INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

HEALTHY AGING OCCURS insidiously, making causal mechanisms difficult to discern (6, 12, 17, 22). However, recent studies suggest that older humans exhibit subtle deterioration of regulatory and integrative hormonal control (22), consistent with an aging hypothesis of impaired neuroendocrine feedback adaptations. The present studies use the male reproductive axis as a dynamic neurointegrative system to examine the latter thesis further. In this networklike (feedback and feedforward coupled) axis, hypothalamic gonadotropin-releasing hormone (GnRH) neurons impose pulsatile drive on luteinizing hormone (LH) secretion (2, 4, 11, 16, 18, 20). Available clinical observations suggest that the GnRH-LH pulsing mechanism offers a possible prototype of early disruption of neurointegrative control in aging (3, 4, 19). To test this notion, we have quantified the daily GnRH-LH pulsing rate (frequency), the LH pulse mass, the regularity of serial LH burst amplitude and interpulse-interval sequences, and the frequency-independent variability of the latter measures in healthy young and older men.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Overview. Hormone concentration profiles in peripheral blood arise from episodic secretory bursts (or pulses), which decay in the circulation due to dilution, distribution, and subsequent metabolic elimination (2, 4, 7-9, 11, 16, 19, 20, 22). We here examine LH pulse generation in 13 young (ages 18-25 yr) and 13 older (ages 60-80 yr) healthy men. Under the assumption that LH pulses reflect corresponding GnRH inputs (see DISCUSSION), we formulate statistical inferences regarding LH secretion pulse timing independently of hormone kinetics.

Clinical protocol. Volunteers underwent blood sampling at 10-min intervals for 24 h. Sera (145 samples/subject) were analyzed in duplicate for LH content (First International Reference Preparation) by two-site monoclonal immunoradiometric assay (IRMA), as reported earlier (13). Observed serum LH concentration time series are illustrated in Fig. 1. In parallel experiments, we infused 50 IU recombinant human LH (Serono Laboratories; equal to 20 IU First International Reference Preparation) every 2 h intravenously as 6-min square-wave pulses in seven young (ages 21-35 yr) and seven older (ages 60-80 yr) men 3-4 wk after downregulation of LH secretion with leuprolide acetate (3.75 mg im). Blood was sampled every 10 min from 0800 to 2400 from a contralateral forearm vein for later assay of LH time series by IRMA, thereby allowing direct validation of model-based estimates of LH "secretory" burst mass.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Illustrative plots of observed serum luteinizing hormone (LH) concentration time series (solid lines) in 4 young (A) and 4 older (B) men. Data were obtained by immunoradiometric assay of blood sampled every 10 min for 24 h from 0800 to 0800. Fitted curves (dashed lines) assume a model of freely varying (analytically estimated) basal (time invariant) LH secretion with superimposed variably skewed LH pulses of individually determinable amplitudes. Biexponential kinetics were estimated simultaneously (METHODS).

Conditional pulse-time model. To quantify LH secretion and elimination simultaneously from available serum LH concentration time series (above), we used a stochastic model of pulsatile and basal hormone release, which allows for two-compartment elimination kinetics and is conditional on pulse times, as validated earlier on statistical and physiological grounds (7-9). This formulation includes burstlike hormone release of variable amplitude and skewness superimposed on an unknown rate of basal (time invariant) secretion. In this construction, the rate of change in the hormone concentration [X(t)] is described by a set of (coupled) differential equations, whose solution is

(1)

Model of pulse waiting times. To compare pulsing patterns requires a statistical model of the GnRH-LH pulse generator, which can accommodate a broad range of pulse-timing behavior. For example, here we wish to quantitate contrasts in the variability in interpulse-interval lengths independently of frequency differences. To this end, we distinguish irregularity (order-dependent reproducibility) from variability (order-independent dispersion about the mean). As a regularity statistic, we applied the approximate entropy (ApEn) measure to quantitate the process randomness of serial interpulse interval and pulse-mass values (14, 15). ApEn is a family of three-parameter regularity statistics, which are specified by m (pattern length), r (tolerance threshold for successive comparisons), and N (total data series length). ApEn specifically quantifies the relative degree of order-dependent subpattern recurrence in numerical or biological time series (14, 15). Second, we examine variability of the set of GnRH (LH) pulse waiting times (interpulse-interval lengths) and pulse mass values, wherein the order of their successive occurrence is irrelevant. Thus regularity and variability provide complementary but distinct insights.

(2)

k1

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Stochastic differential equation-based deconvolution analysis revealed comparable pulsatile, basal, and total daily LH secretory rates and LH elimination kinetics in young and older men, for all three models of basal LH secretion (METHODS; Table 1). However, mean LH pulse frequency (events/24 h) was higher (P < 0.001) and the mass of LH secreted per pulse (IU/l distribution volume) was lower (P = 0.038) in older men in the freely varying basal model, whether pulse mass was normalized to the length of the preceding or following interpulse interval (Fig. 2). The age-related reduction in LH pulse mass was corroborated in the zero basal (3.40 ± 0.62 vs. 5.57 ± 0.63 IU/l) and basal (3.51 ± 0.65 vs. 5.47 ± 0.66 IU/l) LH secretion models (both P < 0.038). In contrast, analyses of pulsatile LH profiles created via controlled intravenous infusions of recombinant human LH in leuprolide-downregulated young and older men (METHODS) yielded comparable (P = NS) estimates of 1) injected LH pulse mass [7.8 ± 0.58 (older) and 6.9 ± 0.82 (young) IU/l]; 2) rapid LH half-lives (7.2 ± 0.08 and 7.2 ± 0.05 min); 3) delayed LH half-lives (97 ± 8.5 and 93 ± 6.6 min); and 4) ApEn values [1.215 ± 0.032 (young) and 1.317 ± 0.045 (older) (P = NS)].

View larger version (12K): [in this window] [in a new window]   Fig. 2.   A: LH secretory burst mass (IU/l distribution volume) in young and older men (n = 13 volunteers each), according to the freely varying model of basal hormone secretion (Fig. 1). B: LH secretory pulse frequencies (events/24 h). Data are the means ± SE (median).

The regularity of observed 24-h serum LH concentration time series and of successive LH pulse-mass or interpulse-interval values was evaluated by the ApEn statistic (METHODS). The latter measure used a vector length of m = 1 and tolerance of r = 20% (for LH concentrations) and r = 75% (for the shorter pulse mass or waiting-time series). ApEn of serum LH concentration profiles was increased markedly in older men (1.579 ± 0.041) compared with young volunteers (1.071 ± 0.071; P < 10³). Because ApEn is a family of statistics defined also by N (number of observations), we compared the ratios of observed to random ApEn (mean of 1,000 randomly shuffled versions of each data series) in the two age cohorts. Parametric and nonparametric (Wilcoxon) statistical testing revealed an elevated ApEn ratio of successive LH pulse mass values (whether or not normalized to the previous or succeeding interpulse interval) in older men (P < 0.01; Fig. 3A). In contrast, the ApEn ratio of serial LH interpulse intervals did not differ by age (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Approximate entropy (ApEn) ratios for observed sequences of LH pulse mass (A) and interpulse-interval values (B) in young and older men (n = 13 subjects each). Individual ApEn ratios are the mean of the ratio of each observed ApEn value to that of 1,000 randomly shuffled versions of the same series. Higher ApEn ratios approaching unity denote greater irregularity. Data are the means ± SE (median).

The Weibull distribution was used to evaluate the behavior of pulse waiting times (interpulse-interval lengths) in the two age groups. Figure 4A illustrates schematically how the mean and SD of a set of interpulse-interval lengths depend upon  (frequency) and  (variability). Figure 4B shows simulations for different values of  (25, 20, 15, 10, and 5 pulses/day) in relation to each of three values of  [1 ( Poisson process), 2, and 5]. Specifically, the Weibull model accommodates both regular (large ) and irregular (small ) pulsing patterns. Figure 4C illustrates the estimated Weibull distributions for the sets of interpulse intervals observed in one young and one older man. This plot highlights how the parameter  mirrors pulsing variability. Figure 5 gives all 26 estimates of  and  and corresponding plots of the estimated Weibull densities (interpulse-interval distributions) and their individual CVs.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   A: illustrative relationship of the Weibull interpulse-interval (IPI) distribution mean and SD to the analytic parameters, gamma (, a measure of interpulse variability) and lambda (, a measure of mean pulse frequency). B: 5 strata of mean probabilistic pulse frequencies (vertical scale) are shown for each of 3 arbitrary degrees of pulsing variability (horizontal groups). * Times of pulse occurrences simulated for a Weibull renewal process. Gamma = 1 conforms to a simple exponential distribution of waiting times (Poisson process), wherein the SD equals the mean (1/) definitionally. C: unequal gamma values allow for more or less interpulse variability at any given pulse frequency, as illustrated in 1 young (left) and 1 older male (right). Top: observed serum LH concentrations (solid line), estimated pulse times (*), and model-predicted fits (dashed line). Middle: reconstructed LH secretion rates. Bottom: * individual IPI lengths (waiting times). Continuous curves give the matching estimated Weibull distributions for the set of IPI values. In this illustration the mean pulsing rates are the same. The lesser variability in pulse intervals in the older male is reflected in the higher  value (gamma parameter).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   A: Weibull plots of IPI distributions estimated in 13 young and 13 older men. The x-axis gives the IPI length (waiting times, min) and the y-axis the corresponding predicted Weibull probability density. B: lambda defines the daily mean LH pulsing frequency (the reciprocal of which is approximately the IPI value; the difference is negligible), and gamma the variability of the waiting-time distribution decorrelated from pulsing frequency (METHODS).

We performed generalized likelihood ratio tests of the two groups of Weibull functions. The full parameter space would consist of four parameters: a  and  for each group. A test of the null hypothesis that both age groups have a common  suggests a trend toward unequal (higher)  in older men (P = 0.08), which denotes reduced interpulse waiting-time variability. Lambda values are highly different in the two age cohorts (P values numerically <10⁶), whether one considers distinct or identical s for the two groups. Elevated  signifies an increased mean LH pulsing rate in the aging male.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present analyses unveiled threefold disruption of the GnRH-LH pulsing system in healthy aging men. First, quantitation of LH secretion by way of a stochastic differential equation-based deconvolution model disclosed an accelerated frequency and reduced amplitude of LH pulses in elderly males. Second, statistical analysis of the regularity of LH pulse-mass sequences by the ApEn measure revealed a significantly more disorderly succession of LH pulse mass over 24 h in older men. And, third, modeling of pulse-generator timing via the Weibull probability distribution pointed to reduced variability of interpulse-interval lengths in aging males. Thus the foregoing comparisons delineate defects in amplitude-dependent LH secretion and in both frequency-dependent and frequency-independent GnRH-LH pulse-generator timing in older men.

The lower LH secretory burst mass estimates and the higher daily GnRH-LH secretory burst frequency inferred here in older men might arise secondarily from a reduction in gonadal sex-steroid negative feedback and/or primarily from an intrinsic hypothalamic defect in GnRH neuronal pulse generation (1, 5, 6, 12, 19, 20, 22). In relation to secondary causes, clinical experiments and biomathematical models indicate that muting of testosterone's negative feedback will reduce LH secretory burst mass, elevate mean GnRH-LH pulse-generator frequency and also heighten the irregularity of LH secretory patterns (20). However, the degree of testosterone deprivation required to initiate such adaptations is not known and may itself be age dependent. In relation to primary dysregulation of the GnRH neuronal pulse generator in aging, an accelerated frequency pulsing could arise from blunted repression and/or heightened activation of relevant neurotransmitter pathways, which govern episodic outflow of the synchronized GnRH neuronal ensemble (16, 19). However, the nature of any imbalance between inhibitory and excitatory control mechanisms inferred here in older men is not known.

The age-dependent increase in daily GnRH-LH pulse frequency was substantial (an ~50% median increment) and mechanistically specific, because total daily LH secretion and LH kinetics were comparable in the two study cohorts. Accelerated GnRH-LH pulse frequency in older men is reminiscent of that observed in ovariprival women. However, in the postmenopausal female, the calculated mass of LH secreted per pulse is elevated significantly (8), in contradistinction to the 50% reduction observed here. From a mechanistic perspective, lower-amplitude LH secretory bursts in the aging male could reflect several pathophysiologies. First, reduced hypothalamic GnRH release has been inferred indirectly in aged animals and humans (1, 5, 19, 20). Second, an excessive feedback action of androgens might repress GnRH-LH output in older men (5, 21). Third, a higher GH stimulus frequency per se can diminish the amount of LH secreted per pulse (4). Fourth, in principle, impaired gonadotrope responsiveness to GnRH could limit LH secretory burst mass in older men. However, measurable pituitary stores of LH are actually increased in older humans (1), and the potency of GnRH is higher (5, 13, 19, 23). Last, systematic alterations in the distribution volume or metabolic clearance rate of LH were excluded as a trivial explanation for reduced LH pulse size in aging men (13, 23). Thus impoverished LH secretory burst mass most likely mirrors reduced hypothalamic GnRH feedforward drive of pulsatile LH output in the elderly male.

The regularity of successive LH pulse-mass values, as quantitated by the ApEn statistic (14, 15), was significantly disrupted in older men. More disorderly LH pulse-mass sequences in aging may denote nonuniform hypothalamic release of GnRH or inconsistent actions of GnRH on gonadotropes over 24 h. Normalizing LH pulse mass to each preceding (or following) interpulse-interval length did not nullify the age-related irregularity in serial LH pulse mass. Thus anomalous regulation of successive LH burst amplitudes in aging is frequency-independent at least on statistical grounds.

The present analysis assumes that the timing of statistically identifiable LH release episodes provides a valid (noncritically censored) index of intermittently synchronized GnRH neuronal secretion (2, 4, 5, 8, 11, 19-22). Under this assumption, we infer that aging alters the probabilistic structure of GnRH pulse generation, as monitored by LH interpulse waiting times (intervals between successive bursts). To decorrelate interpulse-interval variability from the elevated mean GnRH-LH pulse firing rate in older men, we used a Weibull model of a stochastic renewal-like process for the timing behavior of the GnRH pulse generator. The Weibull (member of the generalized Gamma) distribution allows for a full spectrum of interpulse-interval variabilities at any given mean pulsing frequency. Thereby, we show that LH pulse waiting-time variability in both age groups studied is considerably less than that predicted by a simple Poisson process (i.e., Gamma typically exceeded unity in the Weibull formulation; METHODS). The degree of departure of interpulse-interval variability from a Poisson model tended to be greater in older men (P = 0.08). This apparent age distinction would denote relative repression of normal waiting-time variability. The consistency and the cause of such presumptive aging-related (and frequency independent) quenching of normal young-adult variability in the renewal behavior of the GnRH-LH pulse generator in older men are not known.

The present work also highlights an analytic distinction between order-independent variability (Fig. 4) and order-dependent regularity (Fig. 3) of relevant pulse measures. Variability was appraised via the coefficient of variation (percentage ratio of the mean to standard deviation) of the Weibull distribution of interpulse-interval values. Regularity was quantified by the ApEn statistic (14, 15). ApEn measures the reproducibility of subpatterns in time series, with the higher values denoting less orderliness. Here, we used normalized ApEn ratios (defined empirically by the mean ratio of each observed ApEn series to that of 1,000 randomly reordered versions of the same data) to compare series of different N (number of observed LH interpulse-interval or pulse-mass values). ApEn ratios for successive LH pulse-waiting times in young and older men were comparable, whereas ApEn ratios for sequential LH pulse mass values were elevated in older men (above). The foregoing findings suggest rather specific mechanistic changes in aging. For example, GnRH-LH pulse timing is governed by intermittent synchronization of multiple repressive and facilitative inputs to the GnRH neuronal ensemble. In contrast, LH pulse amplitude/mass reflects GnRH stimulus strength, instantaneous gonadotrope-cell responsiveness, the momentary availability of cellular LH stores, de novo LH biosynthesis, and concomitant hypothalamo-pituitary microvascular blood flow (7, 8, 10, 18, 20). Thus more irregular LH pulse mass sequences in older men could mirror age-related disparities in one or more of the foregoing processes, which couple GnRH release to LH secretion.

In summary, the present investigations identify novel facets of altered GnRH-LH pulse regulation in healthy aging men, namely, mean accelerated GnRH-LH pulse frequency, attenuated LH pulse size, reduced serial regularity of LH secretory burst mass and a trend to impaired variability of interpulse waiting times.

Perspectives