoconazole alone or with iv pulsatile vs. continuous testosterone infusions

 
Table 1Go also gives 24-h pooled sera estimates of FSH, estradiol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, sex hormone-binding globulin, GH, and PRL concentrations assessed in respective RIA, IRMA, or chemiluminescence assays (6, 35, 36, 37). PRL concentrations were relatively increased in the control (basal) session. Serum FSH concentrations were higher than baseline (control) in each of the six men during ketoconazole treatment with or without androgen replacement (P < 0.05).

By Cluster analysis, as shown in Fig. 3Go (top panel), LH pulse frequency was similar at baseline and during ketoconazole treatment combined with continuous testosterone add-back, but rose significantly (compared to these two conditions) during treatment with either ketoconazole alone or ketoconazole combined with bolus testosterone injections (P < 0.01).



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  		Figure 3. A (top), Mean (±SEM) serum LH concentration peak frequency (events per 24 h) assessed by Cluster analysis in six men studied at baseline or during treatment with ketoconazole (KTCZ) alone or combined with bolus or continuous iv testosterone replacement. B (Upper), Mean (±SEM) incremental serum LH concentration peak amplitudes in the same six healthy young men. Only continuous iv testosterone add-back yielded mean incremental LH pulse amplitudes higher than those of the other three treatment groups. C (Lower), Influence of ketoconazole, a steroidogenic enzyme inhibitor, without or with bolus or continuous iv testosterone replacement on the mean (±SEM) area of serum LH concentration pulses. D (Bottom), Mean (±SEM) nadir serum LH concentrations in six normal young men treated with ketoconazole to inhibit steroidogenesis without or with concomitant bolus or continuous iv testosterone replacement. Different alphabetic superscripts denote significantly different means, as assessed by ANOVA followed by Duncan’s multiple comparison test. P values correspond to the treatment effect.

 
The maximal serum LH concentration peak height (absolute serum LH pulse amplitude) increased significantly from 5.5 \ 0.29 IU/L basally to 12.1 \ 1.3 IU/L after ketoconazole treatment alone (P = 0.003). During continuous and pulsatile testosterone injections, the maximal LH pulse amplitudes remained elevated at 10.1 \ 0.92 and 11.3 \ 0.97 IU/L, respectively (P = NS vs. each other and vs. ketoconazole treatment alone).

The incremental amplitude (international units per L) of serum LH concentration pulses increased significantly in the continuous testosterone add-back sessions compared to the baseline, ketoconazole treatment alone, or ketoconazole and bolus testosterone values (P < 0.02; Fig. 3Go, upper panel).

The serum LH concentration peak areas (above baseline) were similar during control and ketoconazole treatment (Fig. 3Go, lower panel). Bolus testosterone add-back significantly reduced the LH peak area compared to that after ketoconazole treatment alone. Conversely, continuous testosterone infusions resulted in a nearly 2-fold higher serum LH pulse area compared to bolus injections (P < 0.01).

The interpeak nadir serum LH concentration (see Fig. 3Go, bottom panel) rose approximately 3-fold in response to ketoconazole alone (P < 0.01). Continuous, but not bolus, testosterone add-back significantly reduced this measure (P < 0.01).

Figure 4Go depicts the ApEn (1, 20%) (see Materials and Methods), estimates for each of the six men and four treatment conditions. ApEn rose significantly during treatment with ketoconazole alone, indicating greater disorder or more randomness of LH release over 24 h (P < 0.01). Continuous, but not pulsatile, iv testosterone restored ApEn to the baseline value.



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  		Figure 4. ApEn values as estimates of the quantifiable irregularity or disorderliness of the LH release process over 24 h in each of six young men subjected to acute androgen withdrawal [ketoconazole (KTCZ) treatment alone] without or with iv testosterone replacement (pulsatile vs. continuous delivery). Normalized ApEn (1, 20%) (Materials and Methods) was used as a scale-invariant and model-independent statistic to quantify the relative regularity or orderliness of LH release. Higher ApEn values denote greater disorderliness or more irregularity of LH release patterns over 24 h (see Materials and Methods). Data are individual ApEn values, the means of which were compared by ANOVA (P < 0.01). Different alphabetic superscripts denote significantly different means.

 
Cluster analysis of the 24-h serum total testosterone concentration profiles is summarized in Table 2Go. Pulsatile testosterone add-back yielded periodic testosterone peaks approximately every 90 min. Continuous testosterone delivery produced higher nadir serum testosterone concentrations.


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  		Table 2. Cluster analysis of 24-h serum total testosterone concentration profiles

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have tested the hypothesis that the time course of testosterone’s delivery into the bloodstream can modify its 24-h blood concentrations and/or its feedback actions on LH release. To this end, we used ketoconazole as a steroidogenic enzyme inhibitor (38) to achieve and maintain short term (48-h) hypoandrogenemia in healthy young men. We then replaced testosterone iv via pulses every 90 min or continuously at its expected daily secretion rate (8 mg) (14). Our results show for the first time that continuous iv testosterone replacement in the androgen-depleted human increases mean (24-h) serum free testosterone concentrations, reduces mean serum LH concentrations, and restores the relative orderliness of the LH release process over 24 h more effectively than bolus (pulsatile) testosterone add-back.

The present clinical studies show that 2-day treatment with ketoconazole, a potent inhibitor of steroidogenesis, combined with a replacement dose of glucocorticoid substantially lowers serum total and free testosterone concentrations in healthy young men without significantly altering estradiol and sex hormone-binding globulin levels. Ketoconazole reduced the mean 24-h serum total testosterone concentration determined by 10-min blood sampling by 15- to 20-fold (mean reduction, 17-fold) and serum free testosterone concentrations by 9-fold. Thus, this model achieves effective short term androgen deprivation and, when desired, allows experimentally controlled testosterone add-back. Notably, although concurrent glucocorticoid replacement is essential because ketoconazole markedly inhibits cholesterol side-chain cleavage in both the gonad and the adrenal cortex (38), even a 2-fold higher daily dose of glucocorticoid replacement than that used here does not measurably alter 24-h pulsatile LH release in healthy young men (37).

The present data show that continuous iv testosterone infusion over 24 h elicits 2.5- to 3-fold (P < 0.01) higher mean serum free and total testosterone concentrations than bolus injection of the same total daily dose of androgen (assuming similar adsorptive losses to the syringe and infusion tubing). This difference is 7- to 9-fold if one subject’s values are omitted, whose blood samples were inadvertently withdrawn immediately rather than 10 min after an iv pulse of testosterone. Conversely, the true integrated testosterone difference will be slightly less, because our 10-min sampling schema did not (except in subject 6) capture the momentarily high serum testosterone concentrations that occur after each bolus, as predicted pharmacologically (39). Although human spermatic vein testosterone release is overtly pulsatile (15, 16), our bolus iv injections were given over 1 min, whereas endogenous testosterone secretory bursts are probably prolonged over several minutes. Theoretically, such longer in vivo testosterone secretory bursts would be expected to sustain blood androgen concentrations more effectively, as the duration of a secretory event affects the apparent hormone half-life in the circulation especially in the presence of a high affinity binding protein(s) (39).

Neither temporal mode of iv testosterone replacement at approximately its daily physiological secretion rate in healthy young men normalized mean (24-h) serum LH concentrations, although mean (24-h) serum total and free testosterone levels rose above the normal (pretreatment) range during continuous iv testosterone replacement. Several explanations may be relevant to this initially unanticipated observation. First, testosterone add-back was not initiated until 30 h after ketoconazole treatment was started. Beginning testosterone replacement sooner may have fully normalized the castration-like increase in LH release. Secondly, our bolus iv testosterone infusion protocol was not designed to mimic exactly the complex day-night rhythms of testosterone secretion in healthy young adults (15, 18, 40). Thirdly, the combination of ketoconazole and dexamethasone may have had unexpected effects on the hypothalamo-pituitary-testicular axis, even though this dose of dexamethasone does not affect spontaneous 24-h pulsatile or GnRH-stimulated LH secretion in young men (37).

Based on blood sampling every 10 min for 24 h and model-free Cluster analysis of LH pulsatility (28), we observed that short term hypoandrogenemia increased LH pulse frequency by approximately 36% in healthy men. Testosterone withdrawal also increased the maximal serum LH peak height and the interpeak nadir LH concentration. Despite differences in experimental design, duration of androgen withdrawal, analytical methods, and the choice of volunteers/patient populations, an earlier study also reported a hypoandrogenemia-stimulated increase in LH pulse frequency, albeit in primary hypogonadal men withdrawn for a longer interval from im testosterone ester replacement therapy (41). Likewise, increased LH pulse frequency occurs in men treated with an androgen receptor antagonist to impede testosterone’s negative feedback actions (1, 42, 43). Conversely, slowing of LH pulse frequency results from infusions of pharmacological amounts of 5{alpha}-reduced androgen (6). Here, we note that the increase in LH pulse frequency induced by ketoconazole treatment was reversed by continuous, but not pulsatile, iv testosterone replacement. Earlier kinetic analyses suggested that continuous (vs. intermittent) testosterone delivery would yield higher integrated serum free and total testosterone concentrations (39), as confirmed here by assay of pooled (24-h) serum (above). Thus, significant suppression of LH pulse frequency by continuous, but not pulsatile, androgen add-back probably reflects unequal serum testosterone concentrations achieved by the two iv infusion modes and suggests that higher or more sustained blood testosterone levels may be required to suppress LH pulse frequency.

In refutation of our initial neuroendocrine hypothesis, continuous iv testosterone replacement suppressed mean (24-h) serum LH concentrations significantly more than intermittent (90-min) injections of the same total daily dose of androgen. A similar observation was reported recently in a 4-h sampling study in chronically (5 months or more) castrate male sheep (44). The neuroendocrine basis for this distinction was examined in the present experiments by discrete (Cluster) pulse analysis (29), which is largely model free. Specifically, continuous testosterone add-back significantly reduced LH peak frequency and interpeak nadir serum LH concentrations, which together reduced (mean) serum LH levels. In contrast, pulsatile iv testosterone add-back lowered only the incremental serum LH peak amplitude and thereby reduced the LH peak area. These differences were presumably elicited by the disparate serum testosterone concentrations and/or their distinctive time courses.

Unlike pulsatile iv testosterone replacement, continuous androgen add-back (despite higher 24-h mean serum total and free testosterone concentrations) failed to suppress serum LH peak area or incremental LH pulse amplitude to the baseline level. Peak areas are controlled by hormone half-life, secretory burst duration, and amplitude/mass (45, 28). As different mean serum testosterone concentrations were reached via the two modes of infusion, the foregoing results suggest possible concentration (dose)-dependent feedback actions of testosterone on LH half-life or LH secretory burst duration amplitude (mass). The last-mentioned is controlled by effective GnRH dose (46). Whether the particular kinetic profile of (total or) free testosterone concentrations in blood also modulates androgen’s negative feedback efficacy is not yet determinable.

We used a recently validated ApEn statistic to appraise the androgen dependence of the regularity or orderliness of the LH release pattern over 24 h (30). In response to acute androgen withdrawal, the ApEn of LH release increased significantly, which signifies greater irregularity or disorderliness of LH release, as is visually apparent in Fig. 1Go. Note that this statistical measure of regularity is complementary to pulse analysis, as it quantifies both pulsatile and nonpulsatile pattern recurrence in the profile. Greater disorderliness of LH release has also been observed in healthy aging men (47). An irregularity of hormone secretion also typifies tumoral states, such as acromegaly, Cushing’s disease, and aldosteronoma, and female compared to male GH secretion profiles (31, 32, 33, 34). Of interest, more disorderly GH release also occurs when negative feedback is withdrawn via fasting-induced suppression of plasma insulin-like growth factor I concentrations (31). Here, in studying the LH-Leydig cell axis, we withdrew androgen negative feedback on LH. Thus, decreased orderliness or regularity of (pituitary) hormone release can reflect diminished feedback in either the somatotropin or gonadotropic axes. Importantly, reinstating negative feedback on LH by continuous (high mean serum testosterone concentrations), but not pulsatile, iv testosterone replacement fully reversed the measurable increase in ApEn/disorderliness of LH release. Thus, we infer that the orderliness of minute to minute LH release in young men is endowed by testosterone negative feedback. According to this reasoning, the more disorderly pattern of LH release observed in older men (47) may, therefore, reflect altered androgen negative feedback control of the hypothalamic-pituitary unit in aging individuals.


   Acknowledgments
 
We thank Cindy S. Sites and Patsy Craig for their skillful preparation of the manuscript, Brenda Grisso and Mary Fanon for laboratory assays, and Paula P. Azimi for the artwork.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant RR-00847 to the Clinical Research Center of the University of Virginia, the Baxter Healthcare Corp. (Round Lake, IL; to J.D.V.), the NIH-supported Clinfo Data Reduction Systems, the University of Virginia Pratt Foundation and Academic Enhancement Program, the NSF Center for Biological Timing (Grant DIR89–20162), the NIH P-30 Center for Reproduction Research (NICHHD Grant HD-28934), Postdoctoral Research Training in Diabetes and Hormone Action Grant 5T32-DK-07320 (to A.D.Z.), and Veterans Affairs Merit Review Research Funds (to A.I.). Back

2 Current address: Internal Medicine Endocrinology, CIGNA Health Care of Arizona, 3770 South 16th Avenue, Tucson, Arizona 85713. Back

Received December 30, 1996.

Revised March 10, 1997.

Accepted March 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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