Circadian variation of serum leptin in healthy and diabetic men.

Leptin, from the Greek leptos, meaning thin (in reference to its ability to reduce body fat stores), is a hormone secreted primarily by adipocytes. At one time, leptin was portrayed as a potential means of combating obesity. Recently, leptin has been identified as a potent inhibitor of bone formation, acting through the central nervous system. Since numerous studies clearly show that bone remodeling is circadian rhythmic with peak activity during sleep, it is of interest to explore circadian variability in serum leptin. Accordingly, circadian characteristics of serum leptin were examined in 7 clinically healthy men and 4 obese men with type II diabetes. Blood samples were collected for 24 h at 3 h intervals beginning at 19:00. The dark (sleep) phase of the light-dark cycle extended from 22:30 to 06:30, with brief awakening for sampling at 01:00 and 04:00. Subjects consumed general hospital meals (2400 calories) at 16:30, 07:30, and 13:30. Serum leptin levels were determined by a R&D Systems enzyme immunoassay technique. Data were analyzed by linear least-squares estimation using the population multiple components method. A statistically significant (P < .018) circadian rhythm modeled by a single 24 h cosine curve characterized the data of each group. The 24 h mean leptin level was statistically greater (P < .001) in the obese diabetic men than in the healthy men (9.47 +/- 0.66 ng/mL vs. 24.07 +/- 1.71 ng/mL, respectively). Higher leptin levels occurred between midnight and roughly 02:30, and lowest leptin levels occurred between noon and the early afternoon. The phasing of this rhythm is similar to the circadian rhythm in bone remodeling previously described. Our results suggest the findings from a single morning blood sampling for leptin may be misleading since it may underestimate the mean 24 h and peak concentrations of the hormone.

Circadian rhythm of serum total homocysteine in men.

Serum homocysteine levels were examined in a 24-hour study of 7 healthy and 5 diabetic men, revealing a statistically significant circadian rhythm (p = 0.030), normal concentrations of 11.83 +/- 1.2 vs 12.99 +/- 1.2 micromol/L, with peak values occurring during the evening (10:37 P.M.) and lowest levels occurring during the morning. These findings imply that increased atherosclerotic risk in insulin-resistant diabetics during morning hours does not appear to be explained by differences in homocysteine levels in the normal population.

Circadian interrelationships among levels of plasma fibrinogen, blood platelets, and serum interleukin-6.

Circadian (24 h) rhythms of fibrinogen, interleukin-6 (IL-6), and platelet levels were studied in 11 males ages 46 to 72 years. Since there is a known circadian rhythm for fibrinogen and IL-6, we postulated that the peak level (acrophase) of fibrinogen would follow the acrophase of IL-6, based on the fact that IL-6 is the stimulus for fibrinogen production in the liver. Platelet levels were measured to show whether there was any correlation with the IL-6 acrophase because it has been reported that IL-6 affects megakaryocytes and platelets in dogs. We found that the acrophase for IL-6 occurred at 02:03 h and the acrophase for fibrinogen occurred at 09:16 h. Platelet counts peaked at 16:56 h. Thus, there was a positive correlation between IL-6 and fibrinogen acrophases and a negative correlation of each with the acrophase for platelets. The positive linkage of IL-6 with fibrinogen in this study suggests that suppression of IL-6 production would lower those peak fibrinogen levels that occur in the morning in association with arterial ischemic events. This could result in fewer arterial ischemic events, especially in the morning.

Circadian relationships between circulating atrial natriuretic peptides and serum calcium and phosphate in healthy humans.

Long-acting natriuretic peptide (LANP), vessel dilator (VSDL), and atrial natriuretic factor (ANF) consisting of amino acids (aa) 1 to 30, 31 to 67, and 99 to 126, respectively, of the 126-aa ANF prohormone circulate in humans. Among the biologic properties of these peptides is the ability of ANF to decrease intracellular calcium concentrations. To determine if atrial natriuretic peptides are directly related to serum calcium and/or phosphate in healthy normocalcemic humans, we examined 21 24-hour profiles of VSDL, LANP, ANF, and serum calcium and phosphate in 14 healthy humans. VSDL, LANP, and ANF each had significant (P < .001) circadian rhythms, with peak concentrations late during sleep (at 4:00 AM) being nearly twice the concentrations in the afternoon and evening. Serum calcium and phosphate also had significant circadian rhythms (P < .001) with troughs nearly opposite to those of the atrial natriuretic peptides, suggesting that atrial peptides may be important in the modulation of the circadian rhythms of calcium and phosphate. The nearly identical circadian rhythms of the atrial natriuretic peptides and of parathyroid hormone (PTH) reported by others, along with evidence that PTH may increase atrial peptide release, suggest that some of the effects attributed to PTH may be mediated by atrial natriuretic peptides.

Circadian relationships between circulating atrial natriuretic peptides and serum sodium and chloride in healthy humans.

Long-acting natriuretic peptide (LANP), vessel dilator (VSDL), and atrial natriuretic factor (ANF) consisting of amino acids 1-30, 31-67, and 99-126 of the 126 amino acid ANF prohormone, respectively, circulate in humans and have potent natriuretic properties. To determine whether these peptides have a direct relationship to serum Na and/or Cl, we examined 21 24-hour profiles of these peptides and Na and Cl in 14 healthy humans. LANP, VSDL, ANF, and Cl had significant (p < 0.001) circadian rhythms with peak concentrations at 04.00 h. The circadian rhythm of serum Na was exactly opposite. Sodium correlated negatively with LANP (p = 0.021) and ANF (p = 0.007), while Cl correlated positively with LANP (p = 0.003) and VSDL (p = 0.001). These data suggest that the atrial peptides may be important for the maintenance of serum Na and Cl within their normal ranges and in the modulation of their daily circadian rhythms.

Temporal (circadian) and functional relationship between prostate-specific antigen and testosterone in healthy men.

OBJECTIVES: To study the circadian relationship between serum prostate-specific antigen (PSA) and total testosterone in men without clinically evident prostate disease. METHODS: Blood samples were collected every 3 hours for 24 hours (eight per subject) from 11 clinically healthy men, ages 46 to 72 years. PSA was also monitored once a week for 6 weeks in 16 additional healthy men. PSA, testosterone, and age were correlated by linear regression, and 3-hourly PSA and testosterone values normalized to percent of individual mean were analyzed for circadian rhythm by the least squares fit of a 24-hour cosine. RESULTS: Mean PSA correlated positively (P < 0.001) and testosterone correlated negatively (P = 0.014) with age and inversely with each other (P < 0.001). The mean circadian range of change (ROC) from lowest to highest values for PSA was 0.37 +/- 0.07 ng/mL (28 +/- 9%), and for testosterone it was 202 +/- 23 ng/dL (53 +/- 7%). The mean ROC over 6 weeks was 0.32 +/- 0.04 ng/mL. A significant circadian rhythm was found for PSA (P = 0.011, amplitude = 5.4 +/- 1.8%, acrophase = 5:02 AM; 95% limits, 2:40 to 7:24 PM) and testosterone (P < 0.001, amplitude = 9.4 +/- 1.8%, acrophase = 8:38 AM; 95% limits, 7:12 to 10:04 AM). CONCLUSIONS: The temporal relationship between circadian rhythms in PSA and testosterone suggests different physiologic states over the 24 hours, which may be of chronopharmacologic interest with regard to dosing time of drugs or hormonal treatments intended to affect prostate growth and function. Within-day variation in PSA is of little diagnostic significance and does not prevent accurate clinical classification when a single specimen is used.

Circadian characteristics of interleukin-6 in blood and urine of clinically healthy men.

The cytokine interleukin-6 (IL-6) is a multi-functional small peptide molecule that is produced by various types of lymphoid and non-lymphoid cells and plays a central role in hematopoiesis, host defense mechanisms, and acute phase reactions, including regulation of inflammatory and immune responses. A high-sensitivity ELISA assay was applied to serum (S) and urine (U) samples available from 10 men (median age = 50y, range = 46-71y) in order to compare circadian characteristics of IL-6 between assays and in 2 biological fluids. S and U samples were collected at 3-h intervals for 24hrs beginning at 19:00h on May 14, 1993 (8 samples/subj) and frozen at -25 degrees C until analysis. IL-6 in U was adjusted for time & volume (pg/hr) and assigned to midpoint of collection interval. A significant time-effect was found by ANOVA and a high-amplitude circadian rhythm was detected by the least-squares fit of a 24-hr &/or 24+12-hr cosine for each assay. Higher serum IL-6 values were detected throughout the night, with a peak at 01:00h, and lower values throughout the day, with a nadir at 10:00h. In contrast, IL-6 values in urine were highest during the day, with a major peak in the afternoon at 17:30h and a minor peak at 08:30h, and lowest values overnight, with a nadir at 23:30h. Of interest, the rhythm in urinary IL-6 concentration (pg/ml) was more prominent than hourly excretion rate (pg/hr). Thus, endogenous IL-6 (and possibly other cytokine) levels may be significantly influenced by their large and predictable day-night variations and the biological fluid used.

Blood pressure and atrial natriuretic peptides correlate throughout the day.

Vessel dilator consisting of amino acids (a.a.) 31-67 and atrial natriuretic factor (ANF) composed of a.a. 99-126 of the 126 a.a. ANF prohormone circulate in humans and have potent vasodilatory properties. To determine whether these atrial natriuretic peptides are directly related to blood pressure in healthy normotensive humans, we recently had the unique opportunity to examine the circadian rhythms of vessel dilator, ANF, and blood pressure in seven individuals in 1988 and again in 1993. The changes in mean arterial pressure and systolic and diastolic blood pressure in these individuals during this 5-year hiatus allows comparison in the same individual, if circulating concentrations of atrial natriuretic peptides directly correlate with naturally occurring changes in blood pressure. In both 1988 and in 1993 vessel dilator and ANF each had significant (p < 0.001) circadian rhythms with their peak concentrations at 4:00 AM being nearly twice their concentrations at 4:00 PM. Mean arterial pressure, systolic blood pressure, and diastolic blood pressure also had significant circadian rhythms with peaks and troughs that were exactly opposite to those of ANF and vessel dilator. A significant inverse correlation between 24-hour averages of mean arterial blood pressure and 24-hour averages of vessel dilator (p = 0.05) and ANF (p = 0.02) was also found. These data suggest that vessel dilator and ANF are important for the maintenance of blood pressure within the normotensive range.

Circadian characteristics of circulating interleukin-6 in men.

BACKGROUND: The cytokine interleukin-6 (IL-6) is a multifunctional small-peptide molecule that is produced by various types of lymphoid and nonlymphoid cells. It plays a central role in hematopoiesis, host defense mechanisms, and acute-phase reactions, including regulation of inflammatory and immune responses. METHODS: Because a circadian time structure has been shown to characterize nearly every biologic function tested in human beings, including some cytokines, we sought to investigate the 24-hour pattern of circulating IL-6 in a group of 11 clinically symptom-free men (median age, 50 years; range, 46 to 72 years). Blood samples were obtained every 3 hours for 24 hours (eight samples per subject), and serum was frozen until analysis for IL-6 with a solid-phase ELISA. RESULTS: Average IL-6 values ranged from 1.66 to 5.38 pg/ml, with lowest to highest values within 24 hours ranging from 1.20 to 7.58 pg/ml (120% to 531%) between subjects. On average, values were greater than the mean throughout the night, with a peak at 01:00 hours and less than the mean throughout the day, with a nadir at 10:00 hours. A significant time effect was found by analysis of variance; and a high-amplitude circadian rhythm was described by the least-squares fit of a 24-hour cosine (p < 0.001; amplitude, 41% +/- 5%; acrophase, 02:16 +/- 00:28 hours). In addition, a positive correlation between mean IL-6 levels and age was found (r = 0.63, p = 0.037). CONCLUSIONS: Because monitoring of endogenous cytokine levels is suggested for assessing immune function and pathologic condition, clinical decisions and immunotherapies may be significantly influenced by the large and predictable day-night variations in endogenous cytokine production and bioactivity.

Temporal (circadian) and functional relationship between atrial natriuretic peptides and blood pressure.

Long-acting natriuretic peptide, vessel dilator, and atrial natriuretic factor consisting of amino acids (a.a.) 1 to 30, 31 to 67, and 99 to 126 of the 126-a.a. atrial natriuretic factor (ANF) prohormone, respectively, circulate in humans and have potent vasodilatory properties. To determine if these atrial natriuretic peptides are directly related to blood pressure in clinically healthy normotensive humans, we obtained 24-h profiles of vessel dilator, long-acting natriuretic peptide, ANF, and blood pressure in 10 men in 1988 and 11 men in 1993 (seven men were studied twice) to compare circulating concentrations of atrial natriuretic peptides with naturally occurring changes in blood pressure. Overall, vessel dilator, long-acting natriuretic peptide, and ANF each had significant (p<0.001) circadian rhythms, with peak concentrations late during sleep (at 04:00 h) being nearly twice their concentrations in the afternoon and evening. This high-amplitude circadian change allowed for the refinement of normal limits for ANF peptides by computing 3-hourly tolerance intervals (chronodesms) against which to compare time-specified single samples for normality. Systolic, diastolic, and mean arterial blood pressure also had significant circadian rhythms (p<0.001) with peaks and troughs that were exactly opposite those of the ANF peptides. In addition to this inverse temporal relationship, there was a significant inverse correlation between absolute values for blood pressure and each ANF peptide (p<0.001), implying a functional relationship. These data suggest that in addition to other well-established neurochemical factors, the ANF peptides (vessel dilator, long-acting natriuretic peptide, and ANF) are important for the maintenance of blood pressure and modulation of its circadian rhythm.

Circadian rhythmometry of serum interleukin-2, interleukin-10, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in men.

Serum levels of four cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 (IL-10)] were measured in nine diurnally active healthy adult male subjects at 3-h intervals during a 24-h period. Statistical evaluation by analysis of variance and/or the least- squares fit of a cosine model revealed significant 24-h rhythms for each cytokine. Although the amount of IL-2 in the serum was low, the levels fluctuated to form a single peak at approximately noon. In contrast, the other three cytokines exhibited a biphasic temporal pattern. In subjects with detectable TNF-alpha levels, the first peak occurred at 07:30 and the second at 13:30. IL-10 levels also exhibited a biphasic pattern, with one peak at 07:30 and the second 12 h later at 19:30. GM-CSF levels were last to rise, first peaking at approximately 13:30 and then again at 19:30. These results suggest temporal patterns that are unique for each cytokine, generally with daytime highs and nighttime lows.

Chronobiology as it relates to toxicology, pharmacology, and chemotherapy.

Examples of circadian (daily) rhythms in man are presented and discussed. This is followed by presentation of experimental data indicating how the temporal organization affects the response of the host to potentially toxic agents, namely amphetamine and sodium pentobarbital. Data also are presented indicating that one unequivocally can improve therapeutic efficacy using the L1210 mouse leukemia model. This was accomplished by taking into consideration the circadian host toxicity response to anticancer agents, namely cytosine arabinoside, adriamycin, and cyclophosphamide. The subject monitored was the "cure rate" subsequent to treatment of different tumor loads.

Historical comments on chronobiology with emphasis on cell proliferation, the concept of free running, cancer chemotherapy and experimental design.

A brief historical summary is presented regarding the emergence, over the past several decades, of chronobiology as the newest of the integrating discipline of biology. The emphasis is on the circadian system which normally is synchronized to the 24 h environmental light-dark cycle. In the absence of a suitable synchronizer, the system free runs on its own endogenous genetically determined frequency, which usually only approximates 24 h. Since the metabolic system changes rhythmically in time it follows that an organism such as man is biochemically and physiologically a different entity at different circadian stages; therefore it reacts differently to an identical stimulus given at different times. Different stimuli such as anticancer agents are examples considered clearly timed treatment has been shown to significantly improve therapeutic efficacy, data will be presented using the L1210 mouse leukemic model. Moreover data is presented showing that to ignore such rhythmic fluctuation when designing experiments that such can bring about experimental error and false interpretation. The common "same time of day" sampling does not take care of the rhythmic problem!

Circadian variation in alloxan sensitivity of mice as indicated by mortality and blood glucose alteration.

The objective of this study was to determine in mice if a time-dependent pancreatic beta-cell susceptibility to alloxan could be correlated to daily changes in blood glucose levels and to monitor the pattern of blood glucose at various times of day as mice became diabetic. Food was removed from mice standardized to a 12-h light:dark cycle (lights on at 0600 h CST, during the month of June) at 12 h before subcutaneous injection with 0.27 mg/g of alloxan. Six groups of 30 fasted mice were injected at 4-h intervals. Blood glucose levels were measured from each group immediately prior to injection, and at 2, 4, 8, 12, 24, 48, and 216 h after treatment. Animals receiving alloxan during the early- to middark period had an increase in blood glucose after 2 h, followed by a decline to hypoglycemic levels between 4 and 8 h, and recovery to hyperglycemic levels 48 h after alloxan exposure. Three and 30% of these animals were dead at 8 and 48 h, respectively. Mice treated during the midlight span had decreased blood glucose levels 2 h after alloxan treatment followed by an increase to diabetic hyperglycemia within 48 h. Twenty-three and 70% of the animals treated at 1430 h were dead at 8 and 48 h, respectively. At 216 h, total mortality was 45.6% and 81 of the 98 surviving mice were hyperglycemic. These data suggest a greater sensitivity to alloxan during the midlight resting period of the mice. This may be the result of increased sensitivity to the insulin released from the beta cells when alloxan was given during the light span.

Heparin as a therapy for atherosclerosis: preliminary observations on the intrapulmonary administration of low-dose heparin in the morning versus evening gauged by its effect on blood variables.

Reports on clinical trials with subcutaneous and intrapulmonary administration of low-dose heparin suggest that it may be an attractive therapeutic modality for the treatment of coronary artery disease because of unprecedented reduction in mortality of treated subjects. As a preliminary to a clinical trial with low-dose intrapulmonary heparin, a pilot study was conducted on three subjects. It compares overall circadian responses of 37 blood variables following intrapulmonary administration of heparin (10,500-18,800 U) in the morning (0800 h) and in the evening (2000 h). After each of these times, blood samples, mostly at 3 h intervals for the ensuing 27 h, were analyzed for heparin, APTT, TT, functional fibrinogen, CBC, enzymes, lipids, electrolytes, and hormones. Each time series was analyzed for circadian rhythm by the least-squares fit of a 24 h cosine and circadian mesors were compared by the Bingham test of rhythm parameters. Following heparin in the evening, but not in the morning, a statistically significant increase in circulating heparin levels, as well as directional increases in APTT and TT and decreases in fibrinogen, were observed in all three subjects. Same direction changes in several other variables were also observed. It is concluded that inhalation of heparin in low-dose levels results in variable circadian effects on blood parameters measured, ranging from no changes in their levels to minimal within normal range changes, and that these effects are dependent upon the timing of dose administration. It is suggested that the timed self-administration of low-dose heparin by inhalation be seriously considered for long-term clinical trials in the treatment and prevention of atherosclerosis.

The circadian rhythm of the N-terminus and C-terminus of the atrial natriuretic factor prohormone.

Circadian variation in the circulating concentrations of the N-terminal and C-terminal portions of the atrial natriuretic factor prohormone (pro ANF) was evaluated in 8 men, ages 41-47, who have been followed for 19 years with respect to circadian variation in physiological variables including blood pressure and clinical chemistries. The N-terminus of the ANF prohormone contains two peptides consisting of amino acids 1-30 and 31-67 while the C-terminus contains 1 peptide (amino acids 99-126) of this 126 amino acid prohormone which lower blood pressure and have natriuretic properties. To determine if either the N-terminus and/or the C-terminus of the prohormone have a circadian variation in their circulating plasma concentrations these 8 men had blood samples obtained for radiommunoassay every 3 hr during a 24-hr period. Three radiommunoassays which immunologically recognize (1) the whole N-terminus (i.e. amino acids 1-98), (2) the midportion of the N-terminus (amino acids 31-67) and (3) the C-terminus (amino acids 99-126) of the ANF prohormone were utilized. The whole N-terminus, the midportion of the N-terminus which circulates after being proteolytically cleaved from the rest of the N-terminus, and the C-terminus each had a peak circulating concentration between 0400 and 0700 which were significantly (P less than 0.001) higher than their concentrations at any other time throughout the 24-hr period.(ABSTRACT TRUNCATED AT 250 WORDS)