Acknowledgements

Earl Bakken. During the early 1950s, the cooperation of Earl Bakken, DSc., hon., F.A.C.C., the founder of Medtronic, was second to none. Markers were needed to refer the endocrine and other biochemical changes to some readily measured variable. Earl Bakken built activity monitors for the study of what became the circadian system and has now become a set of yet broader chronomes. Genetically anchored yet socioecologically synchronized chronomes are found in each variable investigated thus far, once a data series is of sufficient density and length. A cosmic influence, long suggested, is becoming amenable to an experimental approach.

In the earliest 1950s, we had the problem of adjusting the sensitivity of the activity-measuring device, so that on the one hand, washing movements of a rodent could be assessed, but on the other hand, the energy-consuming horizontal displacement could be properly quantified. Only in the past few decades has this technology been transferred to humans. Activity meters have now become available to monitor not only the ultradian and circadian systems (7), but also to demonstrate about circasemiseptan and circaseptan components in human activity (8). In viewing periods that were close to 24 hours but statistically significantly different from the precise environmental 24-hour match, and also quite often different from one animal to the next, Franz postulated an endogenous component as the underlying mechanism. Earl Bakken suggested the analogy of a free-running oscillator and built a theoretical model for it (9). The circa-rhythms followed one another (10).

Earl Bakken then proceeded to build the first battery-powered, transistorized cardiac pacemaker in 1957; the following year, he introduced it for long-term fully ambulatory use. Today, thanks to Bakken, much can be done to restore a most basic rhythm, by acting when the heartbeat stops, falters, flutters or fibrillates. Unknown as yet to many who produce and use it, the cardiac pacemaker is a milestone in both chronobioengineering broadly and in applied chronocardiology par excellence.

Patrick Delmore, Head of Communications at Medtronic Inc. and co-author of a series of scientific publications, drew the abstract graphs in this volume. His continued help, intellectual and artistic, and that of his indefatigable assistant Marge Pekula, are gratefully acknowledged, as is the competent and patient artwork of David Lantto of ComputerChrome.

Hurdles. In Figure 1, the dispersion of the single individual glycogen values reveals the very large variability; this scatter may be one reason why Eric Forsgren, who discovered the rhythmicity of liver glycogen, was denied his medical doctorate in Sweden for very many years. Those who determined liver glycogen (or blood eosinophils; see their dispersion in Figure 1) on the basis of single determinations accepted data on rhythms in any and all variables except for the one they worked with. This attitude of ignoring the very large variation on hand for the sake of `simplicity' was and is the major obstacle. The failure to act on (and to accept as fact) a variation of over 60 mm Hg in the (human adult's) blood pressure each day may account for the fact that chronobiology as yet is still not in the mainstream of medicine. Cardiologists do not yet admit to themselves, much less to others, that taking the casual blood pressure, i.e., what they do in everyday practice, has over 40% uncertainty (11). It is easier to accept concepts of constancy and homeostasis, and turn to the next patient in the short time available for the patient's visit.

Complexities. To cite another example, studies in the 1950s resolved the circadian changes of rat liver metabolism and mitoses (9) and led to Figure 2. Such studies were complex, notably in the era before automation. Different techniques were often concomitantly used. Thus, for the Figure 2 problem, the rhythmicities of RNA and DNA, regeneration after partial hepatectomy was first investigated (9, 12, 13). Surgical procedures and techniques of wet chemistry were wedded to radioactive tracer studies, to differential centrifugation, and to more classical physiologic, hematologic and histologic approaches, with 180 partial hepatectomies done within 3.5 hours (12). All of this is background work to the main point in Figure 2, the circadian cell cycle in immature growing liver. Before the necessary teams of scores of investigators and their students working together could be assembled, much existing bias had to be overcome.

Positive attitudes. In 1950, it was customary to relate the results of chemical determinations to the most `constant' material in a tissue: its DNA content. The possibility that DNA formation would undergo a circadian rhythmicity was then viewed as sheer heresy. The late Professor of Biochemistry Cyrus P. Barnum was reluctant to let Franz have a technician paid on a Public Health Service grant to test for such a periodicity. Such a guess was to him wild and unlikely to yield any results. Barnum's straitlaced yet open mind could not justify the spending of taxpayers' money on testing for `rhythms'. He did feel, however, that he could do what he wanted on his own time. He provided much enthusiastic and invaluable help himself, being at the helm of a homogenizer in many 24-hour sample collections that involved more than a sleepless night since the tissues collected had to be processed.

When data on mice given the tracer P³² at 1 hour before killing every few hours for 24 hours were plotted as a function of time (Figure 1 in reference 13), a second increase, 24 hours after a first peak in DNA formation, was barely seen. When the study was repeated on animals injected intraperitoneally 2 hours before killing, there was a second, small peak about 24 hours after the first large peak in the relative specific activity of DNA. This peak in DNA synthesis preceded, as would be anticipated, a much larger second peak in mitotic activity in regenerating liver. To `isolate' this periodicity of RNA and DNA by new biologic data more clearly, separate groups of comparable inbred immature mice were studied. The animals were killed 2 hours after injection of the tracer. Now the rhythmicity both of RNA and DNA came clearly to the fore.

Slow technology transfer. It was long mistakenly believed (14) that a `basic' rhythm such as that in mitosis could not be shifted in its timing by the institution of changes in lighting and that the amenability to shifting of rhythms in motor activity as well as in blood eosinophils (15) was an exception, just as for a very long time rhythmicity, by those believing in homeostasis, was also dealt with as an exception rather than as the rule. By 1958, it was demonstrated that these basic cellular rhythms of mitoses of liver and skin and RNA and DNA formation, and those in circulating glucose and corticosterone and in liver glycogen, could all be changed in their timing along the 24-hour scale by manipulating the lighting regimen. It was concluded: `The possibility to control timing of these rhythms by an easily manipulated environmental factor is of obvious practical interest ...' (16). By the 1950s we also learned that the lighting-induced change applies to humans and that there was an early response of blood eosinophils to sunlight of high intensity. It is interesting today to see patents given for circadian light therapy (with mixed responses by colleagues, discussed at the time of this writing as a hotly debated topic of `Technology transfer' [17]) when the true question revolves around an application of all treatments in the literal and figurative `light' of the entire chronome.

New experiments or metachronanalyses: the economical approach. The studies leading to Figure 2 required teamwork for years and involved the change in model from a murine liver regenerating after partial hepatectomy to the immature growing liver. Rather than changing models, as done in order to map the cycle in growing intact rat liver (Figure 2), it is often much easier today to reanalyze data already in hand and to isolate a sought periodicity from any obscuring `noise'. We faced a choice between preventing, at a cost of millions of U.S. dollars, a periodicity of 3 hours from entering data from a Biosatellite, versus allowing the periodicity to enter the data but then removing it mathematically at the cost of a few hundred dollars. The cheaper approach seemed acceptable as long as the 3-hour input did not gravely alter the animal's circadian behavior under study (18). A similar mathematical approach applied to available data was fruitful in the case of a meta-analysis of the chronome of E. coli and of cyanobacteria, as will be demonstrated below.

From Nathaniel Kleitman and sleep research to the chronome initiative. Chronobiology, like statistics, is first and foremost `common sense applied'. We are all aware of our circadian system. To paraphrase Nathaniel Kleitman, the authority on sleep and wakefulness (19, 20) and on a basic rest-activity cycle (21), eventually we all realize that we have to sleep. (As we have seen in Figures 1 and 2, our RNA and DNA formation, in this order [there is no inverse transcriptase], must also `sleep'.) Kleitman set an example in many ways. He was supportive rather than obstructive when the hypothesis of rhythms being mere conditioned reflexes, `impressed from without and persisting from within' (20), was amended by the demonstration of genetically anchored, objectively quantified and inferentially statistically secured free-running components of a chronome (22-24). Nathaniel Kleitman is ready to set yet another example of self-assessment for science in everyday life, as he now, at 99 years of age, offers to monitor himself as a centenarian. In so doing, Kleitman, it is hoped for a long time and with many others, will be participating in an international endeavor, designed and coordinated by Germaine, involving over 100 investigators. This initiative maps systematically, from womb to tomb, the blood pressure and heart rate chronomes and opportunistically the time structures of other variables.

Basic results from womb to tomb. From this endeavor, we learned that multiseptans, such as the circasemiseptan and circaseptan components, are more prominent than circadians in many physiologic variables of human prematures, Figure 22. Before life adapted to the rotation of the earth around its axis, it may have resonated with effects related to the much slower rotation of the sun around its axis (25). (Religions have recognized our built-in circaseptans and have introduced a day of rest to mark this apparently earliest unit of our natural biochronology.) In adulthood, the multiseptans may decrease only slightly while the circadians greatly increase. There are corresponding changes in the multiseptan/circadian amplitude ratio, shown in Figure 23 and Table 2.

Applied results. Figure 24 suggests an alteration of the adult circasemiseptan/circadian amplitude ratio in patients with occasional undue blood pressure excess. There is a need for long-term monitoring and analysis of infradians as well as of circadians and ultradians, if biologists are to resolve and fully exploit the dynamics within the physiologic range of variation, with applications in particular in health care.

In addition to circadian amplitude-hypertension (26), other new syndromes and yet broader biologic problems may be detected early for the sake of prevention in the broadest sense of the ills of our planet as well as of ourselves.

Primum redigere ad mensuram chronomata, periodicitates vel inclinationes, quæ non unas sed maximas proprietates vitæ sunt; quod est demonstrandum (A hypothesis yet to be further tested is that chronomes, genetically anchored in the genomes of life forms, are the, rather than a fundamental dimension of living matter). The French neurophysiologist Alfred Fessard wrote that periodicity is a fundamental property of living matter (27), and we may add, much more than a clock or calendar. Toward this goal, this volume honors Agostino Carandente best by including, in keeping with his exhortation `Gehen Sie weiter', the most recent results in their historical context. Chronobiology is progressing by applying a spectral approach to smaller and smaller morphologic units. It did so to a time-macroscopic circadian record (28): the 1930 data of Lore A. Rogers (a noted bacteriologist described by a Cosmos Club Vignette of December 1967 as `the bright star in the [U.S. Department of Agriculture's] scientific horizon before World War II'). By 1961, we had demonstrated the circadian free-run of about 21 hours and subsequently the indication of an infradian, in the growth and/or colony advance of E. coli, Figure 25 (29, 30). In his nineties, Rogers (personal communication) had also ascertained, from his technician and co-author G.R. Greenbank, the lack of any known external 21-hour cycle.

Nonetheless, in 1976, a committee reported that the minimal biologic unit capable of exhibiting circadian rhythmicity was the single cell (31): experiments reporting circadian rhythms in Klebsellia (32) and E. coli (33) were criticized on methodological grounds and unpublished negative experiments reported to the committee by several authors (Sweeney, Edmunds, Schweiger, Brinkman, Bünning and Bruce) believed instead (31). It is never easy to rule out periodicity. By 1994, with the early follow-up work (32, 33), a series of more recent added publications explicitly rejects the dogma `eukaryotes only' (34-38), thus fully vindicating our 1961 analyses (29).

Turning to the question of the basic nature of multiseptans, circaseptans and/or circasemiseptans are quantified in the firing of an isolated retina (39, 40), in the beating of an isolated myocardial cell (41), in unicellulars (42-45), in isolated platelets (46) and even in E. coli and cyanobacteria, Figures 25 and 26. The brevity of the time series in many of these models is a grave limitation. The vitality of the isolated platelet gauged by its glutathione content and that of some other models showing a decreasing trend of slow death is another limitation. These circumstances help us the more to emphasize that human prematures are perhaps the most challenging model, since relatively long data series are readily obtained on neonatal critical care units. The promise of chronomes to bear prognostic, diagnostic and therapeutic fruit in health care broadly is thus testable at a critical time when preventive measures may be most effective, an approach currently followed by Elena V. Syutkina of the Institute of Pediatrics in Moscow. Infradians are now documented (in one boy born in the 27th-28th gestational week) by around-the-clock measurements, with interruptions, for 26 months (25). The prominence of circaseptans in the blood pressure and heart rate of prematures is supported by 40 series, Table 2 (47).

There are also limitations to studying prematures. At an age of very active growth, the data have to be detrended for this reason and also because many babies investigated are sick and their disease and treatment can generate added trends. In the light of these complexities, the claim to the relative simplicity of bacterial and unicellular models must seriously be considered. In any event, nobody should want to be an investigator only of one model, if not book (homo unius libri). The proper study of humankind, with Charron (48) and Pope (49) paraphrased, is humans, from womb to tomb, yet indeed our perspective is broadened by unicells and prokaryotes. While a chronome initiative focuses, first and foremost, on humans, other models, under certain conditions, offer relatively inexpensive and more readily manipulable approaches to the time structure of life, leading to testable hypotheses for optimizing both health and the environment relevant to humankind, notably in the neonatal stage and for shift-workers.

Cosmos. On humankind, we have explored effects (upon chronomes) of the moon and the sun. These effects are difficult to separate. We have used the term `circatrigintan' to refer to cycles with a period of about 30 days, rather than terms implying a direct relation to any lunar cycles (50), some of which may turn out to be solar (25). The role of the sun has been debated strongly since early in this century, when Chizhevsky described life on earth as `an echo of the sun' (51). We may have gone a few steps (see chapter 6, Figure 12) beyond previous endeavors reviewed in a scholarly book by Dubrov (52). Furthermore, in Figure 27, Germaine's analysis suggests that circaseptans are not necessarily only a reflection of past adaptation, as a genetic multiseptan anchor, but may still be influenced by the sun's activity: a decrease in the circaseptan prominence of heart rate of a clinically healthy man is observed in association with a drastic reduction in circaseptan features in solar activity, determined by Walsh spectra (53), Figure 27. The approach, involving `remove and replace' experiments broadly, rather than the result on this particular subject itself, deserves further consideration in future investigations on the effects of subtle geophysical factors.

We need not conclude, as did Hobart A. Reimann, a former professor of medicine at the University of Minnesota, in his book on periodic disease (54): `... according [presumably] to Montaigne, whenever a new idea is offered, many say, "It is probably not true." When it is confirmed they say, "Yes, it may be true, but it is not important." When its importance is validated, they say, "Yes, surely it is important, but it is no longer new"'. The chronome was then missing, just as the importance of the genome then went somewhat unrecognized.

The circaseptans are old yet new, as were the circadians in the 1950s. So is the chronome, obvious as the sunrise yet usually in need of data collection and quantification by the computer, the equivalent of both the microscope and the telescope of life in time. This introduction to chronobiology may prevent the next generation from sharing the fate of Molière's M. Jourdain (55), who only late in life learned that he had all along spoken prose (read: chronobiology).

After reading this introduction, we trust that today's bioengineering, biomedical and political communities at large will no longer have to be told that all their lives they have been and are speaking chronobiology, which for Agostino and us is beautiful poetry. All of life, starting in earliest public instruction, in town and gown, awaits quantification by chronobiometry in all disciplines, from sociologists writing about the week to ecologists with blueprints about the environment. With the philosophical and bioethical recognition that all manifestations of life can be meaningfully measured in time, chronobiology becomes the `commune vinculum omnibus artibus, omnibus scientiis et omnibus disciplinis'.

Franz Halberg