Eugene L. Kanabrocki

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.

Circadian relationship of serum uric acid and nitric oxide.

To the Editor: Nitric oxide–mediated damage has been implicated in a number of neurological diseases including stroke1, 2 and multiple sclerosis (MS).3 For instance, monocytes expressing high levels of nitric oxide synthetase have been found in plaques from the brains of patients with MS.4 The proximal agent of neuronal cell damage may be peroxynitrite, which is formed in vivo from the synthesis of nitric oxide and superoxide.

Circadian variation in oxidative stress markers in healthy and type II diabetic men

Seven clinically healthy, nondiabetic (ND) and four Type II diabetic (D) men were assessed for circadian rhythms in oxidative “stress markers.” Blood samples were collected at 3h intervals for ∼27 h beginning at 19:00h. Urine samples were collected every 3 h beginning with the 16:00h–19:00h sample. The dark (sleep) phase of the light–dark cycle extended from 22:30h to 06:30h, with brief awakening for sampling at 01:00h and 04:00h. Subjects were offered general hospital meals at 16:30h, 07:30h, and 13:30h (2400 cal in total/24 h). Serum samples were analyzed for uric acid (UA) and nitrite (NO) concentrations, and urine samples were assayed for 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), and 8-isoprostane (ISP). Data were analyzed statistically both by the population multiple-components method and by the analysis of variance (ANOVA). The 24h mean level of UA and NO was greater in D than in ND subjects (424 vs. 338 μmol/L and 39.2 vs. 12.7 μM, respectively). A significant circadian rhythm in UA (p=0.001) and NO (p=0.048) was evident in ND but not in D (p=0.214 and 0.065). A circadian rhythm (p=0.004, amplitude=8.6 pmol/kgbw/3h urine vol.) was also evident in urine 8-OHdG of ND but not of D. The 24h mean levels of ND and D were comparable (76.8 vs. 65.7 pmol/kgbw/3h urine vol.). No circadian rhythm by population multiple-components was evident in MDA and ISP levels of ND subjects, or in 8-OHdG, MDA, and ISP in D. However, a significant time-effect was demonstrated by ANOVA in all variables and groups. The 24h mean of MDA and ISP in D was significantly greater than in ND (214 vs. 119 nmol/3h urine vol. and 622 vs. 465 ng/3h urine vol.). The peak concentrations of the three oxidative “stress markers” in urine, like those of serum NO, occurred early in the evening in both groups of men. This observation suggests a correlation between increased oxidative damage and increased rate of anabolic–catabolic events as evidenced by similarities in the timing of peak NO production and in parameters relevant to metabolic functions.

Relation between circadian patterns in levels of circulating lipoprotein(a), fibrinogen, platelets, and related lipid variables in men.

BACKGROUND A correlation has been reported between lipoprotein(a) [Lp(a)] concentration and risk for coronary artery disease. High concentrations of Lp(a) might be markers for vascular or tissue injury or might be associated with other genetic or environmental factors that can cause acute myocardial infarction. METHODS We measured the circadian characteristics of circulating Lp(a), fibrinogen, platelets, cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol for a group of adult male volunteers who had no clinical symptoms. We obtained samples every 3 hours around the clock to assess the normal degree of variation within a 24-hour period and to test for similarities in circadian patterns and correlations with level of Lp(a). RESULTS Each variable displayed a highly significant circadian rhythm. Lp(a), fibrinogen, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol peaked in the morning. Cholesterol and platelets peaked in the late afternoon, and triglycerides peaked in the evening. CONCLUSIONS Although peak levels of Lp(a) and fibrinogen coincide with reported morning peak frequencies of myocardial infarction and stroke, the platelet peak appears to coincide with late afternoon peak frequencies of sudden cardiac death and fatal stroke. The data suggest that proper timing of single samples may improve the usefulness and accuracy of diagnosis, risk assessment, and therapy.

REFERENCE VALUES FOR CIRCADIAN RHYTHMS OF 98 VARIABLES IN CLINICALLY HEALTHY MEN IN THE FIFTH DECADE OF LIFE

Nine clinically healthy men, 41-47 yr of age, served as subjects in a 24-hr study conducted at the Edward Hines Jr Veterans Administration Hospital in the Chicago area in May 1988. Physiologic measurements, and blood and urine samples were collected at 3-hr intervals over a single 24-hr period beginning at 1900. The number of variables measured or calculated (total = 98) included: 6 vital signs (oral temperature, pulse, blood- and intraocular pressures); 16 in whole blood (counts and differentials); 50 in serum (SMAC-24, lipids, hormones, electrophoresis of LDH and proteins); and 26 in urine (solids, proteins, creatinine, catecholamines, melatonin, cortisol, electrolytes and metals). Data were analyzed for time effect by analysis of variance (ANOVA) and for circadian rhythm by single cosinor. Individual rhythm characteristics for each variable were summarized for the group by population mean cosinor. The vast majority of variables revealed statistically significant within-day changes in values as validated by one-way ANOVA. All vital signs (except for intraocular pressures) and all serum hormones displayed a prominent circadian rhythm for the group, as did most variables in whole blood, while only about half of the variables in urine demonstrated a significant group rhythm. The results obtained are meant to: (a) document the circadian time structure; and (b) serve as reference values for circadian rhythm characteristics (range of change, mesor, amplitude and acrophase) for a defined group of individuals: clinically-healthy adult men in the prime of life.

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

Circadian (24 h) rhythms of fibrinogen, interleu kin-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 (acro phase) 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 corre lation of each with the acrophase for platelets. The positive linkage of IL-6 with fibrinogen in this study suggests that sup pression 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. Key Words: Circadian— Fibrinogen—Interleukin—6 (IL-6)—Platelets—Arterial ischemia.

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.

Day-night variations in blood levels of nitric oxide, T-TFPI, and E-selectin

Circadian (8/24 hours) variations in serum nitric oxide (NO), total tissue factor pathway inhibitor (T-TFPI), and E-selectin levels were studied in healthy adults and in subjects with type II diabetes. We postulated a possibility a functional relationship between them because vascular endothelium is the primary site of their synthesis and functions. NO is released by the action of eNO synthase isoform and modulates physiologic responses (e.g., vascular dilation, relaxation, increasing blood flow, inhibition of platelet and white blood cell adhesion); T-TFPI, a coagulation inhibitor, is also released from endothelial cells, and is bound to plasma lipoproteins and to glycosaminoglycans; E-selectin is expressed on endothelial cells after activation by inflammatory cytokines (interleukin-1β and tumor necrosis factor-α) and elevated levels have been reported in a variety of pathologic conditions, including diabetes. We found that obese diabetic subjects had greater mean concentrations of NO and E-selectin than healthy men, 39.25 versus 12.71 μM and 81.51 versus 26.03 ng/mL, respectively. The T-TFPI levels were essentially similar in both groups of men, 47.10 versus 48.76 ng/mL. We observed that the time of peak concentrations of T-TFPI and E-selectin was similar to the timing of NO trough levels, suggesting a possible functional relationship. It may be hypothesized, therefore, that the higher concentrations of NO, unbalanced by increases in T-TFPI and E-selectin, may result in increased vascular wall uptake of lipoproteins in diabetic subjects, who are at greater risk than healthy men for developing diffuse atherosclerosis.

Ten-year-replicated circadian profiles for 36 physiological, serological and urinary variables in healthy men

At 3-hr intervals over a 24-hr span, 36 systemic, serologic and urinary variables were examined in 7 men in their mid 20s in the Spring of 1969, and again in the same 7 men in the Spring of 1979 under a similar chronobiologic protocol, using the same chemical and numerical analytical procedures. The variables examined for rhythms by cosinor were: vital signs--blood pressure (systolic, diastolic, pulse pressure and mean arterial pressure), heart rate, intraocular pressure (left and right), oral temperature; serum components--albumin, albumin/globulin ratio, total bilirubin, calcium, carbon dioxide, chlorides, bilirubin, cholesterol, globulin, glucose, potassium, sodium, sodium/potassium ratio, transaminase, triglycerides, total protein, urea nitrogen; and urine components--calcium, calcium/magnesium ratio, creatinine, magnesium, pH, potassium, sodium, sodium/potassium ratio, urea clearance, urea nitrogen, volume and zinc. Although all subjects appeared clinically healthy in 1969 and in 1979, certain inter-study differences were observed in a number of rhythm parameters of different variables. Statistically significant increases in mesor for the group as a whole were observed for serum Ca, cholesterol, Cl, CO2, K, Na, and while statistically significant mesor decreases for a group as a whole were noted in serum glucose and transaminase. Statistically significant increases in amplitude for the group as a whole were observed in serum chloride and urinary Na/K ratio, while statistically significant decreases were observed in amplitude for blood pressure, heart rate, serum albumin, A/G ratio, globulin, glucose, protein, sodium and transaminase.(ABSTRACT TRUNCATED AT 250 WORDS)

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 24h at 3h 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 24h cosine curve characterized the data of each group. The 24h 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 24h and peak concentrations of the hormone. (Chronobiology International, 18(2), 273–283, 2001)