A new approach to biological modeling: Introduction to the biology of functions

Chapter 15

A new approach to biological modeling: Introduction to the biology of functions The complexity of physiologic phenomenon arises from fact that…biological system depends on the coordinated action of each of the constitutive elements. This is why the understanding of a biological system calls for an integrative approach, which ideally consists of the theoretical reconstruction of a given system from its elementary components. This raises a formidable experimental challenge that can only be met by the construction of new mathematical models allowing the numerical simulation of complex biological phenomena. Gilbert A. Chauvet, MD, PhD.1

Introduction In an aphorism, Hippocrates noted that “Life is short/And art long/Opportunity fleeting/Experimentations perilous/ And judgment difficult.”2 The life we perceive to be living is symbolic. All experiences are representational and evanescent. Recounting experiences, then, is a representation of a representation, two steps removed from an objective Truth. You hear the oboe in the opening bars of Mozart’s Requiem hovering above the stirring of cellos. The sound is gone. How can you express the sound of the oboe? You feel wistful and melancholic after listening to it, then the feeling is gone. How to describe it? A patient presents after a myocardial infarction to your office. The moment of ischemia, of neuroendocrine adaptation, of a psychological shift—how can one capture these events? It is only through some representational medium. When we think of symbols, we think of abstract visual representation: cross, peace sign, hand mirror of Venus, etc. In Asian languages, ideograms graphically represent ideas. In Indo-European languages, words have three levels of encoded symbolism. First, each letter represents a sound. Second, word represents a multisyllabic group of sounds. Third, the word-sound combination is arbitrarily associated with an idea. Ideas do not exist because of language. Language exists because of ideas. Symbols require a specific set of knowledge contextualized to space, time, and culture. It excludes more than it includes or communicates, The Theory of Endobiogeny. https://doi.org/10.1016/B978-0-12-816903-2.00015-X © 2019 Elsevier Inc. All rights reserved.

e.g., double entendres, contextual dependence of meaning, punctuation, not to mention tone, accompanying gestures, and facial expressions. It is also true of nonlanguage symbols, which requires induction into a set of shared beliefs and understandings in order to interpret their meaning. Despite the richness of their content, the transmission of ideas feels somewhat perilous as an enterprise of heuristic interpretation. Mathematics is the third type of symbolic representation of abstract concepts. What distinguishes it from the other two is that it is rational and discrete. There is no heuristic effort in understanding the concept of “nine” and presenting it as “9.” As a rational-symbolic language, it is ideal for expressing rational concepts that must be communicated to others by rational means (c.f. Chapter 1). Consider describing the area of two circles of varying radii. One could symbolically represent the circles, but one could not communicate their actual size—making it difficult to know what can fit inside of them. One could sing a song about circles or compose a jazz melody to convey the feeling of each circle, but it would remain highly heuristic. One could use words to describe one as larger than the other, and compare them to other objects, say, a medium-sized apple vs. a basketball, but it is not clear how large a medium-sized apple is, other than to say one knows it when one sees it. The genius of the formula of the A = πr2 where A is the area, π is pi, and r2 is the radius of the circle squared, is that it allows for the area of all possible circles to be described and compared to each other in a rational and clear way. For example, if the area of the first circle is 10 cm2 and that of the second 100 cm2, we all understand the absolute size of each and the relative difference between the first and the second circle. Thus, mathematics represents a clear, universal language offering quantitative and qualitative descriptions. Mathematics has long been applied to physical systems. It works well in part because physical systems are nonvolitional. Forces act on them and they in turn act on other objects. They are in a constant state of entropy with no mechanism of self-maintenance, self-preservation, or 215

216  The Theory of Endobiogeny

s­elf-propagation. Biological systems, while physical in foundation, possess emergent properties. They are selforganizing systems that express life through a process of consuming negentropy in order to resist entropy, a process we refer to as metabolism. They are scaled, multitiered hierarchical systems with levels of integration and interrelation within and across hierarchies. Finally, they have functional organization that is not seen in physical systems. The question arises as to whether one can or should describe biological systems using mathematics. To paraphrase the work of the late Gilbert A. Chauvet, mathematician, physicist, neurologist, and pioneer in mathematical integrative physiology:

a­ ctivities: subcellular, cellular, tissular, global, as well as structure vs function (Chapter 2). 3. Coupling of relative activity of one source (actor) on a sink (recipient) and multiples sources simultaneously on a sink (Chapters 5, 10, 11, and 12).

1. There are a huge number of data points in biology, now more than ever thanks to omics studies. 2. There is no general theory of interpretation for all these data points. 3. A method of integrating this information is required. 4. Most biologists do not know what “integrative” actually means. 5. Biologists and physicians state that there are too many variables in biology for it to be studied mathematically. 6. The study of biology must be done within the constraints of its own theory. 7. Therefore, a general theory of biology is necessary.

The need to use blood tests

In other words, the general theory of biology must attempt to describe biological events mathematically. We have made the argument that the theory of Endobiogeny is a general theory of biology and integrative physiology that creates a framework for explaining (verbally) the qualitative functioning of biological systems. The purpose of this chapter is to demonstrate how this can also be achieved mathematically. Chauvet notes, “Just as physics uses mathematics to provide a general view of the nonliving world, biology will have to rely on mathematical formalism to obtain an integrated vision of living organisms.”3 Dr. Duraffourd developed the biology of functions (BoFs) as an approach to characterizing the permanent dynamism of the organism according to the theory of Endobiogeny. Thus, the BoFs is not a mathematical theory of biology that uses philosophy or clinical empiricism for validation. It is the fruit of a theory of integrative physiology developed as a practical tool by and for clinicians to symbolically represent the most significant factors related to regulation of the terrain. The BoFs, as a series of mathematical formulas, meets Chauvet’s postulates for a biologic theory and addresses a number of concerns cited by researchers in the field: 1. Chosen factors are physiologically relevant upstream regulators of terrain. 2. It recognizes and distinguishes various levels of hierarchical biologic and physiologic organization and

In physics one breaks down complex objects to simpler forms to understand their functioning. In biology one must maintain the integrity of the whole if one is to understand the living system as it functions in and of itself. The question then arises, “How can one model complex physiologic behavior in living biological systems without killing them?” The answer is, “Through the medium of blood.”

From the earliest times of medical practice, physicians sought to look more deeply into the body without directly cutting it open. The goal was to confirm what was obtained by history and physical examination, and, to determine that which the first two could not determine. In the Hellenic tradition, the evaluation of bodily fluids has been the preferred method for over two millennia, primarily blood and urine. The analysis was qualitative in nature, based on the concept of the four humors. The shortcomings of this method include its subjectivity of analysis and lack of a precise assessment of specific neuroendocrine, cellular, and subcellular activities. Evaluation of blood continues to be an important diagnostic tool in modern medicine. The advantages of modern blood tests are many. They are objective, accurate, and reproducible. They are minimally invasive yet allow for the evaluation of complex physiology. They are easily repeated, offering longitudinal assessment of the evolution and devolution of physiologic processes and treatments. The shortcoming of modern lab studies is the binary nature of interpretation. Like the 0s and 1s of digital code, lab results are viewed as having two values and two interpretations: 0: lab test within normal range = no abnormality, ergo: no dysfunction 1: lab test outside the normal range = abnormality, ergo: dysfunction This algorithm is repeated for each individual lab value assuming, in the reductionist model, that each lab value can be viewed in isolation from other lab values. Routine lab testing creates a quandary for the clinician in two situations. The first is a symptomatic patient with normal lab values.4–9 The second is an asymptomatic patient with abnormal lab values.10–12 Both situations call into question the sufficiency of the reductionist model to explain the correlation between individual symptoms and individual biochemical data. This is typically the case for electrolytes, hepatic enzymes such as AST, ALT, and GGT,

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and other common tests. Such a binary system reads with an “error” message for both these situations. The clinician must either ignore abnormal values, ignore symptoms, do further testing without clear guidance of what or how to test, or empirically medicate the patient in hopes that the problem will go away. Other tests such as antibodies associated with autoimmune disease require more complex evaluation and ­decision-making, but pose problems themselves. They have a high degree of specificity but a low degree of sensitivity. Specificity is the percent of patients who have a negative test and do not have a disease. Sensitivity is the number of patients who have a positive test and have the disease.13 When evaluating a patient for lupus, e.g., anti-Smith antibodies (anti-Sm) have a sensitivity of 25%–30% but a high specificity.14 In other words, the presence of anti-Sm antibodies does not rule out disease, but their absence makes it less likely that lupus is present. If a patient is tested for anti-Sm in order to make a diagnosis of lupus in the presence of two clinical symptoms, and the patient is positive for anti-Sm, will they be denied treatment because a total of four criteria have not been met? Over 70% of patients with positive anti-Smith antibodies will not have lupus, but if they do not have lupus, what does it mean that the test was positive? A binary test cannot answer this question. If a patient meets four or more of the criteria associated with lupus, including anti-Smith antibodies, how does that advance an understanding of why they have lupus, or how they will be treated? Regardless of the test results, they will be treated symptomatically based on the organ(s) involved, and the intensity of inflammatory or autoimmune manifestations.15 In that case, what benefit did evaluation of biomarkers bring to the selection of treatment for the patient? Because the human body operates as a system, a method of evaluation is needed that can reflect this complexity while still using serum values as the foundation of its assessment. Such a method should reflect all the properties of a system. The method should be dynamic and individualized, characterizing the function of a single unit of activity in and of itself, relative to other units and relative to the global functioning of the organism in a quantitative and qualitative fashion. If the object of study and method of interpretation are based in a systems approach, serum lab values can be viewed in a nonbinary format, reclaiming their key role in analytical and objective medical practice.

The necessity of using serum biomarkers and their shortcomings The biology of functions] must allow for a synthetic study of the ensemble of functions—specific to the level of activity of each person—separately and in their relative ­interaction

(with each other). Metabolic fluctuations result from the complex fitting of the functionality (or the organism). The blood elements fluctuate unceasingly according to adapted reactions of our bodies to each endogenous and exogenous solicitation. Christian Duraffourd and Jean-Claude Lapraz.16

A biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”17 Biomarkers are used to screen, diagnose, and prognosticate.18 All blood analytes are biomarkers in that they are markers of some biologic process. However, the ability to “screen, diagnose, or prognosticate” arises from a proper analysis of biomarkers that is accurate, valid, and clinically relevant. Numerous biomarkers have been proposed over the years only to be discredited or discarded later. The fundamental shortcoming of these biomarkers is that they continue to be based on reductionist biology rather than systems biology. With respect to the selection and use of biomarkers, there are four common errors that have limited their clinical utility: (1) selection based on an animal model that does not realistically replicate human illness, (2) selection based on taking a clinical problem a priori and using statistical pooling to find an a posteriori correlative relationship, (3) use of biomarkers that are specific but not sensitive, and (4) mistaking downstream effects of pathology to be upstream causes. We will examine each error separately.

Selection based on an animal model of disease Animal models for human illness have long been used to determine single-causative agents of disease. Creating disease in a previously healthy animal is not always a realistic assessment of how the disease develops over time in humans because it fails to replicate the multiple factors within the terrain that are involved. Hepatic encephalopathy and the role of ammonia is a good example. Ammonia was long considered to be the direct cause of hepatic encephalopathy because (a) hepatic injury reduced the metabolic conversion of ammonia to urea, (b) humans with hepatic encephalopathy often had elevated serum ammonia, and (c) ammonia was shown to cause encephalopathy when infused in large amounts in otherwise healthy primates.19, 20 Clinical studies have demonstrated that neither the presence nor the severity of encephalopathy could be predicted solely by the serum ammonia level, nor was the improvement in encephalopathy proportional to the reduction in ammonia levels. Currently, most experts agree that there are multiple variables that play a role in the development of hepatic encephalopathy, of which ammonia is but one.21, 22

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Selection based on statistical pooling Another common method of selecting a biomarker is through epidemiologic studies. In these studies, a clinical condition is selected a priori and numerous biomarkers and epidemiologic data are collected. Patterns of abnormalities in biomarkers are then correlated with the specific condition. Gamma-glutamyl transferase (GGT) is a good example of this with dozens of epidemiologic studies showing strong correlation with various clinical conditions. The GGT is an enzyme that transfers glutamyl residues. An elevation in serum GGT above the norm is seen in hepatobiliary disease, biliary obstruction, and intrahepatic cholestatic disorders. The GGT also plays a key role in glutathione recycling most notably in the liver, but also in bile ducts, small bowel, kidney, brain, pancreas, spleen, and breast. Retrospective analysis of epidemiological studies have associated normal GGT in the upper quartile of normal (40–60; normal = 0–60 IU/L), with the bioaccumulation of heavy metals,23 persistent organic pollutants,24–27 dementia,28–30 hepatic insulin resistance,31 type 2 diabetes mellitus,26, 27, 32–40 hypertension,33, 35, 38, 40–51 and dyslipidemia40 independent of body mass index, lifestyle risk factors, or gender. While GGT has been noted to be elevated in a wide variety of disorders, all these disorders could be best described as having a component of oxidative stress, which would explain the elevation of GGT (even within the upper quartile of the normal range). Oxidation of glucose to make ATP is fundamental to human physiology. A disturbance in oxidation will be implicated in so many disorders that we wonder how it could be predictive of a specific disease state prospectively. If one finds a GGT in the upper range of normal, can one determine from this alone which patient has or will develop diabetes vs. hypertension vs. hyperlipidemia or a combination of these disorders? In a patient with hepatobiliary disease, with a GGT many fold above the norm, GGT can no longer be used to predict the presence of the disorders noted above. How can the risk of these various disorders be evaluated in such patients? There are numerous steps in the oxidation of glucose and in cellular respiration. How does a GGT in the upper quartile of normal guide the clinician in choosing the point of intervention, say, between insulin sensitization vs. oxidants vs. antioxidants vs. the Krebs cycle vs. mitochondrial support with l-carnitine, CoQ10, d-ribose, etc.? All it says is that somewhere in the body, there is or may be an insufficiency of glutathione, without clarifying if it is due to a deficiency in glutathione production, an insufficiency in glutathione recycling, or an excess of glutathione consumption.

Application of a biomarker with high specificity when it has a low sensitivity Prostate-specific antigen (PSA) is the most widely used screening test for prostate cancer in the United States and

Europe. In the United States alone, over 3 billion dollars is spent annually on the test. Discovered in 1970, it was approved by the US food and drug administration (FDA) in 1994 to detect cancer even though its success rate is only 3.8%.52 The PSA is a good screening tool in evaluating the efficacy of treatment of known prostate cancer, and in the surveillance of men with a history of prostate cancer posttreatment.53 In other words, the PSA test is specific for known, active prostate cancer, but not sensitive for cancer, much less predictive of cancer risk. It distinguishes neither benign nor malignant growth. It simply implicates dysregulated growth.54 Dysregulated prostate growth is seen not only in prostate cancer but also in noncancer-related events, such as benign prostatic hypertrophy, injury, use of certain medications, and infection. The PSA levels are low in some men with malignant cancer, and elevated in other men without cancer. Thus, PSA alone is not a good biomarker in screening for prostate cancer. This is not a purely academic discussion because with an abnormal PSA the number-needed-to-treat is 48:1, meaning that in order to save 1 man’s life, 47 men will undergo unnecessary biopsies with loss of sexual and urinary function, based on a test that is being used as an indicator of a specific pathology when it is simply a nonspecific indicator of a disturbed pathophysiologic state within the prostate.55

Studies that mistake the result of a pathologic event as the cause of that event Dysregulation of the immune system is implicated in chronic, low-grade microbial infection, and in altered inflammation/antiinflammation pathways. There was a large body of evidence in the 1980s and 1990s that strongly associated titers of Chlamydia pneumoniae with myocardial infarction.56–65 It was observed that patients who had myocardial infarction had a greater incidence of Chlamydial infection compared to those who did not. Chlamydia was found in biopsies of atherosclerotic tissue, and Chlamydia was found to be atherogenic in vitro. It was hypothesized that treating Chlamydia with the antibiotic Clarithromycin would lower the risk of myocardial infarction. Smaller studies supported this hypothesis66 but larger studies and metaanalysis found no benefit.67, 68 Both Chlamydial infections and arterial disease occur in an environment of immune dysregulation. Thus, they both are downstream effects of altered immunity (Fig.  15.1). The error here was to consider the downstream effects to be sequential, where Chlamydia caused arterial disease, or, contributed in a significant way that it warranted treating with antibiotics. Later studies found that Chlamydia titers and arterial disease also correlated with an elevation of creactive protein, a nonspecific indicator of acute inflammation.69 Thus, it is more accurate to conclude that while all

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FIG.  15.1  Relationship of chlamydia to atherosclerosis according to a global systems approach. Patients who suffer from chronic chlamydial infections and atherosclerosis share a common imbalance in cortico-­ thyrotropic activity. There is a disorganized immune response and chronic inflammation. The immune dysfunction allows for chlamydia to live in the body. The inflammation injures the arterial wall. Chlamydia has a tropism for the nutrients in the fatty plaque. Its presence in the plaque is opportunistic not causative. (© 2015 Systems Biology Research Group.)

these elements are downstream events of altered immunity, they are neither sequentially nor causally related (Fig 15.1). That is to say, the same dysregulation in immune activity that favors inflammation creates a terrain that also favors the installation of chronic, low-grade infections. The greater the inflammatory milieu, the greater the risk of atherosclerotic disease will be. The same type of error was made with respect to vitamin D and heart disease. Low vitamin D levels have been associated with an increased incidence of cardiovascular disease70–72 due to its association with a pro-inflammatory state. However, normalizing vitamin D levels does not change the clinical outcome of patients with CVD even when it improves the inflammatory terrain.73 Because diseases are multifactorial, altering one factor does not necessarily reverse the course of disease. In general, most biomarkers are single variables used to evaluate complex, multisystem disorders. From the Endobiogenic perspective, there are very few cases of “single variable disorders” because the body is a system containing many variables that affect each other’s function. Relying on single biomarkers to screen or diagnose or prognosticate ultimately has limited benefit for the clinician and the patient. Most often, it results in indiscriminate treatment, as in the case of PSA, where men receive potentially harmful biopsies and are prescribed the use of 5α-reductase inhibitors “just to be safe.” Or, it results in excessive treatment because a single biomarker does not allow for the pathophysiologic individuality of the patient (i.e., the terrain) to be determined. For example, in cardiovascular disease, there is a trend to use vitamin D (for low vitamin D), aspirin and fish oil (for elevated CRP), and a statin (for hyperlipidemia) because it is not possible from the current methods of evaluation to determine which aspect(s) of dysregulation is most responsible for the current state of disease.

An exception to the trend of using single biomarkers has been the use of multiple markers simultaneously in critical illness, such as septic shock or multisystem organ failure. Some examples include the Pediatric Risk of Mortality and the Acute Physiology, Age, Chronic Health Evaluation (APACHE).74, 75 These scores use dozens of serum biomarkers and vital signs, such as serum glucose, respiratory rate, heart rate, etc. as well as clinical classifiers, such as surgical status, use of mechanical ventilation on admission, etc. There are two key shortcomings of these multifactor tests with respect to clinical applicability. First, and most fundamentally, these evaluations represent retrospective attempts to find variables that predict mortality in order to stratify patients in research studies. Even within this narrow focus of interest, there is not an attempt to integrate these various factors into a coherent understanding of illness. These scores do not truly integrate physiologic abnormalities in a way that reflects the patient’s terrain. Even if the scores are valid, they do not provide clinical guidance on determining which system(s) is most responsible for the current disorder, to what degree, or in what order interventions should be administered, i.e., cortisol, vasopressors, ventilation, dialysis, etc. In summary, biomarkers are indicators of normal or pathologic activity. Biomarkers are commonly used in medicine and can be useful, but their ability to describe why an abnormality occurred or predict future imbalances is limited by the binary nature of the test. The ideal use of biomarkers would be based on a systems biology approach. In such a system, multiple factors are evaluated simultaneously, relative to each other, describing human physiology in a dynamic fashion. Such an approach can offer specific areas and methods of intervention tailored to the terrain of each individual patient. Endobiogeny offers such a system: the BoFs.

The biology of functions (BoF): A biological modeling system The purpose of the Biology of Functions is to quantify the functional abilities of the organism, before and after the effects of adaptation [to stressors]. Because [the terrain] is in permanent movement, functionality can only be measured by a dynamic, integrated and evolutionary methodology. C. Duraffourd, MD and JC Lapraz, MD.76

The BoFs is a biological modeling system developed by Dr. Duraffourd, based on the theory of Endobiogeny. As with other biological models, it simulates biological activity based on variables assumed to be most representative of the system, and is not a measurement of actual function. It differs from other biological models in three key ways. First, it simulates biological activity using biomarkers related to the direct and indirect effects of neuroendocrine

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a­ ctivity. Second, it evaluates quantitative as well as qualitative ­function. Finally, it evaluates both the potential and functional achievements of the organism. Current research in systems biology is focused on genomic and cellular activity. Many of these mathematical methods have proven to be robust, accurate, and predicative in examining narrow areas of physiologic activity.77–79 However, due to conceptual limitations, they can neither describe the terrain that brought about disease nor suggest the optimal treatment within the context of the global functioning of the individual organism and serve largely as research tools. As a model of the global functioning of the organism, the BoFs evaluates factors both in and of themselves, in relationship to other units of activity and in relationship to the system as a whole. There are numerous indexes evaluating neuroendocrine activity in the BoF. They are derived from 17 serum biomarkers that are linked to the various aspects of this activity, without directly measuring serum hormone levels except for thyroid-stimulating hormone (TSH) (Table 15.1). The biomarker norms are the normative data of the adult, premenopausal female, which is considered to be the null state of human physiology in Endobiogeny. The values of postmenopausal women, of men, and of children

are compared against this normative data. Some exceptions include particular indexes that have grossly different values in various phases of childhood (unpublished data), and well-characterized sexual dimorphisms noted between men and women, and their corresponding variations in serum biomarker values.80 A current shortcoming of the algorithm is the exclusive reliance on normative data from a Western European population. This will need to be addressed to broaden the applicability of the BoFs vis-à-vis men and women,80, 81 non-European populations82, 83 and children.84 Four biomarkers have a high degree of variability in their normative values from lab to lab and during particular phases of life: osteocalcin, total serum alkaline phosphatase, lactate dehydrogenase (LDH), and creatine phosphokinase (CPK). The normative values determined by each lab are standardized to an internal consistency. The indexes evaluate relative neuroendocrine functionality and are derived from 16 direct ratios of the 17 biomarkers. The remaining indexes are indirect ratios: indexes of indexes. Nearly 90% of the indexes describe relative and qualitative function. In other words, they describe the physiologic capability of the organism in a contextual manner. The relativity of the indexes ensures global internal consistency and reproducibility across patients and diseases

TABLE 15.1  Biomarkers used in the biology of functions Origin

Biomarker

Value

Conversion

Bone marrow cellular products

Red blood cell

per μL

÷106

White blood cell, total

per μL

÷103

Neutrophil

%

None

Hemoglobin

g/dL

None

Platelets

per μL

÷103

Bone marrow-serum interaction

Erythrocyte sedimentation rate

mm/h

None

Bone stroma enzymes

Osteocalcin

ng/mL

Proprietary

Alkaline phosphatase bone isoenzyme

%

Proprietary

Lactate dehydrogenase

IU/L

Proprietary

Lymphocytes Eosinophils Monocytes Basophils

General enzymes

Creatine phosphokinase Endocrine

Thyroid stimulating hormone

μIU/mL

None

Electrolytes

Potassium

mmol/L

None

Calcium, total serum

mmol/L

÷2

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with reliable norms. The normal range of each index is determined by two methods. First, the general range is determined from high and low values of each biomarker of which an index is composed. The specific normative range is based on retrospective analysis of unpublished data from clinical practice. The use of such a limited number of biomarkers to derive a large amount of information about human physiology can only be achieved under two conditions. The first is if the body functions as a system and the effects of one event affect other events. The second is if the level of evaluation is sufficiently upstream that a small number of factors are linked to a wide variety of downstream events, having a profound effect on multiple lines of biologic activity. Hormones are secreted in extremely low concentrations (10−9–10−12 g/dL) yet have a profound impact on global physiology at the nuclear, cytoplasmic, cellular, tissue, organ, and system levels. Each subsystem of activity that the endocrine system manages is amplified at the level below it because each system manages or influences increasingly complex subsystems of activity. Thus, small changes at the endocrine level can have a profound and wide-ranging impact on the ensemble of metabolic processes. This is why we believe that such a small number of biomarkers can be used to generate such a large number of indexes.

The logic behind biology of functions indexes Three basic observations are the foundation of this elegant and simple biologic model: (1) the endocrine system is the manager of the terrain, of the biologic system, (2) certain biomarkers are produced as a result of this endocrine management, and (3) the true functionality of any system is based on the relative activity of one factor to another. Because these biomarkers are an indicator of endocrine management, indexing biomarker values as ratios provides an assessment of relative functionality of the endocrine management of the terrain.

Endocrine management We have established that the endocrine system is the true manager of the terrain, and that the effects of endocrine activity cannot be accurately evaluated by direct serum measurement.

Biomarkers and the endocrine system It has been known for nearly 100 years that changes in common biomarkers were associated with specific endocrinopathies.85–88 Through elegant experiments, it has been clarified that the changes in these biomarkers are the result of endocrine management of metabolism. For example, it was observed in the 1950s that androgens cause a proliferation of red blood cells (RBCs).89–95 Thus, RBC levels in the serum are a marker of a certain aspect of androgen function.

It was also observed that estrogens cause a proliferation of white blood cells (WBCs) and the same can be said about WBCs and estrogen activity (cf. discussion below).96, 97

Systems analysis and relative relationships As noted above, newer evidence suggests that the body is a true system, composed of various subsystems that act independently of each other, but in coordination with each other. Because the functioning of each unit is integrated and interrelated to the functioning of the others units and to the whole, it is the relative activity of one unit to another that determines the true state of functionality. The appreciation of relative changes in biomarkers has been present for nearly 100 years and is gaining increasing appreciation once again.86–88, 98–100 The value of relative changes of biomarkers in and of themselves and with respect to other markers is paramount in a systems approach. For example, in and of themselves, normal RBC and WBC counts do not offer actionable information about the state of androgens or estrogens. However, if you relate one to the other, you have a general evaluation of the global activity of androgens relative to estrogens regardless of the absolute value of RBCor WBCs, or the quantitative serum level of androgens or estrogens. The relative imbalance of androgens and estrogens can be clinically significant. Numerous studies have shown that even with normal serum levels of androgens and estrogens, one can develop fibroids, polycystic ovarian disease, infertility, or hair loss.98–103 The ratio of RBC to WBCs, called the “Genital ratio” (cf. below) is a necessary but not sufficient for the evaluation of gonadotropic activity. However, it lays the foundation for increasingly complex evaluations with respect these and other disorders. Normally, the diagnosis of and decision to treat “endocrine” disorders is based solely on quantitative serum concentrations of hormones. If levels are normal, there will be no justifiable basis for treatment, and the patient is condemned to suffer. If an empirical treatment is started out of compassion, there rests no objective reason for the choice of treatment, or a manner in which to understand why the treatment failed if it does not work. In such cases, the patient is considered to have an “idiopathic” disorder, often deemed untreatable. We believe that using ratios of biomarkers may be a more accurate and valid method of determining physiologic functionality, not only in cases of idiopathic disorders, but more broadly when evaluating various disorders, even common ones with atypical courses or unexpected response to treatment.

Precedence of using ratios in clinical medicine The practice of relating biomarker to each other is not new in medicine and there are many examples used on a daily basis (Table 15.2).

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TABLE 15.2  Ratios in medicine System

Ratio

Composition

Indication

Shortcoming

Renal

BUN/creatinine

Blood urea nitrogen/ creatinine

Evaluates the rate of renal perfusion relative to renal clearance

Does not indicate why perfusion or clearance is impaired, if it is due to structural or functional impairments, or both

Microalbumin/ creatinine

Microalbumin/creatinine

Evaluates resorptive integrity of kidney relative to its clearance ability

Does not indicate functional reasons for disruption in renal tubule integrity

A/G ratio

Albumin/globulin

Evaluates the risk of autoimmunity vs. cancer vs. liver failure

Does not evaluate endocrine, GI factors related to protein uptake, distribution or utilization

CD4+/CD8+

Subsets of lymphocytes based on specific cell determinates (CD)

Used to assess relative strength of immune system in HIV seropositive patients; CD4 counts vary day to day, so they are indexed relative to the CD8 count

Does not evaluate the factors related to generation, mobilization and regulation of immune cells

Hematocrit

Red blood cells/whole blood volume

Evaluates the density of blood relative to intravascular volume by indexing the amount of red blood cells produces relative to the total blood volume

Does not evaluate the factors influencing red blood cell production or demargination from the spleen

Immune

Hemato-logic

While these tests are dynamic—they are derived from circulating blood analytes—they do not indicate the relationship of individual units to each other or to the whole system, which is why we do not consider them to be candidates for use in a systems approach to biology. The BoFs is composed of a series of direct and indirect indexes. Direct indexes are composed of individual biomarkers directly related to each by various mathematical relationships. Indirect indexes are composed of direct indexes, indirect indexes, and/or individual biomarkers in various permutations that can contain up to six levels of indexes within indexes. An example of a direct index is the genital ratio, which looks at the impact of androgens relative to estrogens at the tissue level. It is a ratio of 2 of the 17 biomarkers: RBCs and WBCs. Genital ratio = Red blood cells / White blood cells = Androgen tissular activity relative to Estrogen tissular activity An example of an indirect index is the thrombotic index which expresses the risk of sudden thromboembolic phenomenon:

  Thrombogenic index      Thrombotic index =   × Evoked histamine index  / 10     ×Genital ratio     Indirect indexes are complex metaindexes composed of several other indexes. For example, we can open up the thrombogenic index and replace the basic equation with key factors related to thromboembolism:    Bone remodeling     ×Apoptosis × Necrosis  / 10  Thrombotic index =      ×Evoked histamine          × Genital ratio Acute ischemic events that result in sudden cardiac death occur in arteries that often have mild coronary artery luminal occlusion and minimal plaque calcification. Neither calcium score by CT scan nor angiography will be able to identify patients most at risk. Typically these events occur in patients under the age of 60, with minimal classical risk factors, thus general screening factors will not identify them either.104 The ability to aggregate the known factors related to thrombus formation and plaque rupture may help

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i­dentify patients most at risk for sudden cardiac death or acute ischemia based on functional factors rather than structural factors. The mathematical relationships in the index express that the risk of thromboembolism is the result of a triad of factors which are necessary but not sufficient on their own: (1) risk of thrombus formation, which can occur due to necrosis105–107 or apoptosis,104 (2) histamine activity,108–110 and (3) elevated androgens111–119 represented by the Genital ratio (androgens/estrogens) in the numerator, which is consistent with known pathophysiologic mechanisms of thromboembolic phenomenon. In summary, evaluating biologic activity relative to each other has precedence in medicine. It helps contextualize the relevance of one finding to another. The BoFs is composed of direct indexes where individual biomarkers are related to each other, and indirect indexes, where direct indexes and various biomarkers are indexed against each other to express increasingly complex biological activity that is multifactorial in nature. The majority of indexes in the BOFs are indirect indexes that evaluate the function of units of activity relative to other units of activity.

i­nformation, a delivery system of nutrients, and a remover of metabolic waste, but it is the cellular elements: WBCs, RBCs, and platelets that serve to delivery oxygen, defend, heal, and protect the body. The endocrine system as the manager of metabolism determines the rate of production of cellular elements from the bone marrow. Thus, blood is the foundation of life, and the endocrine system, as the manager of blood, is the manager of this foundation. The evaluation of blood cells reveals how the endocrine system manages life. In the BOFs, over 60% of biomarkers used are derived just from the cellular elements of blood, made in the bone marrow (Table  15.1). The complete blood count (CBC), then, is the basis of the BOFs. Androgens and estrogens stimulate the proliferation of RBC and WBCs, respectively. Thus, sex hormones are the foundation of the CBC—hence of life—and the initial point of study in the BOFs. (The bone stroma, discussed below, plays three key roles: protection and nourishment the marrow, regulation and assistance in global energy management, and communication of the state of the peripheral terrain to the central nervous system.)120–122 The roles of androgens, and then later estrogens, as the basis of life are evident from the time of conception. For Experimental and clinical basis for the first 17 days, it is the mother’s hormones that the emthe biomarkers used in the biology of bryo shares. At day 18 of life, the yolk sac becomes the first functions endogenous source of RBCs.123, 124 Rich in androgen recepwhich itself Endobiogeny and the BOFs are based on four scientific con- tors, the yolk sack stimulates erythropoietin, 124 plays a role in yolk sac maturation of RBCs, establishing cepts that are known and generally accepted: (1) human physthe key role of androgens in the foundation of structure.125 iology is complex, multifactorial, and exhibits the properties of RBCs,123 it also of a system, (2) the endocrine system manages metabolism, While the liver is an intermediate source 126 which is the basis of the continuity of life, (3) the metabolic under the management of androgens. By 34 weeks of gesactivity managed by the endocrine system results in the out- tation and throughout the remainder of life, the bone marbecomes the put of biomarkers that reflect the functional achievement of row, stimulated by androgens and estrogens, 127 source of the majority of blood cells. specific aspects of metabolism, and (4) when biomarkers are In summary, the activity of androgens and estrogens is related to each other in ratios, it contextualizes one type of reflected in the production of output of RBC and WBCs function relative to another to which it is linked anatomically, by the bone marrow. The evaluation of this activity, called sequentially, chronologically, biochemically, etc. the Genital Ratio, is used in the majority of indexes of the As shown below, the relationship between various BOFs. To accept the hypothesis that RBCs are a biomarker hormones and particular biomarkers is a long and well-­ of androgen activity and that total WBC count is a biomarker established fact based on modern physiology and the scienof estrogen activity at the level of the tissues, is to accept the tific method. The indexes composed from these biomarkers foundation of the majority of indexes of the BOFs. have been derived through inductive reasoning, and confirmed by over 30 years of clinical practice. The indexes have not been individually validated in peer-reviewed literature. Red blood cells (RBC) However, it stands to reason that if the correlation of each biomarker to endocrine activity is sufficiently demonstrated, Introduction then it is possible that such a biological modeling system Based on studies over the last 50 years, we concluded that RBCs reflect the tissular level activity of androgens. Here, may be a more valid assessment of biological activity. the bone as the site of production of RBCs represents the general assumed level of activity of androgens on other Bone marrow: Complete blood count tissues. This assumption is further refined with complex Life is permanent dynamism and the circulation of blood indexes to account for other factors. Studies have demensures this dynamism. Blood plasma is a conduit of onstrated that the administration of androgens stimulate

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erythropoiesis.89, 93–95, 128–130 Using RBCs as a marker of the functionality of androgens may prove to be more clinically relevant than quantitative measurements for four reasons: contradictory studies regarding serum levels of androgens and clinical effects, the complimentary nature of estrogens, the role of genomic vs. nongenomic effects, and genetic variations in intracellular (IC) conversion of androgens (cf. Chapter 7).

Limits of quantitative measurements of androgens Multiple studies have positively associated elevated levels of serum androgens, RBCs, or both in hypertension,131–134 thrombus formation,111–119 impaired insulin sensitivity,135, 136 and insulin resistance.137 However, low serum levels of androgens have also been positively associated with the same disorders.138–141 For example, while androgens are positively associated with dyslipidemia, they have also been associated with a reduction in triglycerides and LDL.112 Thus, evaluating serum androgen levels may be misleading.

Protective role of estrogens For years, it was believed without strong evidence that delayed cardiovascular mortality in women was due to a protective effect of estrogens. Prospective studies of estrogen supplementation demonstrated that not only supplemental estrogens offered no benefit, but they also elevated the risk of cardiovascular events.142–144 The lack of definitive protective effects of estrogens, and harmful effects of elevated and low serum levels of androgens in some men and not others suggests to us that it is the relative ratio of androgens to estrogens that is clinically relevant, not the absolute quantitative value of either in isolation.

Are androgens harmful in and of themselves? Studies suggest that androgens alone are not predictive of life span or risk of death from cardiovascular disease in men145–147 or women.148–151 Rather, androgens appear to be but one of many factors in a complex interplay of endocrine drivers of metabolism that influence the development, progression, and severity of a wide range of disorders from vascular disease131, 152 to Alzheimer’s disease.153 This may be one reason that assessments relying on serum androgens measurements alone have been inconsistent or contradictory.

Determining androgen function: Genomic and nongenomic effects Androgens, like most other steroidal hormones, have genomic and nongenomic effects.154 The ability to evaluate the relative impact of nongenomic vs. genomic affects in a particular individual may help solve the conundrum of whether high or low androgen activity is protective or harmful.

The genomic effects of androgens are what have been associated with serum levels of androgens. In contrast to the nongenomic effects, these effects take hours to occur, and are linked to many of the classic effects associated with androgens deemed to be harmful when dysregulated. These effects include smooth muscle proliferation, migration and vasorelaxation, increased monocyte migration and foam cell production, and increased apoptosis.154 Nongenomic effects occur within seconds. Mechanisms of action are believed to include a novel membrane-bound receptor, second messenger activation, and sex-hormone binding globulin receptors. Many of the nongenomic effects of androgens are physiologically beneficial and explain the protective effects of androgens observed in studies. They include relaxation of smooth muscle, increased neuromuscular signal transmission by calcium regulation, improved neuroplasticity, cellular proliferation and migration, and modulation of the transcriptional effects of classical androgen receptors.155, 156 What is clinically relevant is that these nongenomic effects cannot be blocked by drugs that block androgen receptor activity. This may explain two observations: (1) the variability of responsiveness to androgen blockers, (2) factors of risk and protection from disease cannot be reliably assessed by quantitative measurement of serum androgens, sex hormone-binding globulin (SHBG), or free androgen levels—because their effects do not rely solely on receptor activity.

Determining androgen function: Metabolic pathways There are a number of other factors adding to the difficulty of equating quantitative levels of testosterone (free or total) with androgen functionality. Recent studies have demonstrated in vitro and in vivo sex-based variability in androgen receptor sensitivity and concentration in various tissues.157 Approximately 5% of testosterone is converted within the cell to either dihydrotestosterone (DHT) or estrogens. In summary, the individual effects of testosterone on the body can vary based on: (1) genomic effects, (2) nongenomic effects, (3) receptor concentration, and (4) IC conversion tendency between DHT and estradiol. The net effect can be an amplification of genomic or nongenomic effects (DHT), or a counterbalancing effect (estrogens). Therefore, we believe that RBCs may be a useful biomarker reflecting the global degree of tissue functionality of androgens, when evaluated relative to other factors (discussed below).

White blood cells (WBC) Introduction The WBCs, also known as leukocytes, are blood elements that mature in the bone marrow then enter the circulation. Leukocytes consist of five types of cells that arise from a

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common hematopoietic precursor. White cells differentiate into neutrophils, monocytes, eosinophils, basophils, and lymphocytes. Estrogen stimulates a proliferation of leukocytes in the bone marrow. 54 Leukocytosis is associated with high estrogen states such as pregnancy158 and autoimmunity,159 as well as during the acute phase of infections. Thus, we believe that total WBC count can be considered to reflect the basic tissue effect of estrogens throughout the body.

Limits of quantitative measurements of estrogens The challenges of evaluating the role of estrogens in human physiology are far greater than for androgens, which is why specific aspects of estrogen activity requires more than a single biomarker (discussed below). Estrogen activity is complex, varied, and fundamental to human life. It involves endocrine and metabolic functions, both genomic and nongenomic in nature. Of all sex steroids, metabolically estrogens require the greatest number of metabolic conversions, being derived as such: Cholesterol à Progesterone à Androgens à Estrogens. Estrogens can be produced in the ovaries, in the adrenals, and by peripheral conversions in various tissues.160, 161 The pattern of estrogen production (central vs peripheral, adrenal vs gonadic vs hepatic) varies based on hereditary factors, age, and parturition status, and is affected by endocrine disrupters.160–163 There are multiple active forms of estrogens (estrone, estradiol, and estriol) as well as varying degrees of activity of estrogen metabolites. There are two types of estrogen receptors (alpha, beta), which have opposing activity with respect to cellular proliferation and various metabolic function. There are genetic polymorphisms in p450 metabolism of estrogens and polymorphisms with respect to receptor sensitivity, concentration, and rate of aromatase activity as well as nongenomic effects, which in sum all impact the effects of estrogens.164–170 In their review of estrogen metabolism, Zhu and Connery conclude: Studies that identify genetic and environmental factors influencing estrogen metabolism at or near estrogen receptors in target cells may be of considerable importance since these factors could profoundly modify the biological effects of estrogens in complex manners depending on the pathways of metabolism that are affected and the biological activities of the metabolites that are formed. Such effects need not be associated with an altered profile of estrogen metabolites in the blood or urine. Ref. 167

Estrogens: Beneficial or harmful? As with androgens, clinical trials are conflicting with respect to the beneficial or harmful role of estrogens in the body. The protective role of estrogens in cardiovascular

disease has come under question, as we have discussed above (cf. androgens).142–144, 160 With respect to cancer, estrogens can promote or lower the risk for cancer in and of themselves and in conjunction with other hormones.171–174 The contradictory nature of estrogen’s effects on telomere length, and the role of telomere length in cancer serve as another good example of the limitations of both quantitative hormone measurement and single-cause theories of disease. Estrogens increase telomere length. Women have the longest telomere length when follicle stimulating hormone and estrogen peak during the menstrual cycle.175 Telomere length is positively correlated with the rate of apoptosis and inversely associated with the risk of cancer. However, estrogens also cause leukocytosis, which is associated with shorter telomere length, less apoptosis, and greater risk of cancer.175, 176 Telomere length alone, like quantitative levels of estrogens, does not appear to be sufficient indicators of the global effects of estrogens on the terrain.

The case for multiple biomarkers of estrogen In conclusion, estrogens have various sources of origin, various rates of metabolism, changing concentrations and receptor densities throughout life, and can be affected by and affect other hormones in the body, as well as being disrupted by endocrine disrupters. Mounting evidence suggests that serum and urinary levels of estrogen and their metabolites may not be sensitive or specific enough measures of the effects of estrogens. We hypothesize, based on experimental evidence and clinical studies, that specific functional effects of estrogens can be inferred through the evaluation of particular serum biomarkers in and of themselves, as well as in conjunction with other biomarkers in increasingly complex ratios. In the BOFs, this assessment of estrogen function is accomplished by evaluating six different biomarkers: (1) total WBC count, (2) percent neutrophil count, (3) percent monocyte count, (4) percent lymphocyte count, (5) thyrotropin-­stimulating hormone (TSH), and (6) serum osteocalcin. Of these, WBCs are used as a general marker of global estrogen effects on tissues, and are the most foundational. Through the use of the genital ratio or its variation, the corrected genital ratio (cf. indirect indexes), WBCs can be used to evaluate the structural, functional, and adaptive role of estrogens in the body.

Neutrophils Neutrophils are a type of leukocyte that arise from granulocytes in the bone marrow. While the total leukocyte count (WBC) reflects global tissue effects of estrogens, we hypothesize that neutrophils can be used to assess particular aspects of estrogen activity, namely immune regulation and anabolism of tissue.

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The role of neutrophils is to participate in the immunologic response of the organism to aggressors. This can occur through inflammation177 or phagocytosis of microbes and cellular debris.178 Neutrophilia, absolute or relative, is associated with the anabolism of tissue, such as during pregnancy,177 wound healing179 autoimmune disease,180–182 and cancer.183–187 Estrogens are associated with the same events: preeclampsia,177 autoimmune diseases,188 and cancer,166 as well as wound healing.189–194 The majority of patients suffering from autoimmune disease are women, which implies a role for estrogens in the etiology of these disorders. Newonset autoimmune disease is frequently diagnosed in the peripartum period, and flare ups of existing disease often occur during pregnancy as estrogen levels rise up to 100fold from nonpregnant levels.195, 196 Neutrophils ordinarily exhibit a short half-life of 3–6 h, requiring a constant production by bone marrow to maintain normal circulating levels. Estrogens affect neutrophil populations in two ways. They increase the total production of neutrophils in bone marrow,197 and they inhibit apoptosis of circulating neutrophils, which increases the relative percentage of neutrophils in the total leukocyte differential, even when the leukocyte count is within normal limits, i.e., in noninfectious states.81 Estrogens manage the production and maintenance of neutrophils, thus estrogens manage a particular aspect of immunity related to inflammation, hostdefense, auto-immunity, and cancer. Therefore, neutrophils may be considered as a biomarker of the role of estrogens in immunologic, inflammatory, and anabolic activity within the body.

Monocytes Monocytes are WBCs derived from monoblasts in the bone marrow. They play an important role in the immune system, combating foreign organisms in the blood through phagocytosis and the release of pro-inflammatory cytokines. They also produce growth factors that aid in fibroblast activity for wound healing. After 24–72 h of circulation, they migrate into extravascular tissue where they differentiate into macrophages or dendritic cells (histocytes). Typically, monocytes represent 3%–8% of the total leukocyte population. The FSH stimulates estrogen production and estrogens suppress monocyte production.198 In the time that FSH is waiting for an estrogen response, monocytes play a role in anabolism by stimulating wound healing by releasing human growth factors.199, 200 During adaptation, as FSH and estrogen levels rise, monocyte levels fall, indicating an anabolic response by estrogens commensurate to the initial antianabolic activity of cortisol, thus reducing the requirement for monocytes. The lower the monocyte count, the greater the influence of FSH and estrogen is on the adaptation response, but this needs to be evaluated relative to the eosinophil count, which reflects

the role of ACTH on adrenal stimulation, as well as other factors (cf. Adaptation index). Conversely, monocytosis is inversely related to the relative efficiency of FSH in stimulating estrogen production. In menopause, monocytosis is observed.201 Monocytosis also reflects a relative or absolute insufficiency of estrogen’s activity during adaptation202 and is associated with increased risk of mortality in multiple diseases marked by dysregulation of the immune system such as lupus,203 autism,204 asthma,205 sepsis,206 atherosclerosis,207 myocardial infarction,208 myeloproliferative disorders, and leukemia.209–214 Thus, monocytosis implicates a terrain that is more favorable to inflammation, and altered immune states; in other words, a terrain of disadaptation of estrogen activity. As the bioavailability of estrogens and androgens are inversely related to each other due to the activity of SHBG,215 and as monocytosis reflects a relative insufficiency of estrogens during adaptation, monocytosis also reflects a more predominant peripheral androgen activity relative to that of estrogens.216

Eosinophils Eosinophils are a subpopulation of WBCs. Fundamentally, the role of the eosinophil is to serve as an indirect method of adaptation and congestion when the adrenal cortical response is not sufficiently adapted to the needs of the organism. While estrogens, as noted above, have a general effect on the proliferation of all leukocytes within the bone marrow, it is ACTH and cortisol that affect the circulating levels of eosinophils. The degree and intensity of ACTH activity on the adrenal cortex is proportional to the level of circulating eosinophils. Thus, the greater the ACTH solicitation of adrenal activity is, the greater the rise in eosinophils.217, 218 Eosinophilia, relative or absolute, is proportional to the degree of adrenal insufficiency, which is proportional to the demand for ACTH and inversely proportional to the efficiency of cortisol.219, 220 On the other hand, cortisol is inversely related to the eosinophil count because it reduces circulating eosinophils in three ways: (1) suppression of eosinophil maturation, recruitment, and survival,221 (2) sequestration of mature eosinophils in lymphoid organs,222 and (3) stimulation of eosinophil apoptosis through transcriptional upregulation.108 The greater the degree of circulating cortisol, the lower the eosinophil count. The lower the circulating cortisol activity, the higher the eosinophil count. While eosinophils cannot replace the complex roles that cortisol plays in the body, they can compensate in part for some of the adaptive functions of cortisol with respect to immune modulation. Eosinophils have direct antimicrobial effects through the production of RNase enzymes223–232 and the generation of reactive oxygen species, and are immunomodulatory through antigen presentation to T-cells.233–239

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Indirectly, they are an indirect source of histamine, which modulates the immune system.240, 241 In summary, eosinophil count is used in the BOFs to assess the intensity of the ACTH solicitation of adrenal activity (positively correlated) and the relative efficiency of cortisol activity (inversely correlated). The less efficient the adaptation response is, the lower the circulating cortisol levels, the greater the role of ACTH in restimulating the adrenal cortex, the higher the circulating eosinophil count will be. Eosinophils also contribute to the evaluation of inflammation, thrombosis, immune, and other activities.

Basophils Basophils are the least populous of all white cells. Basophils have been likened to circulating mast cells and play a role in the innate immune response, particularly against allergens242 and parasites.243 Basophils share similar receptors to eosinophils, such as eotaxin, and may serve as a tertiary method of adapting the adrenal response to aggressors in the face of inadequate cortisol response and insufficient eosinophil response. They are found in high concentration in the circulation and extracellular (EC) spaces of the skin and lungs in patients with atopic disease.244 The percent basophil count on differential is used in only one index in the BOFs, but indirectly in all indexes in which the total WBC count is used.

Lymphocytes Lymphocytes are a subset of leukocytes that are the mediators of immunity. The lymphocytes count is the sum of all three subsets of lymphocytes: natural killer (NK), T, and B cells. The NK cells are part of the innate immune system. They survey and directly attack viruses and tumors. T and B cells comprise the adaptive immune system. T cells manage cell-mediated immunity through the secretion of cytokines, regulate the activity of other immune cells and lyse cells infected by viruses. T cells also play a role in immunoregulation. B cells form antibodies specific to a unique aggressor and retains a memory of the aggressor in case of future aggression. Lymphocytes play a role in cancer surveillance, immunity, and autoimmunity. The concentration of total circulating lymphocytes can be related to three factors: cortisol, estrogen, and TSH. Cortisol is inversely related to lymphocyte counts. It reduces the circulating concentration of all three subtypes of lymphocytes, and augments destruction of lymphocytes.245–248 Estrogens are also inversely related to lymphocytes. There are several lines of evidence and clinical observations related to this. Estrogens directly inhibit the proliferation of lymphocytes.249 In high-estrogen states, such as pregnancy, there is a relative suppression of lymphocyte proliferation in order to reduce the immune

a­ttack by the mother against the fetus.158 Autoimmune disorders occur disproportionally in females who tend to have higher levels of estrogen activity and estrogen variability.157 There is an additional risk of developing autoimmune disease in the peripartum state when there is a terrain of hyperestrogenism and thyroid overstimulation.250, 251 Estrogens augment the infiltration of lymphocytes into various tissues, reducing the level of circulating lymphocytes.157 The relationship between serum TSH and peripheral lymphocytes is positively correlated to the metabolic needs of the body and the degree to which TSH is used to modulate thyroid activity.252, 253 When the lymphocyte counts are elevated, serum TSH levels tend also to be elevated, and the body tends to be in a state of increased need of thyroid activity. For example, in subclinical hypothyroidism, there is an increased appeal to TSH to stimulate the thyroid. These patients have lymphocyte counts that are elevated relative to euthyroid patients and/or in an absolute sense. When the body’s demand for thyroid hormones have been met by exogenous administration of thyroxine, lymphocyte counts reduce from their preintervention levels.254 In disorders of thyroid overactivity, such as Grave’s disease or autoimmunity, there is diminished appeal by the thyroid to TSH for stimulation. One does find diminished peripheral blood lymphocytes in these patients, though not consistently.255 As demonstrated below, other assessments of thyroid function (cf. LDH and CPK) help further contextualize thyroid efficiency. In summary, lymphocytes are inversely related to the degree of cortisol and estrogen activity in adaptation and tissue anabolism. The greater the degree of cortisol expression, and/or the greater the predominance of estrogen activity, the lower the lymphocyte levels. Lymphocytes are directly related to the degree of appeal to TSH to regulate thyroid function. The higher the lymphocyte count, the greater the appeal to TSH is, and often the greater the degree of thyroid insufficiency. Conversely, the lower the lymphocyte count, the more successful TSH has been in modulating thyroid activity regardless of the serum TSH level.

Platelets Platelets are circulating blood cells that arise from megakaryocytes in the bone marrow. Platelets have four primary functions in the body: hemostasis, repair and growth of connective tissues, transport of various factors, and modulation of inflammation. The hemostatic function of platelets has been observed for over 120 years and is well characterized.256 Platelets secrete numerous growth factors for the regeneration of connective tissue once hemostasis has been achieved, including platelet-derived growth factor, insulinlike growth factor-1 (IGF-1), fibroblast growth factor, and others.257, 258

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In general, platelets are absorbers of numerous factors in the blood, such as clotting factors and calcium, which allows them to participate in immediate hemostatic activity.259 In addition, platelets serve as the primary transporter of serotonin from the enteric cells where they are produced. Serotonin aids in intestinal motility and carbohydrate absorption.260 Serotonin also plays constitutive roles in the regulation of bone density.120, 261–263 Thus, platelets contribute to these physiologic activities as a serotonin transporter. Platelets participate in pro-inflammatory activity, adapting innate and adaptive immune mechanisms through the expression of chemokines and cytokines, and receptor-­receptor interaction with leukocytes.264 Platelets also contain histamine, which is secreted before aggregation occurs.265 In the BOFs, after the total WBC and RBC count, platelets are the most important biomarker derived from the bone marrow. Through the Starter index (cf. below), it is used to correct the genital ratio (RBC/WBC) in order to evaluate the role of genital hormones during adaptation. The Genital ratio corrected is used in over 50% of the indexes of the BOFs. Platelets, along with other factors, are used to assess histamine activity, risk of thrombosis, thromboembolic phenomenon, adrenaline activity, and peripheral serotonin activity.

alpha-sympathetic activity regardless of the origin of the anemia (genetic, hemorrhagic, renal, acute, or chronic) resulting in cardiovascular diseases such as cardiac remodeling and coronary ischemia.266–271 Anemia appears to alter the normal adaptive response to stressors, resulting in overadaptation.272 Based on these observations, we have concluded that hemoglobin can be viewed as a marker of the degree of alpha-sympathetic activity in adaptation. Because the general adaptation syndrome is initiated by alpha-sympathetic discharge (i.e., noradrenaline), hemoglobin comes to play an important and pervasive role in the BOFs.

Bone stroma-derived enzymes Two key stroma-derived enzymes are osteocalcin and alkaline phosphatase bone isoenzyme (ALPBi). In addition to their bone-related activity, they have direct effects on nonbone metabolic activity. These biomarkers in particular and the skeletal system in general inform the central nervous system of the state of the internal milieu, helping it modulate basal and adaptive capacities to meet the needs of the organism.120–122

Osteocalcin Hemoglobin Hemoglobin is a metalloprotein found within RBCs. Each RBC contains four hemoglobin subunits with an iron molecule in the center of each hemoglobin subunit. The primary role of hemoglobin is to bind and deliver oxygen from the lungs to the tissues, and bind and deliver carbon dioxide from the tissues back to the lungs. Thus, hemoglobin plays a role in acid-base balance as well as oxygen delivery. Hemoglobin (Hg) is an important determinant of the oxygen content of arterial blood, based on the equation of the calculation of arterial oxygen content (CA). Hg ( g / dL ) × 1.34 × arterial  CA =   saturation of blood ( percent )  + 0.0032 × Partial pressure of oxygen ( torr )  For a given saturation of blood and rate of consumption of oxygen, the lower the hemoglobin content is, the lower the oxygen content will be. Thus, the greater the cardiac output rise must be in order to maintain an equivalent rate of oxygen delivery. This can be expressed in the following equation, based on a rearrangement the Fick equation: Q = (VO2/(CA – CV)) × 100, where Q = cardiac output, VO2 = oxygen consumption, CA = arterial oxygen content, and CV = venous oxygen content. In  vivo and clinical studies demonstrate that in both children and adults, iron-deficiency anemia upregulates

Osteocalcin is a noncollagenous protein. Within the skeletal metabolism, it plays an important role in osteoblasty, fixing ionized calcium to hydroxyapatite crystals. In its nonskeletal role, osteocalcin plays a key role in global energy regulation and adaptation in at least three ways: 1. Glucose regulation: It improves the production and secretion of, and cellular sensitivity to, insulin, as well as the rate of glucose metabolism.121, 122, 273–275 2. Fat regulation: It increases the metabolism of adipocytes.121, 122, 275 3. ATP production: It augments the number and efficiency of mitochondria both in part from its role in glucose regulation and independent of this role.121 Serum osteocalcin measures the inactive carboxylated form. When osteocalcin is decarboxylated to its active form, it enters the tissues. The less active osteocalcin is, the higher the serum levels. The more active a role it plays in global metabolism, the lower the serum level. Osteocalcin regulates and is subject to regulation by various anabolic hormones. Serum osteocalcin is inversely related to insulin-like growth factors (IGFs)276 and estrogen activity. Estrogens stimulate osteoblasts to fix calcium, which requires active, carboxylated osteocalcin, which results in a decrease in serum decarboxylated osteocalcin.277–279 The TSH levels vary inversely with serum osteocalcin levels.280–282 Serum osteocalcin is directly correlated with tumor growth in both hormone-independent and

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h­ ormone-dependent tumors.283, 284 The wide-ranging impact of osteocalcin on the structure (bones) and function (metabolism) of the body cannot be overstated, thus its key role in the BOFs, where it is involved in over 60% of the indices.

Alkaline phosphatase bone isoenzyme Alkaline phosphatases are hydrolytic enzymes that work in an alkaline environment. They hydrolyze phosphates to be (re)used in the formation of proteins and nucleotides, and in the mineralization of bone. Although present in all tissues, they are concentrated in the liver and bile ducts, bone, intestine, and placenta, for which isoenzymes have been identified.285 ALPBi is present in the plasma membrane of osteoblasts. It is an indicator of bone mineralization286 and bone turnover. ALPBi is influenced by thyrotropic hormones in managing bone density.281 ALPBi is inversely associated with the efficiency of IGFs,287, 288 but the strength of this association depends on other factors as well. ALPBi is relationship to IGFs implies a relationship between serum ALPBi and all the activities in which the IGF family plays a role, such as energy production through the regulation of glucose entry into the cell, membrane permeability, free radical production, ATP production, inflammation, etc. ALPBi is also an indicator of dysregulated growth and is associated with acute lymphocytic leukemia, Paget’s disease, and metastasis of cancer to the bone.285

Systemic enzymes Creatine phosphokinase (CPK) CPK is an enzyme that manages the ultra-acute energy needs of the body. It manages the homeostatic state between ATP and ADP and the reservoir of phosphate between creatine and phosphocreatine. Based on computer modeling paradigms and in  vitro experiments, phosphocreatine, not ATP, caries the majority of energy produced by oxidative phosphorylation out of the mitochondria into the cytoplasm.289 When the cell has sufficient ATP, it donates a phosphate to creatine, creating phosphocreatine and ADP. Phosphocreatine is a stable reservoir of phosphate. When the cell needs an immediate augmentation of ATP, phosphocreatine donates a phosphate to ADP, which then becomes ATP. During periods of sudden increases in metabolic demand throughout the body,290 and in tissues with chronically elevated energy requirements, there is increased demand for CPK to transfer phosphate from ADP back to ATP. This allows for instantaneous availability of energy without de novo ATP production.289 The enzyme CPK catalyzes both reactions (Fig. 15.2). Skeletal and cardiac muscles contain the greatest concentration of CPK as they have the greatest needs for ­ultra-acute adaptation of energy. In general, when there is

FIG. 15.2  Interconversion of ADP and ATP. The enzyme creatine kinase allows phosphocreatine to donate a phosphate molecule to ADP, quickly converting it into ATP. The reaction is reversible, allowing ATP to donate a phosphate to phosphocreatine to hold in reserve. (Illustration by Boghog2 [Public domain], from Wikimedia Commons.)

insufficient response to a metabolic demand cells die, either by apoptosis or necrosis,291 resulting, in either case, in elevated amounts of CPK in the serum. This is classically observed during exercise292 and rhabdomyolysis.293 Thus, serum CPK is proportional to the rate of muscle turnover and the metabolic role of androgens (which anabolize muscle), but not in a strictly linear way or as the sole determinant of these functions.294 Elevated CPK levels in the serum is also associated with myocardial infarction,295 but lacks sensitivity and specificity as a sole biomarker of acute myocardial infarction.296 Biomarkers such as total WBC count, total neutrophil count, and platelets increase the sensitivity of the diagnosis and risk of mortality, which is consistent with the Endobiogenic posit that multiple biomarkers are required to accurately assess complex physiologic events.297, 298 CPK levels correlate with the degree of ATP flux due to insufficiency of oxidative phosphorylation, i.e., mitochondrial strain, but again, not in a strictly linear way. As a method of assessing oxidative deficiencies, serum CPK levels alone are neither necessary nor sufficient, but one of many associated factors (cf. Redox index, below),291 as is evidenced in cases of chronic fatigue syndrome where patients have normal cytochrome enzyme activity.299 Subclinical thyroid dysfunction (SCTD) has been associated with elevated morbidity and mortality in diabetes and cardiovascular disease, both of which are disorders of deranged redox states.300–302 CPK is inversely related to thyroid metabolic ­activity303, 304 and may be elevated in hypothyroidism and SCTD. The CPK has been shown to be inversely related to free T3 and free T4 levels, both in the diagnosis and treatment of hypothyroidism.305, 306 However, in any particular patient, the correlation is not linear. This is one of a number of observations (cf. TSH, below) that lead us to conclude that quantitative expression of thyroid hormones is neither sufficiently precise nor reliable to determine the actual metabolic impact of thyroid hormones on cellular metabolism.

Lactate dehydrogenase (LDH) LDH is an enzyme that catalyzes the interconversion of pyruvate and lactate (cf. below). Aerobic respiration, using

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glucose as a substrate, is the most efficient way of ATP production in the cells. The preferential pathway in the cell is to metabolize glycogen to glucose to pyruvate. Pyruvate is then converted to acetyl-CoA, which enters the Krebs cycle. When there is an insufficiency of coenzymes in the Krebs cycle and/or oxidative stress, LDH activity increases in order to convert pyruvate to lactate. Lactate generates ATP by anaerobic metabolism, but at a much lower yield than is attained with aerobic metabolism of glucose. LDH also converts lactate back into pyruvate to produce glycogen as energy storage for future use (Fig. 15.3). LDH is present in large amounts in the liver (the direct storage site of glycogen) and cardiac muscle (a major consumer of glucose) as well as in certain tissues and RBCs, but is found in the serum at low levels. An elevation of LDH in the serum represents a state of impaired oxidation of glucose relative to demands of the organism, as seen in cardiac ischemia,307, 308 muscle turnover,291 rapid cell and tissue growth,309 hemolysis,310, 311 and cancer.312–314

Endocrine Thyroid-stimulating hormone (TSH) TSH is a glycoprotein created in and secreted from the anterior pituitary gland. In clinical medicine, TSH is considered strictly within its intra-thyroid activity of stimulation of thyroxine (T4) and triiodothyronine (T3), i.e., merely as a barometer of thyroid function. Based on more current studies and the Endobiogenic theory of terrain, serum TSH levels have key intra-and extra-thyroid implications that should also be considered if the clinical significance of a serum TSH level is to be properly contextualized. Euthyroidism is defined as normal thyroid function that occurs with normal serum levels of TSH and T4. It has been assumed that TSH and serum levels of T4 have an inverselinear relationship based on classical feedback loops, and that this relationship is a reliable indicator of the sufficiency of thyrotropic regulation of metabolism.

OH

O LDH CH3

C

There are a sufficient number of anomalies to this assumption that raise questions about its validity. For example, euthyroid sick syndrome is defined as a clinical condition with normal thyroid function with a normal TSH levels but low serum T4 and T3. Subclinical hypothyroidism is a condition in which there is a functional hypothyroid state based on an elevated serum TSH, but a normal serum T4. Subclinical hyperthyroidism is a functional hyperthyroid state based on a serum TSH value below the normal limit, but normal T4. Finally, patients with normal serum levels of TSH, T4, and T3 may presents with symptoms consistent with hypo- or hyperthyroidism. See Section “Creatine phosphokinase” for a further discussion of the functional evaluation of thyroid metabolic activity. More recent studies demonstrated that serum TSH lacks a log-linear relationship to thyroid output of free T4 (fT4) and free T3 (fT3) (Fig. 15.4). Hoermann et al. in their evaluation of 3223 untreated patients referred for thyroid testing found poor correlation (R2 = 0.236) between TSH and fT4. For example, a serum TSH of 1.0 mU/L (0.4–4.1 mU/L) was associated with an fT4 anywhere between 4 and 28 pmol/L (9.5–25 pmol/L). Conversely, a free T4 of 14.5 pmol/L was associated with TSH between 0.1 and 100 mU/L.253 In our opinion, the serum level of TSH only reflects the responsiveness of the thyroid to stimulation without determining the final degree of metabolic efficiency of T4 or T3 (cf. “CPK,” and “LDH” above) or the degree to which thyroid catabolic activity has been adapted to anabolic demands from estrogen (cf. Genito thyroid index, antigrowth index, and bone remodeling index, below). TSH has a number of extra-thyroid relationships and functions independent of T4 or T3. TSH receptors are found in divergent tissues throughout the body.315 TSH activity is augmented by estrogen.316–318 TSH is suppressed by somatostatin.319 TSH helps regulate bone density (cf. Estrogen index for a full discussion) as well.280, 281 In summary, in the theory of Endobiogeny, serum TSH is used to evaluate intra- and extra-thyroid activities. Serum TSH is not a sufficient indicator of the efficiency of thyroid regulation of metabolism, but can help contextualize thyroid function relative to the demands of the body.

COOH NADH

Pyruvate

CH3

CH

COOH

Electrolytes Potassium (K+) and calcium (Ca+) are the only two electrolytes used in the BOFs.

NAD+ Lactate

FIG. 15.3  Role of the enzyme lactate dehydrogenase (LDH). Pyruvate is the end-carbohydrate from metabolism of glucose in the cytoplasm. Pyruvate can enter the Krebs cycle. When the rate of pyruvate exceeds the efficiency of any process from the Krebs cycle to mitochondrial oxidative phosphorylation, pyruvate can be converted by LDH to lactate, which has various metabolic roles in the body. (Illustration by Jfdwolff [CC BY-SA 3.0] from Wikimedia Commons.)

Potassium Potassium (K+) is the primary intracellular (IC)IC ion in the body and serves to maintain the resting membrane potential. IC levels are around 140 mmol/L, and extracellular (EC)EC levels 4 mmol/L. It is not the quantitative concentration per

A new approach to biological modeling: Introduction to the biology of functions Chapter | 15  231

100

n = 3223

TSH (mU/L)

10

1

0.1

00.1 4

8

12

20

16

24

28

fT4 (pmoI/L) FIG. 15.4  Relationship of TSH to free T4 (fT4). See text for details (Reproduced from Hoermann R, et al. Complex relationship between free thyroxine and TSH in the regulation of thyroid function. Eur. J. Endocrinol. 2010;162(6):1123–1129. https://doi.org/10.1530/EJE-10-0106, European Society of Endocrinology.)

se but the ratio of IC to EC potassium (35:1) that maintains the resting membrane potential and neuromuscular stability. Serum potassium levels are regulated closely in order to maintain neuromuscular stability. A quantitative increase in serum potassium of 1 mmol/L can have a significant impact on neuromuscular activity.320 One source of EC potassium augmentation is glutamate. The most prominent neurotransmitter in the brain, glutamate is involved in neural plasticity and augments neuronal excitability.320a The egress of potassium from the cell changes the resting membrane potential, allowing for neurons to be more excitable.

Calcium While potassium is the element of membrane and cell stability, calcium is the element of action, movement, and variability. Calcium is the most predominant element in the human body because of its role in skeletal formation. Of total body calcium, 99% is in bones and 1% is bioavailable. Of the 1% that is bioavailable, 99.99999% is in the EC space, maintaining an EC:IC ratio of 12,000:1. Calcium reserves are extremely important to ensuring the proper ­adaptability of the organism during aggressions and programmed changes. Approximately 50% of serum calcium is ionized and bioavailable, and 50% is bound to proteins remaining in reserve. While cytoplasmic calcium levels are kept low, the mitochondrion and endoplasmic reticulum store calcium and make it available to calibrate cell function. Within the blood, calcium is the essential cofactor in the coagulation cascade. Within the interstitium, it is essential as a second messenger in muscle contraction. Calcium augments the rate of neuronal signal transduction and

n­ eurotransmitter secretion through upregulation of vesicle fusion. Within the IC space, calcium serves as a key signal transducer. In summary, both potassium and calcium concentrations are finely regulated at the EC and IC levels. Potassium is the primary IC element and maintains membrane stability. Calcium is a key element of adaptation and stimulates excitation, movement, and activity, both extra- and intracellularly. These two elements have opposing actions and overlapping factors that raise or diminish their serum concentration. Our interest in these elements with respect to the BOFs is how they regulate or are regulated by the adaptation response.

Some examples of direct indices derived from biomarkers Each function is quantified by an index, specified by a level of activity and made precise by a normative range. An index expresses the resultant efficiency of its activity in and of itself and adapted to the metabolic or tissue requirements of the organism. Related to corresponding circulating m ­ etabolites, the indices demonstrate a significant divergence. This divergence allows one to comprehend that the biological constants testify only to themselves and not to their physiological reality. On the contrary, the indices make it possible to evaluate their true level of metabolic activity: that of their production, consumption, and elimination. Their ensemble gives a very precise evolutive evaluation of the functionality: system by system, organ by organ. Christian Duraffourd and Jean-Claude Lapraz.16

232  The Theory of Endobiogeny

Definition of a direct ratio A direct index is an index that is a direct multiplication (product) or division (ratio) of biomarkers measured in the blood. The direct indexes are the basis of all the indirect indexes, which are indexes of indexes, direct or indirect in nature. As the direct indexes form the foundation of the indirect ones, the genital ratio (RBC/WBC) is the starting point of the direct indexes.

Direct indices using red and white blood cells The case for ratios We believe that when the total RBC count is evaluated relative to other factors a more nuanced appreciation of androgens in the body can be obtained. Androgens and estrogens have counterbalancing roles (Chapter  7). The quantity and quality of action as well as the chronology of action is important with respect to various disorders. Furthermore, the bioavailability of estrogens and androgens are inversely related to each other due to the role of SHBG.215, 321 Quantitative levels can be high, low, or normal, but as circulating androgens increase, the relative proportion of bioavailable estrogens decline due to an increase in SHBG, which increases its binding capacity of estrogens. The opposite is true when bioavailable estrogen levels increase in the body. Thus, in the BOFs, WBC count is also used to evaluate (through an inverse relationship) the relative rate of production and efficacy of androgens.321 When evaluating the risk of cardiovascular events, if there is absolute androgen predominance, but estrogens are also elevated, the patient may benefit from a quantitative reduction in androgens and estrogens. If there is androgen predominance but quantitative levels are low, the patient may benefit from an increase in testosterone and estrogen as

a number of large clinical trials have suggested. Regardless of the condition, by evaluating the qualitative and quantitative relationship of gonadic hormones, the BOFs provides guidance as to which clinical intervention may be most beneficial. In summary, three observations create compelling arguments for reconsidering how androgens are evaluated. The first is the contradictory nature of clinical trials with respect to quantitative androgen levels and risk of disease. The second is the multifactorial nature of disease, requiring that androgen activity be evaluated relative to other factors. Finally, the nongenomic effects of androgens may play a larger role in health and disease than previously appreciated, and these effects cannot be reliably predicted by quantitative measurements. Genital Ratio: It expresses the level of activity of tissue androgens relative to tissue estrogens. = RBC / WBC Fig. 15.5 demonstrates the upstream role of androgens, released from the gonads, where they act on the bone. The bone, as a tissue, has a metabolic response to the demands of gonadal androgens, which is to increase the production of RBCs. The output of these erythrocytes is the downstream events. The BOFs measures the quantitative downstream output and uses it as a representation of how effective the upstream regulator was in regulating metabolism, regardless of its quantitative output. Fig. 15.6 demonstrates the role of estrogens in a similar fashion. By relating the effective activity of androgens in relationship to that of estrogens, one quickly obtains an idea of the relative or qualitative achievements of these two anabolic hormones in relationship to each other and the state of metabolism. The clinical advantage was noted earlier in the discussion on the relationship of androgens and estrogens to cardiovascular disease.

FIG. 15.5  Numerator of the genital ratio: Androgen actions on the bone marrow with a downstream output of red blood cells. See text for details. (© 2015 Systems Biology Research Group.)

A new approach to biological modeling: Introduction to the biology of functions Chapter | 15  233

FIG. 15.6  Denominator of the genital ratio: Actions of estrogens on the bone marrow with a downstream output of white blood cells. See text for details. (© 2015 Systems Biology Research Group.)

Direct index using neutrophils and lymphocytes Genito-thyroid (GT) Index: The GT Index expresses the relative activity of the gonads in relationship to that of the thyroid. When elevated, it reflects an efficient thyroid activity. When low, it reflects an augmentation of TSH demand on the thyroid, regardless of absolute thyroid glandular activity. = Neutrophils / Lymphocytes Neutrophils are a biomarker of the direct role of estrogens in immunologic, inflammatory, and anabolic activity within the body.322 Lymphocyte levels are inversely related to the degree of estrogen activity in adaptation and tissue anabolism. Lymphocyte levels are directly related to the degree of appeal to TSH to regulate thyroid function. The higher the lymphocyte count, the greater the appeal to TSH is, and often the greater the degree of thyroid insufficiency. Conversely, the lower the lymphocyte count, the more successful TSH has been in modulating thyroid activity regardless of the serum TSH level.

Direct index using monocytes and eosinophils Adaptation ratio: The adaptation ratio reflects the relative activity of ACTH on cortisol in relationship to FSH’s ­activity on estrogen during the adaptation response. When the adaptation ratio is elevated, FSH activity is effectively predominant. When the adaptation ratio is low, ACTH activity is predominant. = Eosinophils / Monocytes Eosinophil count is used in the BOFs to assess the relative strength of ACTH stimulation on the adrenals (positively correlated) and the relative efficiency of cortisol

activity (inversely correlated). Eosinophilia, relative or absolute, is proportional to the degree of adrenal insufficiency in the adaptation response. The FSH stimulates estrogen production and estrogen suppresses monocyte production. The lower the monocyte count, the greater the influence of FSH and estrogen is on the adaptation response. Conversely, monocytosis reflects a relative or absolute insufficiency of estrogen’s activity during adaptation and the need for monocytes to play a role in anabolism (cf. discussion on monocytes, above).

Direct index using platelets Platelet mobilization: The platelet mobilization index expresses the adaptative liberating capacity of platelets sequestered in splanchnic vs splenic reservoirs. When the platelet mobilization index is elevated, the effects of adrenaline are augmented and favor splanchnic demargination, as the splanchnic vasculature has a greater surface area and platelet capacity than the spleen. When it is low, it reflects a relative insufficiency of adrenaline activity in adaptation. = Platelets / 60 ( RBC ) Of the total mature platelets, some are kept in reserve along the margins of the peripheral vasculature and some in the splenic sinusoids. Because of the role of platelets in serotonin transport and secretion, and the role of serotonin in gastrointestinal motility and digestion, platelets are ­ particularly concentrated in the splanchnic vasculature. During times of adaptation, adrenaline liberates, i.e., demarginates platelets in order to achieve an immediate augmentation of platelet activity without waiting for megakaryocytes to mature.322a–322h The RBCs are in the denominator of the index for a number of reasons. The RBCs, independent of adrenaline mobilization and platelet activation, stimulate the thrombosis process.322i In  vitro studies suggest adrenaline’s

234  The Theory of Endobiogeny

activation of platelets (as opposed to its mobilization) may be mediated in part by increasing the metabolic rate of RBCs, which allows them to increase the activation of platelets.323 Thus, RBCs are in the denominator because they are an activator of platelet activity but not the primary mobilizer. The greater the effects of adrenaline, the more diminished the role of RBCs are as an aid in the aggregation process. Conversely, in anemia, the lower the hematocrit (RBC ÷ whole blood volume), the greater the compensatory rise in platelets must be in order to maintain a normal rate of thrombosis. The greater the anemia, the greater the cardiac output (cf. Hemoglobin, below) to compensate for the diminished oxygen-carrying capacity.324

Direct indexes using osteocalcin, alkaline phosphatase bone isoenzyme, TSH Growth indexes In these indexes, osteocalcin is in the denominator to reflect the inverse relationship between inactive serum osteocalcin and growth. Because ALPBi is associated with growth, it is used in the numerator of indexes evaluating growth, and in the denominator of antigrowth indexes, in contrast to serum osteocalcin, which has an inverse correlation to growth, hence its role in the denominator. Estrogen index: It expresses the endocrinometabolic activity of estrogens, i.e., both the genomic activity of estrogens and the nongenomic metabolic activity within the cells. = TSH / Osteocalcin TSH levels vary inversely with serum osteocalcin l­ evels.280–282 Together, they reflect the endocrinometabolic activity of estrogens. Estrogen activity is directly correlated to the serum level of TSH.316, 325–327 Estrogens relaunch TSH, so that the catabolic activity of thyroid hormones matches the anabolic activity of estrogens.316, 328 The greater the estrogen demand and the less responsive the thyroid, the greater the serum TSH level rises, hence the role of TSH in the numerator of the index. Estrogens increase the conversion of osteocalcin to its active form to increase bone density, thus serum osteocalcin levels are inversely related to estrogen activity, hence the role of osteocalcin in the denominator.277–279 Growth index: The growth index expresses the metabolic activity of growth hormone. = ALPBi / Osteocalcin Chronic growth hormone activity increases ALPBi and reduces serum osteocalcin.287 Turnover index: The turnover index expresses the speed of renewal of tissue; its elevation implies a slowing

down of this renewal; conversely, its reduction signifies the acceleration of tissue renewal. = TSH × ALPBi

Direct index using CPK and LDH Thyroid index: The thyroid index expresses the metabolic activity of the thyroid at the cellular level. = LDH / CPK When assessing the impact of altered CPK levels on cellular metabolism, it is important to relate it to the efficiency of long-term energy production, reflected in the serum level of lactate dehydrogenase (cf. LDH). LDH participates in the conversion of glycogen to glucose for de novo production of ATP (Fig. 15.7). When cells cannot keep up with chronic metabolic needs and necrose, the level of LDH rises in the blood. Thus, LDH can be viewed as a marker of chronic metabolic strain. A person with normal serum levels of LDH and CPK can be, functionally speaking, in one of three states: a relative state of balance between chronic and acute energy management (normal ratio of LDH to CPK), a relative state of metabolic insufficiency (relatively low LDH, relatively elevated CPK), or a relative state of metabolic excess (relatively high LDH, relatively low CPK). For example, in hypothyroidism, both LDH and CPK levels are elevated compared to normal controls, but the more severe the thyroid disease, the greater the rise in CPK relative to LDH (Fig. 15.8).328a Conversely, in hyperthyroid states, the ratio of LDH to CPK is increased, but the greater the degree of hyperthyroidism, the larger the ratio becomes. (Table 15.3). It is interesting to note that in McGrowder et al.’s study, the difference in LDH between subclinical and overt hypothyroidism was not found to be statistically significant, but the difference in CPK was. The significance of the difference is only evaluating the ratio of LDH to CPK, which decreased by 57% between the subclinical and overt hypothyroid states. Other studies have shown more dramatic differences in the ratio of LDH to CPK. For example, Burnett et  al. found LDH levels to be elevated 2 times above the normal serum values, but found CPK levels to be 10–15 times above the norm, reflecting from the Endobiogenic perspective, a greater insufficiency of acute vs. chronic metabolic ­activity.303 Again, in stable coronary artery disease LDH levels are elevated to a greater degree than CPK.308 During metastasis of cancer both LDH and CPK can be elevated 10-fold or more, indicating a significant, supraphysiologic demand on the body. The ratio may be normal, but the actual global metabolic demand is elevated. Thus, both the absolute value of LDH and CPK needs to be evaluated individually and in relationship to each other, as well as other

A new approach to biological modeling: Introduction to the biology of functions Chapter | 15  235

Upstream

Downstream

T4, T3

Increased oxidative demand

Cell

??

O CH3

C

Reduced oxidative demand

OH COOH

LDH

NADH Pyruvate

CH3

CH

COOH

NAD+ Lactate

LDH

LDH

FIG. 15.7  Role of lactate dehydrogenase (LDH) in the numerator of the thyroid index. Thyroid hormones are the upstream regulator that creates a demand for ATP for cellular activity. LDH is the downstream output from the cell. LDH is in the numerator because the more the efficient thyroid hormones are in regulating cell metabolism, the greater the amount of LDH enzyme transcribed and used by the cell. (© 2015 Systems Biology Research Group.)

FIG. 15.8  Role of creatine phosphokinase (CPK) in the denominator of the thyroid index. The less effective the response of the cell to thyroid hormones, the greater the requirement for CPK to recycle ADP to ATP through phosphorylation. (© 2015 Systems Biology Research Group.)

TABLE 15.3  Relationship of LDH and CPK to thyroid activity Condition

LDH

CPK

Ratio

Hyperthyroidism

233.80

88.37

2.65

Subclinical hyperthyroidism

227.81

105.98

2.15

Normal controls

202.85

102.19

1.99

Subclinical hypothyroidism

340.38

179.80

1.89

Hypothyroidism

421.00

389.90

1.08

CPK, creatine phosphokinase; LDH, lactate dehydrogenase. Modified from McGrowder DA, et al., Serum creatine kinase and lactate dehydrogenase activities in patients with thyroid disorders. Nig J Clin Pract. 2011:14(4).

236  The Theory of Endobiogeny

determinants of cellular energy production (cf. ALPBi, LDH, and osteocalcin). In summary, the enzyme CPK is directly related to the degree of insufficiency of thyroid activity, muscle turnover, the metabolic activity of androgens, and oxidative insufficiency/mitochondrial strain. The ratio of LDH/CPK evaluates the final functional achievement of thyroid hormones in regulating the metabolic activity of the cell.

Some examples of indirect indices derived from direct indices and biomarkers The new mathematics...is one of relationships and patterns. It is qualitative rather than quantitative and thus ­embodies the shift of emphasis that is characteristic of systems thinking—from objects to relationships, from quantity to quality, from substance to pattern. Fritjof Capra.

= Genital ratio × Starter index Genital ratio = RBC / WBC = RBC × Starter index / WBC Comment: The starter index looks at the starting energy for adaptation in the splanchnic bed (c.f. Chapter  9: Somatotropic Axis, Glucagon, and, The theory of Endobiogeny, volume 2, chapter 1: The Autonomic Nervous System, BoF Indexes) (The genital ratio is corrected for the role of androgens in the sequestration vs. mobilization of elements of adaptation. Musculotrope index: expresses the relative level of endocrine and metabolic (i.e., genomic and nongenomic) activity of androgen receptors according to the balance orientation of sex hormones in osteomuscular metabolism. = Genital ratio corrected × ( CPK / ALPBi )

329

Definition of an indirect index Direct indexes evaluate specific aspects of basic physiologic relationships, such as the genital ratio (RBC/WBC). Indirect indexes are metaindexes, composed of both direct indexes, and indirect indexes—in other words, indexes of indexes. By comparing the activity of numerous factors in relationship to others simultaneously, we find several advantages. It allows for a more sophisticated evaluation of an individual’s terrain. For example, through these indirect metaindexes, one can weigh both exacerbating and protective factors related to a disorder. By opening up the metaindex and evaluating the indexes from which it is composed, one can evaluate the particular variables most implicated in the abnormal activity. Finally, one can evaluate the cumulative effect of all variables in toto. Such an approach may allow for a more precise stratification of patients based not on clinical symptoms, but on pathophysiology. This allows for a more precise treatment to be devised based on the neuroendocrine factors most responsible for an individual’s symptoms as opposed to tissue pathology of clinical symptoms alone. In summary, indirect indexes allow one to model increasingly complex aspects of metabolism based on a systems analysis approach of the terrain.

Some indirect indexes using RBCs and other factors to evaluate androgens Genital ratio corrected: expresses the basic level of activity of tissue androgens relative to tissue estrogens during the phenomenon of acute adaptation.

Comment: CPK reflects testosterone’s role on muscle turnover.294 ALPBi reflects the rate of bone turnover.286 The greater the effects of androgens, the lower the rate of bone turnover, the lower the serum level of ALPBi, thus the greater the musculotropic index value will be.330, 331

Some indirect indexes using WBCs and other factors to evaluate estrogen activity Genital ratio corrected: expresses the basic level of activity of tissue androgens relative to tissue estrogens during the phenomenon of acute adaptation. = Genital ratio × Starter index. Refer to the discussion above. Aromatization of adrenal estrogens: expresses the relative part of aromatizing activity of adrenal cortex hormones into estrogens relative to the adrenal cortex’s other activities. = Permissive cortisol index / Genital ratio corrected Permissive cortisol index = 1 / Androgenic index = 1 / ( Genital ratio corrected × Androgenic index ) The formula states that the rate of aromatization of adrenal products to estrogens is inversely related to rate of production of androgens in the body. The adrenal gland produces androgens (i.e., 17-ketosteroids) that can be converted to testosterone, dihydrotestosterone, or estrogens. The adrenal cortex’s contribution to androgens and ­estrogens are inversely related to each other. The greater the rate of conversion of these products is to estrogens, the less the availability of precursors for peripheral androgen activity. Conversely, the greater the uptake of adrenal androgens is by androgen-sensitive tissues, the less the availability of adrenal androgens is to be converted to estrogens.169, 332–334

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Some indirect indexes using neutrophils and other factors to evaluate estrogen activity Throughout the various indexes, estrogen activity is evaluated in relationship to adrenal activity (discussed above; c.f. RBC, WBC, monocytes, and eosinophils), thyroid activity (cf. lymphocytes, TSH, LDH, CPK, and osteocalcin), as well as its competitive, cooperative, and additive function with respect to anabolic factors such as progesterone, androgens (cf. RBC, monocytes, CPK), and somatotropic growth factors (cf. TSH, osteocalcin, alkaline phosphatase). Cortisol index: expresses the functional activity of cortisol from the adrenal cortex and its excretion during syndromes of adaptation.

production, and adrenal androgen production.339, 340 Due to the extensive role of cortisol in human physiology, the eosinophil count contributes to the assessment of atopic disorders, various aspects of cellular metabolism such as apoptosis, necrosis, and membrane permeability, as well as histamine expression,108, 341–349 inflammation,350 coagulation,350 immune function, carcinogenesis, and cancer survival.351–364 As always, the role of eosinophils needs to be evaluated relative to other factors. Cortisol index: We have already discussed this index, but present it from a different perspective to reveal the role of eosinophils in the formula. = ( Catabolism / Anabolism index ) /

= ( Catabolism / Anabolism index ) / Adaptation index In the numerator is the catabolism/anabolism index which evaluates the relative rate of catabolic activity in relationship to that of anabolism. Cortisol primarily favors catabolism thus this index is in the numerator. In the denominator is the adaptation index, which is eosinophils/ monocytes (cf. adaptation index). Recall that cortisol stimulates the apoptosis of eosinophils. Thus, the lower the adaptation index, the greater the activity of cortisol is effectively, and the higher the index will be. Rate of anabolism: expresses the rate of anabolic activity of the body. = Catabolism index / ( Catabolism / Anabolism index ) Catabolism index = Thyroid index / Corticoadrenal index Comment: Recall how the catabolism/anabolism index evaluates the relative balance of metabolic activity. It is in the denominator because the smaller the index, the more it favors anabolism. However, in order for anabolism to occur, there must be some catabolism which provides material to “nourish anabolism” as we say in Endobiogeny. Thus, the numerator is the catabolism index. The catabolism index is itself composed of two indexes: thyroid and corticoadrenal index (i.e., index of global adrenal cortex function). The thyroid index was discussed earlier. The greater the activity of peripheral thyroid hormones are, the greater the provision of catabolic material for anabolism. However, this is truly only in the case that adrenal cortex activity is not too excessive. In this case, it blocks anabolism, which is reflected in a smaller value of the index.

Some indirect indexes using eosinophils and other factors Eosinophil count contributes to the evaluation of various aspects of adrenal physiology, such as circulating cortisol, permissive and adaptive cortisol activity,219, 335–338 DHEA

Adaptation index Adaptation = Eosinophils / Monocytes = ( Catabolism / Anabolism index ) /

( Eosinophils / Monocytes ) = ([ Catabolism / Anabolism index ] × Monocytes ) / Eosinophils Comment: The lower the eosinophil count is, the greater the role of cortisol secretion during adaptation, the more efficacious cortisol is in its antiinflammatory and antiallergic capacity, which is consistent with the clinical literature, as noted above, hence the placement of eosinophils in the denominator. Evoked histamine index: expresses the circulating rate of active histamine. = ( Eosinophils × Platelets × Adaptation index ) / Adrenal Cortex index Adaptation index = Eosinophils / Monocytes =  Eosinophils × Platelets × ( Eosinophils / Monocytes )  / Adrenal cortex index

(

)

= Eosinophils2 × Platelets /

( Adrenal cortex index × Monocytes ) Adrenal cortex index = Cortisol index / Androgenic index

(

= Eosinophils2 × Platelets × Androgenic

)

/ ( Cortisol index × Monocytes ) As noted above, eosinophils are an indirect source of histamine secretion; the greater the relative or absolute percent eosinophils, the lower the rate of circulating cortisol, the greater the rate of circulating histamine. As the formula suggests, other factors modulate the threshold of histamine

238  The Theory of Endobiogeny

production, thus eosinophilia alone is not sufficient to account for the total amount of circulating histamine. The greater the estrogen activity (low monocytes), the greater the release of histamine independent of the antihistaminic effects of cortisol.365 Neither estrogen nor testosterone alone have been sufficient to account for histamine secretion in human models, thus suggesting that a multifactorial assessment of factors related to histamine secretion will be more accurate in assessing the histamine burden in the body.366

Some indirect indexes using lymphocytes and other factors Catabolism/Anabolism index: It expresses the relative part of activity of catabolism of the organism in relationship to its anabolic activity. = Genito  thyroid index / Genital ratio corrected Genito  thyroid index = Neutrophils / Lymphocytes = Neutrophils / ( Genital ratio corrected × Lymphocytes ) Anabolism index: It expresses the level of anabolic activity of the organism. = Catabolism index / ( Catabolism / Anabolism index )  Catabolism index × Genital ratio corrected  =   ×Lymphocytes  / Neutrophils The anabolism index evaluates the absolute rate of anabolism as a result of corticotropic, gonadotropic, and thyrotropic considerations of relative and absolute activity. (cf. catabolism-anabolism index under “Indirect indexes using neutrophils” and the catabolism index under “Indirect indexes using LDH or CPK” for a further discussion). A low rate of catabolism in and of itself does not mean that the rate of anabolism is low. Each level of activity can be elevated, low, or normal. The anabolism index seeks to evaluate the quantitative rate of anabolisms. The catabolism index as a quantitative assessment of catabolism is in the numerator. The lower the absolute rate of catabolism, the greater the predominance of anabolism may be. However, the relative rate of catabolism to anabolism rate the greater the predominance of anabolism. As noted above, the higher the lymphocyte levels, the less well adapted the thyroid is in its catabolic activity, thus the lower the rate of catabolism will be. The greater the genital ratio corrected, the greater the predominance of ­androgens relative to estrogens in adaptation, which favors the completion of anabolism. Apoptosis index: It expresses the general level of apoptotic activity of the organism in its entirety.

= Structural expansion index / Membrane expansion index Structure expansion index = Anabolism index × Nucleomembrane activity index Membrane expansion = Catabolism index × Growth index corrected = ( Anabolism × Nucleomembrane activity index ) / ( Catabolism index × Growth index corrected ) Apoptosis was first described in 1847. For 140 years (1847–1987), the study of apoptosis was morphologic in nature. From 1988, with the discovery of bcl-2 protein, the genetic mechanisms of apoptosis have been the primary focus of study.367 From the Endobiogenic perspective, because the endocrine system manages the rate of metabolism of the cell, it mediates the life of the cell and the time of apoptosis or necrosis or lack thereof, such as with cancer cells. The plethora of pro- and antiapoptotic signaling factors are the means of regulating apoptosis and while interesting, are not the determinant of when and to what degree of intensity apoptosis occurs (or does not). The validity of such an index would allow for a global approach to managing apoptosis that is concordant with the general scheme of factors related to cancer growth, and away from the endless search for “silver bullets” in pharmacotherapy—natural or synthetic—that are highly target with respect to specific mechanisms of apoptosis, but carry the risk of potentially more serious side effects. The numerator is composed of the Anabolism index and the Nucleomembrane index. The greater the numerator, the greater the rate of apoptosis is. Cell growth occurs as a result of anabolism, which requires increased activity at the level of the nucleus with respect protein transcription (represented by the Nucleomembrane index) relative to membrane activity. The greater the anabolic activity of the cell, the sooner it will reach the end of its programmed number of division, and hence die by apoptosis. The denominator is composed of the membrane expansion index, which is itself composed of the product of the catabolism and the growth index corrected indices. When there is catabolic predominance,368, 369 and/or elevated IGF activity370, 371 the membrane expands.372 A greater rate of membrane expansion relative to that of structural activity implies that more energy is devoted to cellular hyperplasia than to cellular divisions, hence the longer it takes for the cell to die due to reaching its programmed time of death. In summary, the endocrine system is the regulator of apoptosis, while pro-apoptotic proteins are the mechanism of apoptotic cell death. From the Endobiogenic perspective, an endocrine approach to the evaluation of the global physiologic rate of apoptosis allows one to evaluate the reason for apoptosis (or its insufficiency) and to pinpoint the causative

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factors, and thus allows for a clinical plan to address these particular imbalances. In contrast, merely enumerating the number of pro- or anti-apoptosis factors active does not at this time offer a path of clinical intervention.

Some indirect indexes using platelets and other factors Evoked histamine index: We reproduce the formula again to discuss the role of platelets. = ( Eosinophils × Platelets × Adaptation index ) / Adrenal cortex index Adaptation index = Eosinophils / Monocytes = ( Eosinophils × Platelets × Eosinophils ) / ( Adrenal cortex × Monocytes )

(

)

= Eosinophils2 × Platelets / ( Adrenal cortex × Monocytes ) The quantitative amount of histamine in platelets is less than that found in leukocytes.373 However, because platelets activate neutrophils and monocytes on the endothelial surface of the vasculature, and due to their 25-fold numerical superiority to leukocytes, they play an amplifying role in inflammatory disorders374 well characterized by the actions of eosinophils and basophils.375 This is reflected in the formula of the histamine index. The greater the numerator is, the greater the numerical value of the index and thus the greater role of pro-histaminic elements relative to antihistaminic elements. In the numerator is the product of two factors: Eosinophils2 × Platelets The normal value of percent eosinophils is 0.1–7, square of the norm is 0.12–72, or 0.01–49. Conversely, the value of platelets is 100–400 (cf. Table 15.1 for conversion factor). Thus, mathematically, and it is proposed that biologically, the role of platelets in histamine expression is 2–10,000fold greater than that of eosinophils, balanced of course by the relative effects of antihistaminic factors expressed in the denominator.

Some indirect indexes using osteocalcin Antigrowth indexes In these indexes, osteocalcin is in the numerator to reflect the relationship between inactive serum osteocalcin and antigrowth activity. Pro-amyloid index: expresses the level of IC hypometabolism. By extension, it evaluates the degree of insufficiency of cellular respiration (i.e., mitochondrial efficiency in the production of ATP by oxidative phosphorylation).

By extension, it evaluates the degree of cellular nutritional insufficiency. = Index of reduction × Insulin resistance index Comment: Osteocalcin is in the denominator of the insulin resistance index, itself in the denominator of the formula, placing osteocalcin in the numerator of the global formula. Recall that serum osteocalcin is inactive osteocalcin, and that the greater the level of inactive osteocalcin, the less optimized mitochondrial function and insulin activity will be. With respect to the composition of the formula, the index of reduction is in the numerator. The greater the rate of reductive capacity, the less the relative potential of oxidation will be of carbohydrates or fats.376 The index of insulin resistance is in the denominator. The greater the degree of insulin resistance, the less glucose is available for oxidation.377 Thus, the pro-amyloid index evaluates the degree of cellular nutritional insufficiency (insulin resistance) and insufficiency of material for cellular respiration (index of reduction). It is called the pro-amyloid index because the greater the degree of mitochondrial insufficiency, the more likely the organism will be to rely on proteins such as amyloid proteins as an alternate form of energy.378 Antigrowth index: expresses the global level of activity of the ensemble of antigrowth factors (cf. TSH for a full discussion). = 1 / Growth index corrected Growth index corrected = Growth indeex / Turnover Growth index = Alk Phos bone isoenzyme [ ABPi ] / Osteocalcin Turnover = APBi × TSH = ( ABPi / Osteocalcin ) / ( APBi × TSH ) = Osteocalcin × TSH Comment: Osteocalcin is as a pro-growth factor at the tissue level. Serum osteocalcin is inversely related to its activity at the tissue level because it measures the inactive form of osteocalcin. The higher the serum osteocalcin level, the less pro-growth activity at the tissue level. Thyroid hormones favor growth by increasing the metabolic rate of the cell. The greater the serum TSH, the less responsive the thyroid is to stimulation thus the less well-calibrated growth activity is at the cellular level. While this index evaluates the relative insufficiency of pro-growth factors (it is the inverse of the growth index corrected), it can be considered as an indirect evaluation of antigrowth factors such as leptin, resistin, and other antigrowth factors that are known to directly oppose the effects of alkaline phosphatase and osteocalcin.379, 380

240  The Theory of Endobiogeny

Somatostatin index: expresses the level of activity of somatostatin; indirectly, it witnesses the relative level of activity of the exocrine pancreas. = Antigrowth index / Cortisol index Comment: Somatostatin inhibits growth hormone381–384 and cortisol inhibits somatostatin. This index is a good example of how one type of endocrine activity—in this cortisol—is contextualized and oriented toward a completely different hormonal activity—somatostatin—but its evaluation relative to another series of activities—antigrowth factors. The lower the degree of circulating cortisol in the face of antigrowth activity, the less inhibition of somatostatin there must be, and therefore the greater the relative predominance of somatostatin activity is presumed to be.385–388

An indirect indexes using CPK and LDH Catabolism index: This was discussed earlier under section “Anabolism index.” It expresses the level of catabolic activity of the organism. = Thyroid index / Adrenal cortex index Thyroid index = LDH / CPK = LDH / ( Adrenal cortex index × CPK )

Two indirect indexes using TSH and other biomarkers Thyroid yield: It expresses the relative part of thyroid metabolic activity in relation to its level of solicitation by the pituitary. By extension, it contributes to an evaluation of the threshold of response of the thyroid to pituitary solicitation. = Thyroid index / TSH Thyroid index = LDH / CPK = ( LDH / CPK ) / TSH = LDH / ( TSH × CPK ) Comment: The thyroid index evaluates the functional metabolic impact of thyroid hormones on the rate of cell metabolism, as discussed under section “Creatine Phosphokinase.” By assessing this activity relative to the serum TSH level, the thyroid yield index evaluates how readily the pituitary can adapt the thyroid. For example, a patient with a normal thyroid index (irrespective of serum fT4 and fT3) but a very low TSH (i.e., 0.1 mU/L) has a thyroid that is quickly regulated by the pituitary and is at risk of overadapting thyroid activity relative to the degree of solicitation. Consider a terrain in which the effects of thyroid hormone activity are 150% optimal. The thyroid index is 8.25 (3.5–5.5). When TSH stimulates the thyroid gland, the gland responds quickly resulting in a rapidly inhibited TSH

at 2 (0.5–4.5 μIU/mL). The thyroid yield is 8.25/2 = 4.125 (2–3). What is being evaluated is the functional IC yield of thyroid hormones relative to the efficiency of response to TSH. The interpretation is that thyroid activity is elevated and the gland is sensitive to stimulation and responds 2–3 times too quickly. In other words, the current TSH value is normal but too elevated for this patient. Despite having a normal serum TSH, a medicinal plant that slows down TSH stimulation of the thyroid, such as Lycopus europaeus (Gipsywort), would be recommended. Now consider a situation where the thyroid activity is 80% of optimal with a thyroid index value of 2.8. When TSH stimulates the thyroid gland, it also responds too quickly resulting in a rapidly inhibited TSH at 0.66. The thyroid yield is 2.8/0.678 = 4.125, the same elevated thyroid yield as in the first case. Here, the interpretation is that the thyroid gland responds quickly to stimulation but there is something at the peripheral level that impairs optimal regulation of cellular metabolism regardless of the quantitative output of thyroid hormones from the gland. In this case, while inhibition of TSH with Lycopus europaeus is recommended, so is improvement of peripheral sensitivity to thyroid hormones, such as Zingiber officinale (Ginger). The thyroid yield index can offer clinicians new insights into how to approach clinical symptoms and how to reconcile it with laboratory data. It relies not on serum levels of T4 and T3, but on this subtler functional assessment, regardless of the quantitative output of thyroid hormones. Bone remodeling index: It expresses the level of bone remodeling and the degree of alteration of bone and cartilage; it also testifies to the general level of metabolism and, in particular, its activity in adaptation. = Turnover index / Osteocalcin = ( TSH × ALPBi ) / Osteocalcin Comment: As discussed under section “Bone stromaderived enzymes,” the bone plays a significant role in energy regulation. The rate of bone turnover can be viewed, according to the theory of Endobiogeny, as a reflection of global rate of cell turnover. The higher the turnover index, the lower the rate of peripheral cell turnover is in the body. Elevated serum TSH implies a lack of thyroid activity at the tissue level, and may implicate inefficient thyroid regulation of the metabolic rate of the cell (cf. “thyroid yield,” above). Elevated ALPBi is directly related to the degree of bone turnover from osteoblastic activity.286 It indirectly implicates according to the theory of Endobiogeny, an increased demand on the bone stroma to assist in the regulation of global energetic requirements of the organism.120–122 Osteocalcin regulates mitochondrial activity, estrogen sensitivity, and insulin sensitivity,121, 122, 275 which can augment the energetic and anabolic capacity of the cells. Serum osteocalcin is inversely related to these effects of

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o­ steocalcin, hence its role in the denominator [cf. Estrogen index (Osteocalcin) for a full discussion).

Structure and function values Because the terrain operates under both basal and adaptive demands, Dr. Duraffourd developed a method for distinguishing the effects of function (adaptation) vs. structural (basal) metabolic activity. All lab values and initial results of indexes represent functional values. Using the starter index, which evaluates how the organism adapts to aggressions, the values of function can be extrapolated back into structure. Some indexes, such as the thyroid index, have only function values. Most, though, have structure and function values. This is valuable because it allows the Endobiogenist to evaluate the modalities of function and determine if the origin of the problem related to the maintenance of structure of functional adaptation demands. It can also help offer more precise recommendations for lifestyle modification, such as meditation vs. aerobic exercise. One may find indexes in which the structure value is absolutely high and function value absolutely low, or vice versa. From this, two questions often arise. First, “which value is ‘correct?,’” and “Which value should be treated? The elevated or diminished value?” Assuming that the data input is correct and within reasonable variation form the normal value (cf. below), both values are “correct.” They reflect serious discrepancies in the capabilities of the organism in structural and functional achievements. The area of emphasis in the evaluation and the choice of treatment will be based on an assessment of whether the disorder is one of a structural or functional disadaptation. When in doubt, use adaptogenic plants.

Comparison to historical and physical exam findings If the Endobiogenist has performed a detailed history and physical examination and has interpreted it correctly, their findings will correlate very well with the BOFs. However, it should be understood that each method of evaluation has different parameters and offers different levels of information. Therefore, discrepancies can be seen. For the novice Endobiogenist, whenever there is doubt between the conclusions derived from these three methods, treat based on the conclusions of the BoF. This maxim by no means contradicts or diminishes the value or necessity of history and physical exam. Ideally, vis-à-vis the BOF, they will contextualize the findings of the BoF and allow for a more precise selection of treatment. Recall that history and ­physical examination produce unique information that cannot be derived from the BoF, such as past history or emunctory function.

The optimal integration of history, physical examination, and BoF are illustrated in two cases of hypothyroidism. Two 48-year-old premenopausal women develop Hashimoto’s thyroiditis. The first woman developed it after a single, intense traumatic experience: the death of her beloved husband. The duration and intensity of treatment will be relatively shorter as long as she has worked through the grief of her husband’s death. The second woman developed her thyroiditis at the culmination of a divorce from her verbally abusive husband of 28 and 18 years of living with her verbally abusive father before that. In this case, thyroid oversolicitation is more entrained. The second patient will likely require a longer, more intensive treatment that addresses central and peripheral factors. The role of the BOFs is to determine precisely the therapies most targeted to the terrain. The role of the history and physical examination will be to determine the duration of treatment and buffering capacity of the organism.

Some words of caution about values of indices The BOFs is a modeling system that characterizes particular aspects of the management of the terrain—namely neuroendocrine management. It does not directly measure physiologic activity. There are many other aspects of biologic existence that it does not measure: electrochemical, electromagnetic, communication networks, van der Waal charge interaction, conformational binding efficiency of proteins, etc. What the BoF does do is evaluate and calculate neuroendocrine management based on three very particular assumptions: (1) The endocrine system is the true manager of the terrain and the autonomic nervous system modulates the endocrine system. (2) Biomarkers reflect the downstream metabolic output of upstream neuroendocrine management of terrain.1 (3) Biomarker values input into the system do not deviate significantly from their normative values. Stipulation 3 is most crucial when the patient has a TSH <0.5 μIU/mL or >5 μIU/mL. In these cases—often due to iatrogenic dosing of thyroid medication—a large percentage of indexes may be accurate in the general picture they present, but not precise with respect to their numerical value. For example, consider the estrogen index whose formula is TSH/osteocalcin (adult female 0.2–0.4). Note how as the TSH varies for the same osteocalcin,2 the results deviate from the normative value (Table 15.4). In the particular case of the estrogen index, there are compensatory formulas for calculating aspects of estrogen

1. The only exception to this point is serum TSH. It has both upstream and downstream activities.

242  The Theory of Endobiogeny

TABLE 15.4  Evaluation of the estrogen index with variable TSH values and stable osteocalcin value Estrogen index (0.2–0.4) TSH

Osteocalcin

High

7.5

5

1.5

3

5

0.6

1.5

5

0.5

5

0.1

0.1

5

0.02

activity that do not involve TSH or osteocalcin. Because the BOFs is composed of formulas, the output is only as accurate as the input data. ● ● ●

When TSH values are <0.5 μIU/mL enter a value of 0.5 When TSH values are >5 μIU/mL enter a value of 5 On follow-up studies, for the prior evaluation, reenter the original measured TSH in order to evaluate the relative change in terrain. This is because numerous other biomarkers will also change as the TSH has changed.

Some advice on the interpretation of indices The BOFs is a tool to aid the physician in the assessment of the terrain and its evolution or devolution over time based on the effects of time itself, treatment, or both. It is not a stand-alone method of diagnosing or treating patients. It is not a binary system where it is sufficient to see how many results are high, low, or normal. When using the BOFs in the Endobiogenic evaluation remember two basic principles in order to do no harm: 1. Interpret a single index relative to one or more other indices. 2. When in doubt as to the root cause of illness, treat symptoms. With respect to point one, we will illustrate an evaluation of thyroid function. The thyroid metabolic index evaluates the metabolic impact of peripheral thyroid hormones (T4, T3). What does it mean if the value is within the normal range and what are the therapeutic implications? One must evaluate this level of peripheral thyroid hormone function relative to the thyroid yield, as we discussed above. Avena sativa (Wild oat) and Zingiber officinale (Ginger) are two possible plants that can improve the thyroid yield. But, which of these two is most targeted for this patient?

Normal

Low

0.3

One may expand the evaluation to look at genital function. If the GT index (neutrophil/lymphocytes) is low, Avena sativa is more targeted. If there are insufficient genital androgens, Zingiber officinale is more targeted. Let us say that the thyroid index is normal and you wish to contextualize its meaning to two other indexes: thyroid yield and Genitothyroid. There are 9 possible combinations of interpretations. Table 15.5 demonstrates an Endobiogenic method of analysis of three indexes, assuming that one is normal and the other two vary between elevated, normal or low. The evaluation can be further expanded to look at the role of the thyroid in general metabolic activity relative to the adrenal cortex. Based on this evaluation, further considerations can be made in the assessment of terrain and the choice of treatments. The algorithm will result in a different combination of therapeutic choices based on if the thyroid metabolic index is low or elevated. If we consider three possible values for each index (normal, high, low), then an evaluation of three indices vis-à-vis each other has 27 possible c­ ombinations: 9 for each permutation of the thyroid metabolic index TABLE 15.5  Evaluation of three indexes where one (Thyroid index) is normal Thyroid yield index

Genito-thyroid index High

Normal

Low

High

Lycopus europaeus

Lycopus europaeus

Zea mais

Normal

Depends on other factors

No treatment indicated

Vitex agnus castus

Low

Depends on other factors

Avena sativa Zingiber officinale

Avena sativa Zingiber officinale

2. The osteocalcin value is the adjusted value based on a proprietary formula that accounts for variations in the osteocalcin value between various laboratories.

A new approach to biological modeling: Introduction to the biology of functions Chapter | 15  243

(low, normal, or elevated). Because of the polyvalent action of medicinal plants, there are typically <27 possible ­combinations of medicinal plants. For example, if the thyroid metabolic index is normal, and thyroid yield is low and there is insufficient adrenal cortex activity and there are insufficient gonadal androgens and digestive issues and/or inflammation, Zingiber officinale as a single plant would be highly efficient in addressing all these issues in the same patient.

BoF and therapy The biology of functions makes it possible to determine the pathogenic tendencies of the organism, the stage of evolution and the eventual degree of pathology. It makes it possible to follow spontaneous evolution and evolution under therapy and to adjust this treatment as well as possible. Christian Duraffourd and Jean-Claude Lapraz.16

The outcome of every consultation is precise medical guidance with regard to therapy, lifestyle, alimentation, disease risk, prognosis, and disease prevention. The BOFs allows for an evolutive assessment of all these factors because it evaluates the terrain of each individual in its basal structural and functional activities in-and-of-themselves and vis-à-vis each other. Along with an Endobiogenic history and physical examination, the BOFs offers for the first time in the history of medicine, an approach to treatment that is precise and targeted to each individual at the particular time of evaluation. It is rooted in an objective yet qualitative assessment of terrain and is no longer limited to symptomatic considerations. Consider a condition as “simple” as acne. While there are common elements in the terrain that bring out acne, there are different reasons for these structural factors of initiation to occur, and different secondary factors that influence the location and type of acne. For example, the treatment of acne that appears across the forehead and upper back in a 16-year-old boy will be different from that appearing on the nasolabial folds in a 40-year-old woman. In both cases, there is hepatobiliary and colonic congestion and a relative insufficiency of gonadal androgens with hyperluteal response. In the case of the boy, there is an appeal to the adrenal cortex by ACTH to assist in puberty by aromatizing DHEA to gonadal androgens. The appearance of acne on the forehead and upper back witnesses overexpression of ACTH to achieve this goal. In the case of the woman, as she is undergoing a genital recycling at 40, the organism has developed a relative genital insufficiency. There is an appeal by FSH to relaunch estrogens. The estrogens are relaunching LH to stimulate a compensatory ­gonadal ­androgen response, which does not occur. This causes a further FSH activity for two reasons. The first is to further increase estrogens to stimulate LH. The second reason is a direct horizontal pituitary

stimulation of LH by FSH. The appearance of the acne around the nasolabial folds witnesses this FSH activity (cf. The Theory of Endobiogeny, Volume 3 Chapter 8 and Volume 4)

Conclusions There is nothing new in the tendency to take obvious things for granted and to postpone logical thought…For many centuries we were satisfied to accept life itself without questioning and without inquiring as to its beginnings, variations and potentialities. Now we have some desire of understanding how life began, of its continuation and limitations. Manfred Sakel, MD.389

Mathematics is a universal language that provides a quantitative, rational, objective description of events. Mathematics has been used since antiquity but was not applied to human physiology until contemporary times. The use of ratios allows for a qualitative element of description to add more depth of information to quantitative data. The BOFs is derived from the theory of Endobiogeny and its integrating vision of physiology. Endobiogeny differs from other systems-biology approaches in three key areas. First, it maintains a global vision of the organism in toto, rather than focusing on activity exclusively at the cellular or subcellular levels. Second, it considers the endocrine system rather than genes to be the manager of the body. Third it seeks to characterize both the reason and mechanisms of disease, the why and the how, rather than exclusively focusing on the mechanisms. The BOFs is a modeling system that evaluates the quantitative and qualitative aspects of management of the terrain by deducing upstream actors from the downstream output that enters from tissues into the blood. Since the neuroendocrine system is the effective manager of the global terrain, the elements selected to model functioning of the terrain are neuroendocrine determinants of metabolism. It differs from current approaches to biological modeling in key ways. It models, it does not measure actual biological activity. It is not focused exclusively on cellular activity—it integrates general cell function into the global management of the terrain. It does not evaluate genetics, but expressed phenotypic activity in space and time. Finally, it was created by clinicians with a particular concern for clinical relevance. The relationship between downstream biomarkers and neuroendocrine elements has been present in a scattered way throughout medical literature for nearly 100 years. The absence of a comprehensive, coherent, and internally consistent theory presented researchers and clinicians alike from integrating this information to create a system such as the BOFs. In effect, everyone saw the pearls, but no one knew how to string them together before the theory of Endobiogeny. There is emerging clinical evidence of the robustness of the BOFs indexes in describing the pathophysiology

244  The Theory of Endobiogeny

of well-characterized states.322, 390–393 Further research is required to determine the predictive and prognostic capabilities of the system. In clinical practice, when a trained Endobiogenist is able to take a full history and perform an in-depth physical examination, the BOFs can confirm the first two, and yet offer new information not available using any other method described from antiquity to present. The BOFs offers a new approach to integrative physiology that is complex, dynamic, and completely relevant to the clinical management of patients3.

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17. Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001;69(3):89–95. 18. Vasan  RS. Biomarkers of cardiovascular disease: molecular basis and practical considerations. Circulation. 2006;113(19):2335–2362. 19. Cole M, Rutherford RB, Smith FO. Experimental ammonia encephalopathy in the primate. Trans Am Neurol Assoc. 1970;95:82–85. 20. Cole M, Rutherford RB, Smith FO. Experimental ammonia encephalopathy in the primate. Arch Neurol. 1972;26(2):130–136. 21. Bernal W, Hall C, Karvellas CJ, Auzinger G, Sizer E, Wendon J. Arterial ammonia and clinical risk factors for encephalopathy and intracranial hypertension in acute liver failure. Hepatology. 2007;46(6):1844–1852. 22. Haussinger D, Schliess F. Pathogenetic mechanisms of hepatic encephalopathy. Gut. 2008;57(8):1156–1165. 23. Lee  DH, Lim  JS, Song  K, Boo  Y, Jacobs Jr. DR. Graded associations of blood lead and urinary cadmium concentrations with ­oxidative-stress-related markers in the U.S. population: results from the third National Health and nutrition examination survey. Environ Health Perspect. 2006;114(3):350–354. 24. Shi  M, Gozal  E, Choy  HA, Forman  HJ. Extracellular glutathione and gamma-glutamyl transpeptidase prevent H2O2-induced injury by 2,3-dimethoxy-1,4-naphthoquinone. Free Radic Biol Med. 1993;15(1):57–67. 25. Lee DH, Jacobs Jr. DR. Association between serum concentrations of persistent organic pollutants and gamma glutamyltransferase: results from the National Health and examination survey 1999-2002. Clin Chem. 2006;52(9):1825–1827. 26. Lee DH, Steffes MW, Jacobs Jr. DR. Can persistent organic pollutants explain the association between serum gamma-­glutamyltransferase and type 2 diabetes? Diabetologia. 2008;51(3):402–407. 27. Lee DH, Silventoinen K, Jacobs Jr. DR, Jousilahti P, Tuomileto J. Gamma-Glutamyltransferase, obesity, and the risk of type 2 diabetes: observational cohort study among 20,158 middle-aged men and women. J Clin Endocrinol Metab. 2004;89(11):5410–5414. 28. Nemes  Z, Devreese  B, Steinert  PM, Van Beeumen  J, Fesus  L. Cross-linking of ubiquitin, HSP27, parkin, and alpha-synuclein by gamma-glutamyl-epsilon-lysine bonds in Alzheimer's neurofibrillary tangles. FASEB J. 2004;18(10):1135–1137. 29. Yates CM, Butterworth J, Gordon A. Gamma-glutamyl transpeptidase in dementia. Neurobiol Aging. 1989;10(1):107–108. 30. Butterworth  J, Yates  CM, Reynolds  GP. Distribution of phosphate-­ activated glutaminase, succinic dehydrogenase, pyruvate dehydrogenase and gamma-glutamyl transpeptidase in post-mortem brain from Huntington's disease and agonal cases. J Neurol Sci. 1985;67(2):161–171. 31. Loomba  R, Rao  F, Zhang  L, et  al. Genetic covariance between ­ gamma-glutamyl transpeptidase and fatty liver risk factors: role of beta2-adrenergic receptor genetic variation in twins. Gastroenterology. 2010;139(3):836–845 [845 e831]. 32. Bidel S, Silventoinen K, Hu G, Lee DH, Kaprio J, Tuomilehto J. Coffee consumption, serum gamma-glutamyltransferase and risk of type II diabetes. Eur J Clin Nutr. 2008;62(2):178–185. 33. Lee D, Jacobs DR, Gross M, et al. Gamma-Glutamyltransferase is a predictor of incident diabetes and hypertension: the coronary artery risk development in young adults (CARDIA) study. Clin Chem. 2003;49(8):1358–1366.

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