Follicle-stimulating hormone is secreted more irregularly than luteinizing hormone in both humans and sheep.

Recently introduced statistical tools capable of discerning differences between the pattern of luteinizing hormone (LH) secretion and that of follicle-stimulating hormone (FSH) could be valuable in understanding ovulation and menopause, and ultimately in making diagnostic decisions and treating infertility and polycystic ovary syndrome. We assessed the validity and scope of the hypothesis that FSH is secreted more irregularly than LH in ewes and fertile women. We compared secretory irregularity of LH to that of FSH in both ovariectomized ewes (n = 7) and women of proven fertility (n = 5) during the follicular and luteal phases of their reproductive cycles. In each sheep, time series from both hypophyseal portal blood (HPB) and peripheral blood were evaluated in 72 samples obtained every 5 min; in each human, both luteal and follicular periods were studied in 192 samples obtained every 7.5 min. To quantify serial irregularity, we used approximate entropy (ApEn), a scale- and model-independent statistic. FSH secretion was consistently more irregular than that of LH in each subject. For sheep HPB, ApEn(FSH) = 1.415+/-0.097 was larger than ApEn(LH) = 0. 822+/-0.213, P < 0.0001 (mean+/-SD, paired t test). This difference persisted peripherally: ApEn(FSHper) = 1.431+/-0.101 > ApEn(LHper) = 1.252+/-0.086, P = 0.024. In women, ApEn(FSH) = 1.467+/-0.217 > ApEn(LH) = 0.923+/- 0.305, P < 0.0001. ApEn(FSH) > ApEn(LH) in 100% of women (peripheral) and sheep HPB. Secretion during the follicular phase was more irregular than during the luteal phase for both FSH and LH (P < 0.01). LH mean level secretion showed a wake/sleep difference in women, P < 0.005, with higher values awake. The consistency and statistical significance of these findings suggest that this LH/FSH difference may be broadly based within higher mammals. Ranges of normative and abnormal regularity values of LH, FSH, and their difference can be used in a number of settings, both (currently) research and (potentially ultimately) clinical milieus.

Enhanced basal and disorderly growth hormone secretion distinguish acromegalic from normal pulsatile growth hormone release.

Pulses of growth hormone (GH) release in acromegaly may arise from hypothalamic regulation or from random events intrinsic to adenomatous tissue. To distinguish between these possibilities, serum GH concentrations were measured at 5-min intervals for 24 h in acromegalic men and women with active (n = 19) and inactive (n = 9) disease and in normal young adults in the fed (n = 20) and fasted (n = 16) states. Daily GH secretion rates, calculated by deconvolution analysis, were greater in patients with active acromegaly than in fed (P < 0.05) but not fasted normal subjects. Significant basal (nonpulsatile) GH secretion was present in virtually all active acromegalics but not those in remission or in fed and fasted normal subjects. A recently introduced scale- and model-independent statistic, approximate entropy (ApEn), was used to test for regularity (orderliness) in the GH data. All but one acromegalic had ApEn values greater than the absolute range in normal subjects, indicating reduced orderliness of GH release; ApEn distinguished acromegalic from normal GH secretion (fed, P < 10(-12); fasted, P < 10(-7)) with high sensitivity (95%) and specificity (100%). Acromegalics in remission had ApEn scores larger than those of normal subjects (P < 0.0001) but smaller than those of active acromegalics (P < 0.001). The coefficient of variation of successive incremental changes in GH concentrations was significantly lower in acromegalics than in normal subjects (P < 0.001). Fourier analysis in acromegalics revealed reduced fractional amplitudes compared to normal subjects (P < 0.05). We conclude that GH secretion in acromegaly is highly irregular with disorderly release accompanying significant basal secretion.

Acute and short-term administration of a sulfonylurea (gliclazide) increases pulsatile insulin secretion in type 2 diabetes.

The high-frequency oscillatory pattern of insulin release is disturbed in type 2 diabetes. Although sulfonylurea drugs are widely used for the treatment of this disease, their effect on insulin release patterns is not well established. The aim of the present study was to assess the impact of acute treatment and 5 weeks of sulfonylurea (gliclazide) treatment on insulin secretory dynamics in type 2 diabetic patients. To this end, 10 patients with type 2 diabetes (age 53 +/- 2 years, BMI 27.5 +/- 1.1 kg/m(2), fasting plasma glucose 9.8 +/- 0.8 mmol/l, HbA(1c) 7.5 +/- 0.3%) were studied in a double-blind placebo-controlled prospective crossover design. Patients received 40-80 mg gliclazide/placebo twice daily for 5 weeks with a 6-week washout period intervening. Insulin pulsatility was assessed by 1-min interval blood sampling for 75 min 1) under baseline conditions (baseline), 2) 3 h after the first dose (80 mg) of gliclazide (acute) with the plasma glucose concentration clamped at the baseline value, 3) after 5 weeks of treatment (5 weeks), and 4) after 5 weeks of treatment with the plasma glucose concentration clamped during the sampling at the value of the baseline assessment (5 weeks-elevated). Serum insulin concentration time series were analyzed by deconvolution, approximate entropy (ApEn), and spectral and autocorrelation methods to quantitate pulsatility and regularity. The P values given are gliclazide versus placebo; results are means +/- SE. Fasting plasma glucose was reduced after gliclazide treatment (baseline vs. 5 weeks: gliclazide, 10.0 +/- 0.9 vs. 7.8 +/- 0.6 mmol/l; placebo, 10.0 +/- 0.8 vs. 11.0 +/- 0.9 mmol/l, P = 0.001). Insulin secretory burst mass was increased (baseline vs. acute: gliclazide, 43.0 +/- 12.0 vs. 61.0 +/- 17.0 pmol. l(-1). pulse(-1); placebo, 36.1 +/- 8.4 vs. 30.3 +/- 7.4 pmol. l(-1). pulse(-1), P = 0.047; 5 weeks-elevated: gliclazide vs. placebo, 49.7 +/- 13.3 vs. 37.1 +/- 9.5 pmol. l(-1). pulse(-1), P < 0.05) with a similar rise in burst amplitude. Basal (i.e., nonoscillatory) insulin secretion also increased (baseline vs. acute: gliclazide, 8.5 +/- 2.2 vs. 16.7 +/- 4.3 pmol. l(-1). pulse(-1); placebo, 5.9 +/- 0.9 vs. 7.2 +/- 0.9 pmol. l(-1). pulse(-1), P = 0.03; 5 weeks-elevated: gliclazide vs. placebo, 12.2 +/- 2.5 vs. 9.4 +/- 2.1 pmol. l(-1). pulse(-1), P = 0.016). The frequency and regularity of insulin pulses were not modified significantly by the antidiabetic therapy. There was, however, a correlation between individual values for the acute improvement of regularity, as measured by ApEn, and the decrease in fasting plasma glucose during short-term (5-week) gliclazide treatment (r = 0.74, P = 0.014, and r = 0.77, P = 0.009, for fine and coarse ApEn, respectively). In conclusion, the sulfonylurea agent gliclazide augments insulin secretion by concurrently increasing pulse mass and basal insulin secretion without changing secretory burst frequency or regularity. The data suggest a possible relationship between the improvement in short-term glycemic control and the acute improvement of regularity of the in vivo insulin release process.

Failure of physiological plasma glucose excursions to entrain high-frequency pulsatile insulin secretion in type 2 diabetes.

Insulin is released in high-frequency pulsatile bursts at intervals of 6-13 min. Intrapancreatic mechanisms are assumed to coordinate pulsatile insulin release, but small oscillations in plasma glucose concentrations may contribute further. To gain additional insight into beta-cell (patho)physiology, we explored the ability of repetitive small glucose infusions (6 mg/kg over 1 min every 10 min) to modify rapid pulsatile insulin secretion in 10 type 2 diabetic individuals (plasma glucose 9.3 +/- 1.0 mmol/l, HbA1c 7.9 +/- 0.5%, mean +/- SE) and 10 healthy subjects. All subjects were investigated twice in randomly assigned order: during saline and during glucose exposure. Blood was collected every minute for 90 min to create a plasma insulin concentration time-series for analysis using 3 complementary algorithms: namely, spectral analysis, autocorrelation analysis, and approximate entropy (ApEn). During saline infusion, none of the algorithms were able to discriminate between diabetic and control subjects (P > 0.20). During glucose entrainment, spectral density peaks (SP) and autocorrelation coefficients (AC) increased significantly (P < 0.001), and ApEn decreased (P < 0.01), indicating more regular insulin time-series in the healthy volunteers. However, no differences were observed in the diabetic individuals between the glucose and saline conditions. Furthermore, in spite of identical absolute glucose excursions (approximately 0.3 mmol/l) glucose pulse entrainment led to a complete (SP: 4.76 +/- 0.62 [range 2.08-7.60] vs. 17.24 +/- 0.93 [11.70-20.58], P < 0.001; AC: 0.01 +/- 0.05 [0.33-0.24] vs. 0.64 +/- 0.05 [0.35-0.83], P < 0.001) or almost complete (ApEn: 1.59 +/- 0.02 [1.48-1.67] vs. 1.42 +/- 0.05 [1.26-1.74], P < 0.005) separation of the insulin time-series in diabetic and control subjects. Even elevating the glucose infusion rate in the diabetic subjects to achieve comparable relative (and hence higher absolute) glucose excursions (approximately 4.9%) failed to entrain pulsatile insulin secretion in this group. In conclusion, the present study demonstrates that failure to respond adequately with regular oscillatory insulin secretion to recurrent high-frequency and (near)-physiological glucose excursion is a manifest feature of beta-cell malfunction in type 2 diabetes. Whether the model will be useful in unmasking subtle (possible prediabetic) defects in beta-cell sensitivity to glucose drive remains to be determined.

Glucagon-like peptide 1 increases mass but not frequency or orderliness of pulsatile insulin secretion.

Glucagon-like peptide 1 (GLP-1) is a peptide hormone that is released from the gut after luminal stimulation. The hormone is a potent insulin secretagogue and is a potential novel pharmaceutical adjuvant in the treatment of NIDDM. Insulin is secreted as a series of punctuated secretory bursts superimposed on a basal insulin release. Recently, the contribution of these secretory bursts to overall insulin secretion has been evaluated, and studies using catheterization across the pancreas in a canine model and studies using deconvolution in humans have revealed that the majority of insulin is released during these secretory bursts. Moreover, the main regulation of insulin secretion is through perturbation of mass and frequency of these secretory bursts. The mode of delivery of insulin into the circulation seems important for insulin action, and it is therefore important to know the impact of a potential therapeutic insulin secretagogue on the mode of insulin secretion. To assess the effects of GLP-1 on the mass, frequency, amplitude, and overall contribution of pulsatile insulin secretion, we used a recently validated deconvolution model to examine these variables before and during infusion of GLP-1 in eight healthy men (age 28 +/- 2 years; BMI 24 +/- 2 kg/m2). At a constant glucose infusion (2.5 mg x kg-1 x min-1), near-steady state was reached at 75 min, and sampling was performed every minute at t = 75-115 and 145-185 min. At t = 115 min, an infusion of saline or GLP-1 (50 pmol x kg-1 x min-1) was given. The regularity of insulin secretion was measured by approximate entropy, a recently developed mathematical statistic, applied herein to assess the regularity in a hormone concentration time series. After GLP-1 infusion, there was an abrupt increase in the peripheral concentrations of serum C-peptide (696 +/- 65 vs. 1,538 +/- 165 pmol/l) and insulin (49 +/- 8 vs. 138 +/- 21 pmol/l) concentrations. This increase was mainly due to an increase in the pulsatile component of insulin secretion that was achieved by a fourfold increase in secretory burst mass (28.2 +/- 4.4 vs. 100.1 +/- 15.8 pmol x l-1 x pulse-1; P < 0.001), and amplitude (12.7 +/- 2.2 vs. 4.3 +/- 7.7 pmol x l-1 x min-1; P < 0.002), whereas the secretory burst frequency was not affected by GLP-1 (11.5 +/- 0.7 vs. 12.6 +/- 0.6 pulses/h; P = 0.4). As a consequence, the detected contribution of pulsatile to overall insulin secretion was increased from 56 +/- 4 to 77 +/- 4% (P < 0.005). The orderliness of the insulin release process was not deteriorated by short-term GLP-1 infusion as assessed by approximate entropy (1.19 +/- 0.04 vs. 1.18 +/- 0.04; P = 0.7).

Gender- and age-related differences in heart rate dynamics: are women more complex than men?

OBJECTIVES: This study aimed to quantify the complex dynamics of beat-to-beat sinus rhythm heart rate fluctuations and to determine their differences as a function of gender and age. BACKGROUND: Recently, measures of heart rate variability and the nonlinear "complexity" of heart rate dynamics have been used as indicators of cardiovascular health. Because women have lower cardiovascular risk and greater longevity than men, we postulated that there are important gender-related differences in beat-to-beat heart rate dynamics. METHODS: We analyzed heart rate dynamics during 8-min segments of continuous electrocardiographic recording in healthy young (20 to 39 years old), middle-aged (40 to 64 years old) and elderly (65 to 90 years old) men (n = 40) and women (n = 27) while they performed spontaneous and metronomic (15 breaths/min) breathing. Relatively high (0.15 to 0.40 Hz) and low (0.01 to 0.15 Hz) frequency components of heart rate variability were computed using spectral analysis. The overall "complexity" of each heart rate time series was quantified by its approximate entropy, a measure of regularity derived from nonlinear dynamics ("chaos" theory). RESULTS: Mean heart rate did not differ between the age groups or genders. High frequency heart rate power and the high/low frequency power ratio decreased with age in both men and women (p < 0.05). The high/low frequency power ratio during spontaneous and metronomic breathing was greater in women than men (p < 0.05). Heart rate approximate entropy decreased with age and was higher in women than men (p < 0.05). CONCLUSIONS: High frequency heart rate spectral power (associated with parasympathetic activity) and the overall complexity of heart rate dynamics are higher in women than men. These complementary findings indicate the need to account for gender-as well as age-related differences in heart rate dynamics. Whether these gender differences are related to lower cardiovascular disease risk and greater longevity in women requires further study.

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period.

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.

Patients with Cushing's disease secrete adrenocorticotropin and cortisol jointly more asynchronously than healthy subjects.

We examined serum concentration time-series for ACTH and cortisol in 20 patients with pituitary-dependent ACTH excess (Cushing's disease) and in 29 age- and gender-matched controls. For each subject, blood samples were obtained at 10-min intervals for 24 h. Joint ACTH-cortisol synchrony was quantified using the recently introduced cross-approximate entropy (cross-ApEn) statistic. In patients, cross-ApEn was greater than in controls (1.686 +/- 0.051 vs. 1.077 +/- 0.039, P = 3.45 x 10(-16), giving a sensitivity of 85%. In control subjects, but not in patients, cross-ApEn was correlated positively with age (r = 0.465, P = 0.011) There was no gender difference in cross-ApEn, nor a relationship between cross-ApEn and the 24-h ACTH and cortisol secretion, in patients or controls. In contrast, the maximal cross-correlation coefficient for the ACTH and cortisol series after detrending the series was 0.394 +/- 0.033 in controls and 0.297 +/- 0.034 in patients with considerable overlap of the subgroups, giving a sensitivity for this index of only 5%. In addition to previous findings of increased individual irregularity of ACTH and cortisol release in Cushing's disease, we can now also demonstrate greater joint asynchrony of the circulating concentrations of these hormones. Thus, Cushing's disease disrupts ensemble network secretory dynamics over individual hormone output. We conclude that, like GH-secreting pituitary and aldosterone-secreting adrenal tumors, ACTH-secreting pituitary tumors exhibit significant loss of orderly hormone release patterns. Moreover, Cushing's disease is marked further by deterioriation of bihormonal synchrony between ACTH and cortisol release, thus suggesting further erosion of within-axis feedback control.

Disruption of the young-adult synchrony between luteinizing hormone release and oscillations in follicle-stimulating hormone, prolactin, and nocturnal penile tumescence (NPT) in healthy older men.

The healthy human male hypothalamo-pituitary-gonadal axis exhibits age-dependent loss of coordinate LH-testosterone secretion. A putative independent defect in Leydig-cell steroidogenesis with aging would confound the attribution of such LH-testosterone asynchrony to a hypothalamo-pituitary locus per se. Accordingly, here we appraise by sampling every 2.5 min overnight the joint synchrony of moment-to-moment LH release with simultaneously monitored pituitary FSH secretion, prolactin release, and nocturnal penile tumescence (NPT) oscillations, as a neurophysiological correlate of sleep regulation) in 10 young (ages 21-34) and 8 older (ages 62-72) healthy men. Joint synchrony for paired LH-FSH, LH-prolactin, and LH-NPT observations in young vs. older individuals was quantified by the cross-approximate entropy (cross-ApEn) statistic, with larger cross-ApEn values indicating greater two-variable asynchrony. Concomitantly, we assessed (possible) univariate changes with age for each of LH, FSH, prolactin, and NPT, as quantified by approximate entropy (ApEn). Hormone assays were performed by random-access direct chemiluminescence analyzer. Overnight mean (+/- SEM) serum LH concentrations (IU/L) were equivalent in older (3.1 +/- 0.31 IU/L) and younger (2.9 +/- 0.29) men, as were their serum total testosterone concentrations; viz., 425 +/- 48 (older) and 523 +/- 40 (younger) ng/dL. However, all three sets of paired time-series were significantly more asynchronous in the older cohort. First, cross-ApEn of paired LH-FSH release was significantly higher (or more asynchronous) in older subjects; viz., 1.902 +/- 0.022 in older men vs. 1.607 +/- 0.058 in younger individuals (P = 0.0005). Second, cross-ApEn of paired LH and prolactin release was 1.744 +/- 0.085 in older volunteers vs. 1.346 +/- 0.084 in younger subjects (P = 0.0046). Third, and most notably, cross-ApEn for the joint LH-NPT observation time-series was significantly greater in older subjects at 1.771 +/- 0.056 vs. 1.223 +/- 0.086 (young) (P = 0.0001), thereby denoting loss of coordination between (neural) signals directing intermittent LH secretion and those governing sleep-associated penile tumescence in older men. Among one-variable results, only ApEn of LH release was significantly higher in older individuals at 1.323 +/- 0.058 vs. 0.897 +/- 0.089 in younger subjects (P = 0.0019), signifying greater disorderliness of the LH secretory process in aged men. Individual ApEn values of FSH and prolactin release and NPT were age-invariant. In ensemble, the present clinical experiments indicate that, within the aging male reproductive axis, bihormonal network disruption is more pronounced than individual signal disruption. We suggest that abrogation of joint synchrony among hypothalamically directed pituitary hormones and a neurogenically organized sexual response (nocturnal penile tumescence) can be unified thematically under an hypothesis of disrupted central nervous system hypothalamo-pituitary network coordination in human aging. Such implicit disarray of multinodal communication is of consequence both scientifically and clinically, especially in proposing aging theories and intervention strategies.

Apparently complete restoration of normal daily adrenocorticotropin, cortisol, growth hormone, and prolactin secretory dynamics in adults with Cushing's disease after clinically successful transsphenoidal adenomectomy.

ACTH production in Cushing's disease is characterized by a markedly elevated rate of basal (nonpulsatile) secretion, an increased mass of ACTH released per burst and an unremarkable pulse frequency. In addition, the ACTH secretory process and that of GH and PRL exhibit profoundly disordered patterns. Whether some or all of these disturbances can be reversed or normalized by transsphenoidal microadenomectomy remains unknown. We therefore investigated the detailed dynamics of ACTH, GH, and PRL in eight patients (aged 38.9+/-4.2 yr) with pituitary-dependent Cushing's disease who were in long-term (8.2+/-1.7 yr) clinical remission following transsphenoidal surgery and eight controls matched for age, gender, and body mass index. To this end, blood was sampled at 10-min intervals for 24 h for the later assay of ACTH, cortisol, GH, and PRL. Secretory activity was quantitated by deconvolution methods, and the pattern orderliness (regularity) of hormone release was determined by the approximate entropy (ApEn) statistic. The joint synchrony of ACTH and cortisol secretion was monitored by the cognate bivariate statistic, cross-ApEn. Diurnal properties of the hormonal release were appraised by cosinor analysis. Based on deconvolution analysis, postsurgical patients exhibited a normal frequency, half-life, duration, and mass of ACTH and cortisol secretory bursts. Accordingly, the 24-h production rates of both ACTH (2.5+/-0.7 microg/L in patients vs. 2.9+/-0.7 microg/L in controls; P = 0.755) and cortisol (49+/-11 micromol/L in patients vs. 73+/-15 micromol/L in controls; P = 0.217) were normal also. The acrophase of the diurnal rhythm of ACTH (patients, 0817 h +/- 37 min; controls, 0850 h +/- 38 min; P = 0.629) and cortisol (patients, 1000 h +/- 24 min; controls, 0855 h +/- 30 min; P = 0.175) was also restored by surgery. ApEn values of ACTH (patients, 1.168 +/- 0.090; controls, 0.864+/-0.122; P = 0.133) and cross-ApEn of ACTH-cortisol (patients, 1.396+/-0.087; controls, 1.170+/-0.076; P = 0.140) secretion were both normal in this cohort, denoting restoration of the secretory process regularity. Cortisol ApEn was slightly higher in patients (patients, 1.034+/-0.084; controls, 0.831+/-0.038; P = 0.048). Both GH and PRL time series manifested full reconstitution of pulsatile, 24-h rhythmic, and entropic properties. In summary, clinically successful transsphenoidal microadenomectomy in adults with Cushing's disease can fully normalize virtually all quantitative features of regulated ACTH, cortisol, GH, and PRL secretion. Further studies will be needed to establish the consistency of these findings in larger cohorts of adults with Cushing's disease and in children with this disorder and to delineate the significance, if any, of a residual, minimally detectable disruption of orderly cortisol secretion in this patient population.

Joint growth hormone and cortisol spontaneous secretion is more asynchronous in older females than in their male counterparts.

In humans, cortisol and GH are secreted in a pulsatile manner, and an interaction between GH and the hypothalamic-pituitary-adrenal axis has been established. In view of the sexually dimorphic pattern in GH secretion, we investigated the GH-cortisol bihormonal secretory dynamics in male and female healthy older individuals. We studied the GH and cortisol secretory patterns in 83 healthy subjects (45 men and 38 women; age range, 59.4-73.0 yr) by determining serum GH and cortisol concentrations at 20-min intervals for 24 h. The irregularity of GH and cortisol secretion was assessed using approximate entropy (ApEn), a scale- and model-independent statistic. The synchrony of joint GH-cortisol spontaneous secretion was quantified using the cross-ApEn statistic. Cross-correlation analysis of GH and cortisol patterns was computed at various time lags covering the 24-h period. Mean 24-h serum GH concentrations were significantly higher in females (mean, 1.31 mU/L; SD, 0.87) than in males (mean, 0.88 mU/L; SD, 0.42; P = 0.009), whereas mean 24-h serum total cortisol concentrations were higher in males (mean, 9.0 microg/dL; SD, 1.4) than in females (mean, 7.3 microg/dL; SD, 1.4; P = 0.0001). GH secretion was more irregular in females (mean ApEn, 0.81; SD, 0.23) than in males (mean ApEn, 0.60; SD, 0.20; P < 0.001). No significant difference in the regularity of cortisol secretion was noted between sexes. Cross-ApEn values of paired GH-cortisol were higher in females (mean, 1.15; SD, 0.18) than in males (mean, 1.01; SD, 0.16; P = 0.0003). Stepwise multiple linear regression analysis indicated that estradiol and insulin-like growth factor-binding protein-3 concentrations were independently related to GH ApEn values (r(2) = 0.14; P = 0.01), whereas cross-ApEn values of paired GH-cortisol were best predicted by FSH concentrations (r(2) = 0.37; P = 0.003). Cross-correlation analysis revealed a significant positive correlation between GH and cortisol, peaking at lag time of 4.7 h in males (r = 0.30; P < 0.0001) and 4.3 h in females (r = 0.14; P < 0.0001), with GH leading cortisol by these time intervals. In addition, a significant negative correlation between the two hormones was noted over time, peaking at 4.7 h in males (r = -0.21; P < 0.0001) and 6.3 h in females (r = -0.25; P < 0.0001), with cortisol leading GH by these time intervals. The above results indicate that in the elderly, females have a more disordered GH secretory pattern and a more asynchronous joint GH-cortisol secretion than their male counterparts. These observations most likely reflect bidirectional interactions between the GH and hypothalamic-pituitary-adrenal axis in humans as well as diminution of subsystem integrity and synchronous control of interconnected hormonal systems with advancing age.

Basal, pulsatile, entropic, and 24-hour rhythmic features of secondary hyperprolactinemia due to functional pituitary stalk disconnection mimic tumoral (primary) hyperprolactinemia.

Under physiological conditions, PRL secretion is regulated precisely by various stimulating and inhibiting factors. Hyperprolactinemia may arise as a primary consequence of a PRL-secreting pituitary adenoma. Secondary hyperprolactinemia (SH) may emerge in patients with hypothalamic disease, hypophyseal stalk compression, or suprasellar extension of a (nonlactotrope) pituitary adenoma. The latter may reflect diminished delivery of dopamine or other inhibitory factors to normal lactotropes. We hypothesized that diurnal and ultradian rhythms of PRL secretion would differ in secondary (e.g. hypothalamic) and primary (e.g. tumoral states) hyperprolactinemia (PH), assuming that the underlying pathophysiologies differ. To test this clinical postulate, we investigated the patterns of 24-h PRL release in eight patients with SH associated with functional hypothalamo-pituitary disconnection and in eight patients with PH attributable to microprolactinoma. Data in each group were compared with values in healthy gender-matched controls. PRL time series were obtained by repetitive 10-min blood sampling, followed by high- precision immunofluorometric assay. PRL concentration profiles were analyzed by the complementary tools of model-free discrete peak detection, waveform-independent deconvolution analysis, cosinor regression, and the approximate entropy metric to quantitate pulsatile, basal, 24-h rhythmic, and pattern-dependent (entropic) PRL secretion. Patients with tumoral hyperprolactinemia (PH) showed a 2-fold higher 24-h mean serum PRL concentration than patients with SH (62 +/- 13 microg /L vs. 30 +/- 6.9 microg/L, respectively, P = 0.029). Estimated PRL pulse frequency (events/24 h) was similar in the two patient groups (18.5 +/- 0.7 vs. 17.6 +/- 0.8; P = 0.395) but elevated over that in euprolactinemic controls (P < 0.0001 for both). Deconvolution analysis disclosed a mean daily PRL secretion rate of 790 +/- 170 microg in PH patients vs. 380 +/- 85 microg in SH patients (P = 0.030). Nonpulsatile PRL secretion comprised nearly 70% of total secretion in both patient groups and 50% in controls (P < 0.0001). Cosinor analysis revealed similar acrophases in all three study cohorts. The mean skewness of the statistical distribution of the individual PRL sample secretory rates was reduced, compared with controls (P < 10 (-5) for each), but equivalent in SH and PH patients (0.83 +/- 0.12 vs. 0.78 +/- 0.08, respectively), denoting a loss of the normal spectrum of low- and higher-amplitude secretion rates. Approximate entropy, a regularity statistic, was markedly elevated in both patient groups over controls (P < 10 (-6) for each) and was slightly higher in PH patients than in SH patients (1.639 +/- 0.029 vs. 1.482 +/- 0.067, P = 0.048). In summary, patterns of PRL secretion in PH and SH states exhibit an equivalently increased frequency of PRL pulses, a comparably marked rise in nonpulsatile (basal) PRL secretion. Despite overlap, the regularity of PRL release patterns is disrupted even more profoundly in PH (tumoral), compared with SH. Assuming that the orderliness of serial PRL output monitors normal integration within a feedback-controlled neurohormone axis, then the more disorderly patterns of tumoral PRL secretion point to greater regulatory disruption in PH. The latter may reflect abnormal secretory behavior associated with lactotrope neoplastic transformation and/or isolation of the tumor cell mass from normal hypothalamic controls.

Estrogen and testosterone, but not a nonaromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-steroid hormone replacement.

Activation of the gonadotropic and somatotropic axes in puberty is marked by striking amplification of pulsatile neurohormone secretion. In addition, each axis, as a whole, constitutes a regulated network whose feedback relationships are likely to manifest important changes at the time of puberty. Here, we use the regularity statistic, approximate entropy (ApEn), to assess feedback activity within the somatotropic (hypothalamo-pituitary/GH-insulin-like growth factor I) axis indirectly. To this end, we studied pubertal boys and prepubertal girls or boys with sex-steroid hormone deficiency treated short-term with estrogen, testosterone, or a nonaromatizable androgen in a total of 3 paradigms. First, our cross-sectional analysis of 53 boys at various stages of puberty or young adulthood revealed that mean ApEn, taken as a measure of feedback complexity, of 24-h serum GH concentration profiles is maximal in pre- and mid-late puberty, followed by a significant decline in postpubertal adolescence and young adulthood (P = 0.0008 by ANOVA). This indicates that marked disorderliness of the GH release process occurs in mid-late puberty at or near the time of peak growth velocity, with a return to maximal orderliness thereafter at reproductive maturity. Second, oral administration of ethinyl estradiol for 5 weeks to 7 prepubertal girls with Turner's syndrome also augmented ApEn significantly (P = 0.018), thus showing that estrogen per se can induce greater irregularity of GH secretion. Third, in 5 boys with constitutionally delayed puberty, im testosterone administration also significantly increased ApEn of 24-h GH time series (P = 0.0045). In counterpoint, 5 alpha-dihydrotestosterone, a nonaromatizable androgen, failed to produce a significant ApEn increase (P > 0.43). We conclude from these three distinct experimental contexts that aromatization of testosterone to estrogen in boys, or estrogen itself in girls, is likely the proximate sex-steroid stimulus amplifying secretory activity of the GH axis in puberty. In addition, based on inferences derived from mathematical models that mechanistically link increased disorderliness (higher ApEn) to network changes, we suggest that sex-steroid hormones in normal puberty modulate feedback within, and hence network function of, the hypothalamo-pituitary/GH-insulin-like growth factor I axis.

A sexually dimorphic pattern of growth hormone secretion in the elderly.

In rodents, the sexually dimorphic pattern of pulsatile GH secretion is an important determinant of growth, liver enzyme function and insulin-like growth factor I (IGF-I) expression. Whether this difference is present in humans at different ages is unclear. We studied GH secretory patterns in the elderly by constructing 24-h serum GH profiles in 45 male and 38 female (age, 59.4-73.0 yr) volunteers and related patterns to IGF-I, IGF-binding protein-3 (IGFBP-3), and GH-binding protein levels; body mass index; and waist/hip ratio. Serum GH concentrations were measured in samples drawn at 20-min intervals and analyzed using a sensitive chemiluminescent assay (Nichols Institute Diagnostics: sensitivity, 0.036 mU/L). The 24-h serum GH profiles were analyzed using a concentration distribution method to determine GH peak and trough levels, spectral analysis, and assessment of serial irregularity by approximate entropy (ApEn). There was a highly significant difference in mean 24-h serum GH concentrations in females compared to males (males, 0.88 mU/L; females, 1.31 mU/L; P = 0.009) as a result of significantly higher trough GH levels (males, 0.04 mU/L; females, 0.16 mU/L; P < 0.001). Peak values were not significantly different. Serum IGF-I levels were significantly higher in males (males, 162.4 ng/mL; females, 87.8 ng/ mL; P < 0.001). Peak GH values were related to serum IGF-I levels (males: r = 0.39; P = 0.009; females: r = 0.5; P = 0.002), whereas trough GH levels were not. IGFBP-3 levels were similar and related to GH peaks only in males (r = 0.32; P = 0.03). GH was secreted with a dominant periodicity of 200 min in males and 280 min in females (P < 0.025). The proportion of time taken up by regular oscillatory activity was less in females (females, 11.1%; males, 14.7%; P = 0.01). GH secretion assessed by ApEn was more disordered in females (males, 0.60; females, 0.81; P < 0.001), and increasing disorder was associated with lower IGF-I levels. Body mass index was negatively related to GH in both sexes. In males, trough values were the major determinant (r = -0.31; P = 0.04), whereas in females, the peak value was the major determinant (r = 0.35; P = 0.04). Trough GH levels were inversely related in both sexes to waist/hip ratio (males: r = -0.40; P = 0.006; females: r = -0.44; P = 0.006) and to increasing secretory disorder (ApEn; r = -0.46; P < 0.001). These data demonstrate a sexually dimorphic pattern of GH secretion in the elderly.

Disruption of the joint synchrony of luteinizing hormone, testosterone, and androstenedione secretion in adolescents with polycystic ovarian syndrome.

The present study explores the postulate that the polycystic ovarian syndrome (PCOS) is marked by failure of physiological feedforward and feedback signaling between pituitary LH and ovarian androgens. To this end, we appraised the 3-fold simultaneous overnight release of LH (assayed by high precision immunofluorometry), testosterone (RIA), and androstenedione (RIA) in 12 an- or oligoovulatory adolescents with PCOS (mean +/- SEM age, 16.4 +/- 0.47 yr) and 10 eumenorrheic girls (age, 16.5 +/- 0.45 yr). Gynecological (postmenarchal) ages (years) were also comparable at 4.8 +/- 0.39 (PCOS) and 4.0 +/- 3.6 (control; P = NS). Body mass index and fasting serum insulin and estradiol concentrations were indistinguishable in the two study cohorts. Mean overnight serum concentrations of LH (assayed by both immunofluorometry and Leydig cell bioassay), testosterone, androstenedione, and 17alpha-hydroxyprogesterone were each elevated significantly in patients with PCOS (all P

Developmentally delimited emergence of more orderly luteinizing hormone and testosterone secretion during late prepuberty in boys.

To quantitate changing feedback control in the GnRH-LH/FSH-testosterone axis in male puberty, we here quantitate the orderliness of hormone release patterns using the regularity (pattern-sensitive) statistic, approximate entropy (ApEn), in 46 eugonadal boys representing 6 genitally defined stages of normal puberty. ApEn is a single variable, model-free, and scale-independent barometer of coordinate signaling or integrative regulation within a coupled neuroendocrine axis. Accordingly, we quantitated ApEn of LH profiles obtained by immunofluorometric assay of sera sampled every 20 min for 24 h. LH ApEn declined remarkably between early prepuberty (genital stage I-A: mean bone age, 4.6 +/- 1.6 yr; testis volume, <3 mL for at least 3 succeeding yr) and late prepuberty (genital stage I-C: bone age, 8.7 +/- 1.8 yr; testis volume, <3 mL for up to 1 yr thereafter; P: = 0.00019), which indicates the acquisition of more regular LH release patterns in late prepuberty. Maximal LH orderliness occurred in puberty stage II (bone age, 10.7 +/- 1.0 yr; testis volume, 2.8 +/- 0.4 mL). The LH secretory process was more disorderly in mid- and later puberty (Tanner stages III and IV). Transpubertal variations in testosterone ApEn manifested a similar tempo, i.e. the greatest regularity of testosterone secretion (lowest ApEn) emerged in Tanner genital stage II (P: < 10(-)(7)), with less orderly patterns evident both earlier and later in sexual development. In contrast, FSH ApEn values remained invariant of pubertal status. Analysis of bihormonal coupling using the theoretically related bivariate cross-ApEn statistic disclosed maximal 2-hormone synchrony for LH and testosterone secretion in genital stage II (P: = 0.031), with relative deterioration of coordinate LH and testosterone release patterns both before and after. LH and FSH release became maximally synchronous at the end of prepuberty (genital stage I-C; P: = 0.029), and FSH and testosterone synchrony peaked in pubertal stage III (P: = 0.037). As mean 24-h serum concentrations of LH, FSH, and testosterone rose transpubertally by 35-fold (LH), 68-fold (FSH), and 70-fold (testosterone), respectively, we infer that pubertal developmental stage per se rather than level of hormone output dictates coordinate GnRH-LH/FSH-testosterone secretion. In summary, in eugonadal boys, the regularity of 24-h LH and testosterone secretory patterns undergoes well defined pubertal stage-specific control. No sexually developmentally delimited regulation is inferable for FSH. The concept of temporally biphasic puberty-dependent variations in neurohormone secretory regularity contrasts with the unidirectional rise in daily hormone output. Accordingly, we infer that late prepuberty and early puberty (Tanner genital stages IC and II) embody a physiologically unique sexual developmental window, marked by transiently enhanced LH and testosterone feedback stability in boys. Whether analogous plasticity of hypothalamo-pituitary-gonadal interactions unfolds during female adolescence is not known.

Disruption of the synchronous secretion of leptin, LH, and ovarian androgens in nonobese adolescents with the polycystic ovarian syndrome.

The present study probes putative disruption of hypothalamic control of multihormone outflow in polycystic ovarian syndrome by quantitating the joint synchrony of leptin and LH release in adolescents with this syndrome and eumenorrheic controls. To this end, hyperandrogenemic oligo- or anovulatory patients with polycystic ovarian syndrome (n = 11) and healthy girls (n = 9) underwent overnight blood sampling every 20 min for 12 h to monitor simultaneous secretion of leptin (immuno-radiometric assay), LH (immunofluorometry), and androstenedione and T (RIA). Synchronicity of paired leptin-LH, leptin-androstenedione, and leptin-T profiles was appraised by two independent bivariate statistics; viz., lag-specific cross-correlation analysis and pattern-sensitive cross-approximate entropy. The study groups were comparable in chronological and postmenarchal age, body mass index, fasting plasma insulin/glucose ratios, and serum E2 concentrations. Overnight mean (+/- SEM) serum leptin concentrations were not distinguishable in the two study groups at 30 +/- 4.8 (polycystic ovarian syndrome) and 32 +/- 7.4 microg/liter (control). Serum LH concentrations were elevated at 9.5 +/- 1.4 in girls with polycystic ovarian syndrome vs. 2.8 +/- 0.36 IU/liter in healthy subjects (P = 0.0015), androstenedione at 2.8 +/- 0.30 (polycystic ovarian syndrome) vs. 1.2 +/- 0.11 ng/ml (control) (P = 0.0002), and T at 1.56 +/- 0.29 (polycystic ovarian syndrome) vs. 0.42 +/- 0.06 ng/ml (P < 0.0001). Cross-correlation analysis shows that healthy adolescents maintained a positive relationship between leptin and LH release, wherein the latter lagged by 20 min (P < 0.01). No such association emerged in girls with polycystic ovarian syndrome. In eumenorrheic volunteers, leptin and androstenedione concentrations also covaried in a lag-specific manner (0.0001 < P < 0.01), but this linkage was disrupted in patients with polycystic ovarian syndrome. Anovulatory adolescents further failed to sustain normal time-lagged coupling between leptin and T (P < 0.01). Approximate entropy calculations revealed erosion of orderly patterns of leptin release in polycystic ovarian syndrome (P = 0.012 vs. control). Cross-entropy analysis of two-hormone pattern regularity disclosed marked disruption of leptin and LH (P = 0.0099), androstenedione and leptin (P = 0.0075) and T-leptin (P = 0.019) synchrony in girls with polycystic ovarian syndrome. In summary, hyperandrogenemic nonobese adolescents with oligo- or anovulatory polycystic ovarian syndrome manifest: 1) abrogation of the regularity of monohormonal leptin secretory patterns, despite normal mean serum leptin concentrations; 2) loss of the bihormonal synchrony between leptin and LH release; and 3) attenuation of coordinate leptin and androstenedione as well as leptin and T output. In ensemble, polycystic ovarian syndrome pathophysiology in lean adolescents is marked by vivid impairment of the synchronous outflow of leptin, LH and androgens. Whether analogous disruption of leptin-gonadal axis integration is ameliorated by therapy and/or persists into adulthood is not known.

Quantification of evolution from order to randomness in practical time series analysis.

The principal focus of this chapter is the description of a recently developed, readily usable regularity statistic, ApEn, that quantifies the continuum from perfectly orderly to completely random in time series data. Several properties of ApEn facilitate its utility for practical time series analysis: (1) ApEn is nearly unaffected by noise of magnitude below a de facto specified filter level; (2) ApEn is robust to outliers; (3) ApEn can be applied to time series of 100 or more points, with good confidence (established by standard deviation calculations); (4) ApEn is finite for stochastic, noisy deterministic, and composite (mixed) processes, the last of which are likely models for complicated biological systems; (5) increasing ApEn corresponds to intuitively increasing process complexity in the settings of (4). This applicability to medium-sized data sets and general stochastic processes is in marked contrast to capabilities of "chaos" algorithms such as the correlation dimension, which are properly applied to low-dimensional iterated deterministic dynamical systems. The potential uses of ApEn to provide new insights in biological settings are thus myriad, from a perspective complementary to that given by classic statistical methods. The ApEn statistic is typically calculated by a computer program, with a FORTRAN listing for a "basic" code referenced above. It is imperative to view ApEn as a family of statistics, each of which is a relative measure of process regularity. For proper implementation, the two input parameters m (window length) and r (tolerance width, de facto filter) must remain fixed in all calculations, as must N, the data length, to ensure meaningful comparisons. Guidelines for m and r selection are indicated above. We have found normalized regularity to be especially useful; "r" is chosen as a fixed percentage (often 15 or 20%) of the SD of the subject rather than of a group SD. This version of ApEn has the property that it is decorrelated from process SD, in that it remains unchanged under uniform process magnification or reduction; thus we can entirely separate the questions of SD change and regularity change in data analysis. Because regularity questions are thematically orthogonal to the type of information that, for example, moment statistics ascertain, we highly recommend that ApEn be used in conjunction with other such statistics, rather than as a sole indicator of process typicality. Last and yet foremost, we recommend the following order of detail in Practical analysis. First, either visually or algorithmically, eliminate outliers.(ABSTRACT TRUNCATED AT 400 WORDS)

Irregularity and asynchrony in biologic network signals.

The principal focus of this chapter has been the description of both ApEn, a quantification of serial irregularity, and of cross-ApEn, a thematically similar measure of two-variable asynchrony (conditional irregularity). Several properties of ApEn facilitate its utility for biological time series analysis: (1) ApEn is nearly unaffected by noise of magnitude below a de facto specified filter level; (2) ApEn is robust to outliers; (3) ApEn can be applied to time series of 50 or more points, with good reproducibility; (4) ApEn is finite for stochastic, noisy deterministic, and composite (mixed) processes, these last of which are likely models for complicated biological systems; (5) increasing ApEn corresponds to intuitively increasing process complexity in the settings of (4); and (6) changes in ApEn have been shown mathematically to correspond to mechanistic inferences concerning subsystem autonomy, feedback, and coupling, in diverse model settings. The applicability to medium-sized data sets and general stochastic processes is in marked contrast to capabilities of "chaos" algorithms such as the correlation dimension, which are properly applied to low-dimensional iterated deterministic dynamical systems. The potential uses of ApEn to provide new insights in biological settings are thus myriad, from a complementary perspective to that given by classical statistical methods. ApEn is typically calculated by a computer program, with a FORTRAN listing for a "basic" code referenced above. It is imperative to view ApEn as a family of statistics, each of which is a relative measure of process regularity. For proper implementation, the two input parameters m (window length) and r (tolerance width, de facto filter) must remain fixed in all calculations, as must N, the data length, to ensure meaningful comparisons. Guidelines for m and r selection are indicated above. We have found normalized regularity to be especially useful, as in the growth hormone studies discussed above; "r" is chosen as a fixed percentage (often 20%) of the subject's SD. This version of ApEn has the property that it is decorrelated from process SD--it remains unchanged under uniform process magnification, reduction, and translation (shift by a constant). Cross-ApEn is generally applied to compare sequences from two distinct yet interwined variables in a network. Thus we can directly assess network, and not just nodal, evolution, under different settings--e.g., to directly evaluate uncoupling and/or changes in feedback and control. Hence, cross-ApEn facilitates analyses of output from myriad complicated networks, avoiding the requirement to fully model the underlying system. This is especially important, since accurate modeling of (biological) networks is often nearly impossible. Algorithmically and insofar as implementation and reproducibility properties are concerned, cross-ApEn is thematically similar to ApEn. Furthermore, cross-ApEn is shown to be complementary to the two most prominent statistical means of assessing multivariate series, correlation and power spectral methodologies. In particular, we highlight, both theoretically and by case study examples, the many physiological feedback and/or control systems and models for which cross-ApEn can detect significant changes in bivariate asynchrony, yet for which cross-correlation and cross-spectral methods fail to clearly highlight markedly changing features of the data sets under consideration. Finally, we introduce spatial ApEn, which appears to have considerable potential, both theoretically and empirically, in evaluating multidimensional lattice structures, to discern and quantify the extent of changing patterns, and for the emergence and dissolution of traveling waves, throughout multiple contexts within biology and chemistry.

Orderliness of hormone release.

ApEn, approximate entropy, is a recently formulated family of parameters and statistics quantifying regularity (orderliness) in serial data, with developments both within theoretical mathematics, as well as numerous applications to multiple biological contexts. ApEn appears to have broad application to hormone pulsatility analysis within endocrinology, bringing a new perspective to the assessment of secretory patterns. ApEn is complementary to pulse detection algorithms widely employed to evaluate hormone secretion time-series--it is scale-invariant and model-independent, evaluates both dominant and subordinant patterns in data, discriminates series for which clear pulse recognition is difficult, and often provides a direct barometer of feedback between subsystems. ApEn is applicable to systems with at least 50 data points and to broad classes of models: it can be applied to discriminate both general classes of correlated stochastic processes, as well as noisy deterministic systems. Moreover, ApEn is complementary to spectral and autocorrelation analyses, providing effective discriminatory capability in instances in which the aforementioned measures exhibit minimal distinctions. We present some basic background on the above, and illustrate various facets of ApEn utility via several representative endocrinological studies.