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 20's 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 and other variation in epinephrine and norepinephrine among several human populations, including healthy blinded and sighted subjects and patients with leprosy.

Urinary levels of epinephrine and norepinephrine were studied in three populations: presumably healthy young men who were studied twice, 8 years apart, and in the same month to avoid seasonal influences; blinded but otherwise healthy males and females; and patients with leprosy. Determinations of epinephrine and norepinephrine were done on urine samples collected at 3-hr intervals for 72 hr from the first two groups and for 10 days from patients with leprosy. The same chemist, method, and protocol were used in all studies. In one study of the group of healthy young men, the effect of meal timing and fasting on the rhythms was investigated. From these studies several generalizations can be made. Highly statistically significant population circadian rhythms characterize both epinephrine and norepinephrine in all three groups. In general, the rhythm in norepinephrine was "noisier" than that in epinephrine; in the blind subjects, both variables were characterized by noisier rhythms than in the sighted. The overall levels of epinephrine were much higher in blind than in sighted subjects. The overall levels of norepinephrine were much higher in blind males than in blind females. In the studies done with the group of healthy young men, the overall levels of epinephrine and norepinephrine increased significantly over a 10-year period. The amplitude of the rhythm also increased significantly with time. The average age of the group was 27 years at the time of the first study and 40 years at the time of the second. The patients with leprosy also showed strong circadian variation in both epinephrine and norepinephrine, but, because many of the subjects were on medication, comparisons with the other groups are questionable. In general the data raise some questions about, but do not refute, the commonly held view that the amplitude and mesor of the circadian rhythm in epinephrine decrease with age. Additional work is needed to resolve this question completely.

Circadian variations in eleven radioimmunoassay variables in the serum of clinically healthy men.

Radioimmunoassay studies were conducted on 13 clinically healthy male subjects. Ten ranged in age between 23 and 44 years, two were age 51, and one was age 58. Blood samples were collected at 3-hr intervals over a period of 27 hr. Each serum sample was analyzed for the following hormones: insulin, gastrin, melatonin, prolactin, triiodothyronine (uptake), thyroid-stimulating hormone, triiodothyronine, thyroxine, luteinizing hormone, growth hormone, and follicle-stimulating hormone. Group data for each hormone were fitted to a 24-hr cosine curve. A statistically significant fit to this curve was evident in the six italicized variables. Those that did not yield a statistically significant fit frequently revealed a statistically significant variation along the 27-hr span. Chronogram and cosinor plots are presented.

The effects of various lighting schedules upon the circadian rhythms of 23 liver or brain enzymes of C57BL/6J mice.

The activities of 23 brain or liver enzymes were studied in 5-6 week old C57BL/6JNctr male and female mice that had been fed ad libitum and standardized for 2 weeks to either (1) 12 hr of light (0600-1800) alternating with 12 hr of darkness (1800-0600) (LD 12:12), (2) staggered sequences of 12 hr of light and 12 hr of dark (SLD 12:12) or (3) continuous illumination (LL 12:12) for 2 weeks. Mice in the LD 12:12 and LL 12:12 experiments were killed at 4 hr intervals along a 24-hr span in order to sample at six different circadian stages. Lighting schedules for mice in the SLD 12:12 experiment were organized such that six different circadian stages were sampled when all mice were killed at one time of day. All 23 enzymes demonstrated a prominent circadian rhythm in at least one of the experiments. Moreover, about two-thirds of the enzymes in LD and SLD 12:12 had a statistically significant fit to a 24-hr cosine curve, while only one-third of the enzymes in LL 12:12 had significant fits to cosine curves. Peak activities of enzymes from mice in LD 12:12 were clustered at the time of transition from light to dark. This was also the trend for the activities of enzymes from mice in SLD 12:12, but resynchronization did not appear completed within the 2-week span. This, along with the observation that mesors (mean 24-hr activity) were reduced and amplitudes altered, indicated that the 2-week standardization period was not sufficient for some enzymes. Times of peak activities, mesors and amplitudes were affected for most enzymes from mice in the LL 12:12 environment. This suggests that individual mice became desynchronized from one another with respect to the original light-dark schedule and that rhythms were altered or lost because individual mice were free running with frequencies different from 24 hr.

Sex differences in circadian rhythms of several variables in lymphoreticular organs, liver, kidney, and corneal epithelium in adult CD2F1 mice.

In this paper data resulting from an investigation into the influence of sex on selected circadian rhythms in lymphoreticular organs, liver, kidney, and corneal epithelium of adult CD2F1 mice are reported. Increased organ weight, total DNA and RNA in the spleen, total DNA and [3H thymidine [( 3H]TdR) incorporation into DNA in the thymus, and [3H]TdR incorporation into DNA in the liver and bone marrow in female mice compared to male littermates is demonstrated. In contrast, kidney weight and [3H]TdR incorporation into DNA as well as the corneal epithelium mitotic index are greater in male mice. Except for the corneal epithelium mitotic index and total splenic RNA and DNA, circadian rhythmicity in the variables studied is validated using the cosinor method of rhythmometric analysis in male but not in female mice. The lack of sinusoidal rhythmicity in female mice is presumed to be due to asynchrony of estrous cycling between mice within this group. Moreover, a differential organ response to exogenous testosterone enthanate is reported. The administration of this hormone suppresses [3H]TdR incorporation into DNA in the thymus and liver but not in the spleen or bone marrow at 18, 42, or 72 hr after injection.

Effect of fasting on circadian rhythmicity in deoxyribonucleic acid synthesis of several murine tissues.

These studies were done with CD2F1 adult mice that had been standardized to 12 hours of light alternating with 12 hours of darkness to determine what effect a 24-hour fast had on circadian rhythms in DNA synthesis of 10 different regions of the gut, as well as in the pancreas, liver, thymus, spleen, bone marrow, lung and testis. The mitotic index of the corneal epithelium also was studied. The overall responses varied rather dramatically. For example, in the different regions of the gut the response ranged from no statistically significant change in the colon to a strong statistically significant decrease in DNA synthesis in the cecum. In short, one cannot generalize about the effect of short-term fasting on the entire gut, but when there was any statistically significant effect, it always was a decrease. The spleen was the only tissue that showed no statistically significant response in DNA synthesis. In the bone marrow, however, a statistically significant increase in DNA synthesis was recorded at 8 and 24 hours after fasting began. In the lung there was a rather dramatic increase in DNA synthesis at 8 hours, but this was followed by decreases of 37 and 55% at 16 and 24 hours, respectively. There was a statistically significant increase of 83% in the mitotic index 24 hours after the fasting began. The data clearly demonstrate the necessity of considering circadian variation when evaluating the effects of fasting on cell proliferation.

Effects of epidermal growth factor on deoxyribonucleic acid synthesis and stomach weight in hypophysectomized adult mice.

Epidermal growth factor (EGF) or saline was administered intraperitoneally to hypophysectomized adult male CD2F1 mice or intact controls at 0700 hr. Subgroups of mice were killed at 4, 8, or 12 hr after injection. EGF was shown to stimulate [3H]TdR incorporation into DNA into several organs as previously reported. The response to EGF was found to be enhanced in both hypophysectomized and fasted mice. Differences in [3H]TdR incorporation into DNA, corneal epithelium mitotic index, RNA in pancreas and kidney of hypophysectomized and intact mice are reported. EGF was shown to result in stomach enlargement due to increased luminal contents in both hypophysectomized and intact mice.

Effect of ACTH 1-17 at different circadian stages on [3H]TdR incorporation into DNA.

The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [3H]TdR into DNA (DNA synthesis) in the tongue, esophagus and stomach of CD2F1 mice standardized to 12 hours of light alternating with 12 hours of darkness. A question asked was whether the time of administration along the 24-hour time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [3H]TdR into DNA at certain times, decreases at other times, or no response at still another time. In general the most marked effects of 20 IU/kg of ACTH 1-17 when compared to controls, was to decrease DNA synthesis of as much as 60% 4 hours after administration at the end of the dark or beginning of the light span. A 2- and 3-way analysis of variance supported the conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo.

Circadian ACTH 1-17 effects on murine tritiated thymidine incorporation into DNA of thymus, bone marrow and spleen; also on total splenic RNA and DNA.

The importance of administration time along the 24-h scale is shown for a potent corticosteroidogenic adrenocorticotropin analogue, ACTH 1-17 (Synchrodyn 1-17). This molecule affects the incorporation of [3H]TdR into DNA (DNA synthesis) in the thymus, bone marrow and spleen, and total RNA and DNA of spleen in CD2F1 mice, standardized in light alternating with darkness at 12-h intervals. As a function of timing, the same dose of ACTH 1-17 at one time increases, at another time decreases (in each case with statistical significance) and at still another time elicits no response in DNA synthesis or in total RNA and DNA of spleen. Effects upon DNA synthesis are recorded with doses of 0.02 IU/kg body weight. The most marked effect with 20 IU/kg body weight is a decrease of DNA synthesis seen (4 h) after administration of ACTH 1-17 late in the dark span and early in the light span. The effect of ACTH 1-17 on the thymus is more prominent than that on bone marrow and spleen. Time-dependence also characterizes placebo effects by comparison to values in untreated controls. At the cellular level responses to ACTH 1-17 or placebo are characterized by critical interactions of treatment kind with treatment timing as well as interval-to-kill-time. The study documents the need to time-specify, in several ways, responses to ACTH 1-17 and suggests more broadly that 'increases' and 'decreases' may have to be complemented by changes in endpoints of rhythms in all those endocrine studies that involve rhythmic variables and rhythm-dependent effects upon these variables.

Circadian-stage dependent ACTH 1-17 effect on DNA synthesis in murine duodenum, colon and rectum.

The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [3H]TdR into DNA (DNA synthesis) in the duodenum, colon and rectum of CD2F1 mice standardised to 12 hr of light alternating with 12 hr of darkness. A question asked was whether the difference in times of administration along the 24-hr time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [3H]TdR into DNA at certain times, decreases at other times, or no response at still another time. A generalization that can be made from all these tissues is that ACTH 1-17 had a greater influence in bringing about a decrease in DNA synthesis when it was administered around the time of transition from dark to light. A similar finding was made earlier for the ACTH 1-17 effect upon the tongue, esophagus and stomach. A 2- and 3-way analysis of variance supports our conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo.

Circadian dependent effect of epidermal growth factor, insulin and glucagon on hepatic pyruvate kinase and malic enzyme of mice.

The influence of epidermal growth factor (EGF), 0.75 micrograms g1; insulin, 1.5 micrograms g-1; glucagon, 1.25 micrograms g-1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F1 mice were treated toward the end of the light or dark periods, 9 or 23 hours after lights on (9 or 23 HALO), and subgroups of six mice were killed at 4, 8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian-stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronobiology: anatomy in time.

Chronobiology is that branch of science that objectively explores and quantifies mechanisms of biological time structure, including the important rhythmic manifestations of life. It is the study of biological rhythms. This paper introduces chronobiology and some of its vocabulary, principles, and techniques. A circadian rhythm is a regularly repetitive, quantitative physiological change with a period of about 24 hr (20-28), but the spectrum of rhythms includes those with periods less than 20 hr (ultradian) and longer than 28 hr (infradian). These rhythms are ubiquitous among the eukaryotes, innate and endogenous; their periods are precisely controlled by synchronizers in the environment. Rhythms can be manipulated by altering their synchronizers or by introducing more dominant ones. When organisms are removed from their environment and placed in constant conditions, rhythms revert to their natural frequencies and free-run. All of an organism's rhythms operate simultaneously, but their peaks and troughs do not necessarily occur at the same time. There are rhythms in susceptibility to drugs; a fixed dose may have a therapeutic effect at one point along the 24 hr time scale and a harmful one at another. Knowledge of these rhythms can be important when designing experimental or treatment protocols and interpreting results. Examples are provided to show that single-time-point sampling can lead to erroneous results, unless biological periodicity is taken into consideration.

Circadian effect of ACTH 1-17 on mitotic index of the corneal epithelium of BALB/C mice.

The objective was to determine the effect of ACTH 1-17, an adrenocorticotropin analogue, on the mitotic index in the corneal epithelium of mice standardized in 12 hr of light alternating with 12 hr darkness. A question asked was whether the time of administration along the 24-hr time scale influenced any response found. The findings showed that ACTH 1-17 could, depending upon when it was administered, bring about a statistically significant decrease, an increase or even no such change in the mitotic index. The greatest responses found were increases, especially when ACTH 1-17 was administered during the dark span. Also the time after injection when the responses occurred varied. The greatest response recorded was at 12 hr after injection when ACTH 1-17 was given at 2 hr into the dark with a 641% and a 718% increase with a low (0.02 IU/kg) and a higher (20 IU/kg) dose, respectively. A 3-way analysis of variance supported the conclusion that the kind-of-treatment, time-of-treatment and treatment-to-kill interval (sampling time) are important factors when determining any response to ACTH 1-17 on the mitotic index.

Circadian variation in the urinary excretion of electrolytes and trace elements in men.

Three-hour urine specimens were collected over a period of 27 hours from 11 healthy adult male subjects. Each specimen was analyzed for Na, K, Ca, Mg, and Zn using atomic absorption spectrophotometry. Each sample was also dialyzed, pH 7.35, and subsequently analyzed for Na, K, P, Ca, Mg, Zn, Fe, Pb, Al, Ni, Cu, Mo, Hg, Cr, Cd, and Mn using a multielemental argon-plasma emission system. The data were evaluated on conventional time plots (chronograms) and as computer-determined "cosinor" plots. A population circadian rhythm with a statistical significance was detected for total Na, K, Ca, and Mg, and for nondialyzable Na, K, P, Ca, Zn, and Mo. For almost every element studied the increase from lowest to highest 3-hour group mean along the 24-hour time scale was more than 100%. The 24-hour excretion of Na, K, Ca, Mg, and Zn appeared in good agreement with the so-called "normals." The nondialyzable levels of Fe, Pb, Al, Ni, Cu, Mo, Hg, Cr, Cd, and Mn were similar to the total urinary excretions reported in the literature.

Circadian stage-dependent effects of insulin and glucagon on incorporation of [3H]thymidine into deoxyribonucleic acid in the esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, and spleen of the adult female mouse.

Insulin and glucagon were injected ip at four different circadian states into separate subgroups of female adult BALB/Cann mice that had been standardized to 12 h of light alternating with 12 h of darkness. Comparable control subgroups were injected with saline. Four, 8, 12, and 18 h after each of the four injections, subgroups of seven mice that had been injected 30 min earlier with tritiated thymidine ([4H]TdR) were killed; a total of 336 mice were used. The incorporation of [3H]TdR into DNA of the esophagus, stomach, duodenum, jejunum, caecum, colon, rectum, and spleen was subsequently determined. The results demonstrate for the first time that both hormones affect the incorporation of [3H]TdR into DNA in all of the examined organs but in different ways and at different circadian stages. The effects of these hormones were complex, but several generalizations emerged. 1) Insulin tended to increase the incorporation of [3H]TdR into DNA in the examined organs, whereas glucagon tended to decrease it. 2) Insulin was more effective in stimulating the incorporation of [3H]TdR into DNA when injected either at the end of the dark span or the beginning of the light span, as opposed to the end of the light span or the beginning of the dark span. 3) Insulin had its greatest effect on [3H]TdR incorporation into DNA in the glandular stomach and rectum, whereas glucagon had its greatest effect on the colon and spleen. 4) The effects of both insulin and glucagon were different from those of epidermal growth factor, as revealed in a similar study done by us. Our results suggest that insulin, glucagon, and epidermal growth factor play important roles in the control of growth of various endodermally derived organs.