Predict disease with WES – ask your doctor to request this DNA lab test

Dear Health Consumer,

When you are ready to have a better understanding of your human genome responsible for a majority of known disease-related variants that can help you and your doctor monitor and predict your health and be proactive with disease related challenges in the future to achieve a better health outcome, you may use this letter.

A letter for your doctor to request the WES DNA lab test



Connie Dello Buono


1708 Hallmark Lane San Jose CA 95124


Disease prediction, senior care and health concierge



Exome sequencing provides a cost-effective alternative to whole genome sequencing as it targets only the protein coding region of the human genome responsible for a majority of known disease related variants. Whether you are conducting studies in rare Mendelian disorders, complex disease, cancer research, or human population studies, Novogene’s comprehensive human whole exome sequencing service provides a high-quality, affordable and convenient solution.

Novogene’s bioinformatics analysis includes data QC, mapping with reference genome, SNP/InDel, somatic SNP/InDel calling, statistics and annotation. Novogene utilizes internationally recognized software in bioinformatics analysis, e.g. BWA, SAMtools, GATK, etc.

In particular, Novogene bioinformatics pipeline includes annotation with the exome aggregation consortium (ExAC). ExAC dataset spans 60,706 unrelated individuals sequenced as part of various disease-specific and population genetic studies. This population scale database greatly facilitates research of disease pathogenesis.

The Novogene Advantage

  • Unsurpassed data quality: We guarantee a Q30 score ≥80%, exceeding Illumina’s official guarantee of ≥75%. See our data example.
  • State-of-the-art exome capture: Agilent SureSelect Human All Exome V6 (58 M) is used.
  • Accurate variant calling with longer read length up to 150 bp.
  • Extraordinary informatics expertise: Novogene uses its cutting-edge bioinformatics pipeline and internationally recognized best-in-class software to provide customers with “publication-ready data”.

Project Workflow

Exome Sequencing Service Project Workflow

Exome Capture

  • Agilent SureSelect Human All Exon V6 Kit

Sequencing Strategy

  • 180~280 bp insert DNA library
  • HiSeq platform, paired-end 150 bp

Data Quality Guarantee

  • We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, which exceeds Illumina’s official guarantee of ≥ 75%.

Sample Requirements

  • Input DNA:
    • For fresh sample: ≥ 1.0 μg (a minimum of 200 ng can be accepted with risk)
    • For FFPE sample: ≥ 1.5 μg
  • DNA concentration: ≥ 20 ng/μl
  • DNA Volume: ≥ 10 μl
    • OD260/280 = 1.8 – 2.0 without degradation or RNA contamination

Turnaround Time

  • Within 22 working days after verification of sample quality (without data analysis)
  • Additional 5 working days for data analysis

Recommended Sequencing Depth

  • For Mendelian disorder/rare disease: effective sequencing depth above 50×
  • For tumor sample: effective sequencing depth above 100×

Analysis pipeline

Exome Sequencing Service Analysis Pipeline

Advanced Analysis

Monogenic disorders

1. Variant filtering
2. Analysis under dominant/recessive model (Pedigree information is needed)
2.1 Analysis under dominant model
2.2 Analysis under recessive model
3. Functional annotation of candidate genes
4. Pathway enrichment analysis of candidate genes
5. Linkage analysis
6. Regions of homozygosity (ROH) analysis

Complex/multifactorial disorders

1. Variant filtering
2. Analysis under dominant/recessive model (Pedigree information is needed)
2.1 Analysis under dominant model
2.2 Analysis under recessive model
3. Functional annotation of candidate genes
4. Pathway enrichment analysis of candidate genes
5. De novo mutation analysis (Trio/Quartet)
5.1 De novo SNP/InDel detection
5.2 Calculation of de novo mutation rates
6. Protein-protein interaction (PPI) analysis
7. Association analysis of candidate genes (at least 20 trios or case/control pairs)

Cancer (for tumor-normal pair samples)

1. Screening for predisposing genes
2. Mutation spectrum & mutation signature analyses
3. Screening for known driver genes
4. Analyses of tumor significantly mutated genes
5. Analysis of copy number variations (CNV)
5.1. Analysis of CNV distribution
5.2.Analysis of CNV recurrence
6. Fusion gene detection (for WGS porject only)
7. Purity & ploidy analyses of tumor samples
8. Tumor heterogeneity analyses
9. Tumor evolution analysis
10. Display of genomic variants with Circos

Pain is Not Just a Matter of Nerves

Summary: Researchers reveal the role glial cells play in the sensation of pain.

Source: Medical University of Vienna.

The sensation of pain occurs when neural pathways conduct excitation generated by tissue damage to the spinal cord, where the nociceptive information is extensively pre-processed. From there, the information is transmitted to the human brain, where the sensation of “pain” is finally created. This is the general belief. However, researchers from the Division of Neurophysiology at MedUni Vienna’s Center for Brain Research have now discovered that pain is not just a matter of nerves but that non-neuronal cells, the glial cells, are also involved in clinically relevant pain models and their activation is sufficient to amplify pain. The study has now been published in the leading journal “Science”.

Glial cells are the commonest type of cells in the human brain and spinal cord. They surround neurons but are distinct from them and play an important supporting role – for example, in material transport and metabolism or the fluid balance in the brain and spinal cord.

Novel explanation for puzzling pain phenomena

At the same time, however, when they are activated – by pain processes, for example ­– glial cells are themselves able to release messenger substances, such as inflammatory cytokines. Glial cells therefore have two modes: a protective and a pro-inflammatory mode. “The activation of glial cells results in a pain-amplifying effect, as well as spreading the pain to previously unaffected parts of the body. For the very first time, our study provides a biological explanation for this and for other hitherto unexplained pain phenomena in medicine,” says Jürgen Sandkühler, Head of the Division of Neurophysiology at MedUni Vienna’s Center for Brain Research.

Image shows neurons.

Over-activation of glial cells in the spinal cord can, for example, be caused by strong pain stimuli from a wound or surgical intervention, or even by opiates. Sandkühler: “This could also explain why opiates are initially very good at relieving pain but then often cease to be effective. Another example is the phenomenon of “withdrawal” in drug addicts, where activated glial cells cause severe pain throughout the body.”

A healthy lifestyle can beneficially impact the glial cell system

According to Sandkühler, neuroinflammatory diseases of the brain, environmental factors and even the person’s own lifestyle can lead to activation of glial cells. Examples from the current literature are: depression, anxiety disorders and chronic stress, multiple sclerosis or Alzheimer’s and diabetes, as well as lack of exercise and poor diet. Sandkühler: “Glial cells are an important factor in ensuring the equilibrium of a person’s neuroinflammatory system.” The study results give grounds for speculation that improvements in a person’s lifestyle could have a beneficial impact upon this system and ensure that they generally suffer less pain or “minor niggles”, says Sandkühler: “It is therefore in our own hands: thirty minutes of moderate exercise three or four times a week, a healthy diet and avoiding putting on excess weight can make a huge difference.”


Source: Medical University of Vienna
Image Source: This image is adapted from the Medical University of Vienna press release.
Original Research: Abstract for “Gliogenic LTP Spreads Widely in Nociceptive Pathways” by M.T. Kronschläger, R. Drdla-Schutting, M. Gassner, S.D. Honsek, H.L. Teuchmann, and J. Sandkühler in Science. Published online November 10 2016 doi:10.1002/da.22577

Medical University of Vienna. “Pain is Not Just a Matter of Nerves.” NeuroscienceNews. NeuroscienceNews, 11 November 2016.


Gliogenic LTP Spreads Widely in Nociceptive Pathways

Learning and memory formation involve long-term potentiation of synaptic strength (LTP). A fundamental feature of LTP induction in the brain is the need for coincident pre- and postsynaptic activity. This restricts LTP expression to activated synapses only (homosynaptic LTP) and leads to its input specificity. In the spinal cord, we discovered a fundamentally different form of LTP that is induced by glial cell activation and mediated by diffusible, extracellular messengers, including D-serine and tumor necrosis factor (TNF), and that travel long distances via the cerebrospinal fluid, thereby affecting susceptible synapses at remote sites. The properties of this gliogenic LTP resolve unexplained findings of memory traces in nociceptive pathways and may underlie forms of widespread pain hypersensitivity.

“Gliogenic LTP Spreads Widely in Nociceptive Pathways” by M.T. Kronschläger, R. Drdla-Schutting, M. Gassner, S.D. Honsek, H.L. Teuchmann, and J. Sandkühler in Science. Published online November 10 2016 doi:10.1002/da.22577

Artificially intelligent nanoarray analyzes 17 diseases from breaths


Schematic representation of the concept and design of the study. It involved collection of breath samples from 1404 subjects in 14 departments in nine clinical centers in five different countries (Israel, France, USA, Latvia, and China). The population included 591 healthy controls and 813 patients diagnosed with one of 17 different diseases: lung cancer, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, prostate cancer, kidney cancer, gastric cancer, Crohn’s disease, ulcerative colitis, irritable bowel syndrome, idiopathic Parkinson’s, atypical Parkinsonism, multiple sclerosis, pulmonary arterial hypertension, pre-eclampsia, and chronic kidney disease. One breath sample obtained from each subject was analyzed with the artificially intelligent nanoarray for disease diagnosis and classification, and a second was analyzed with GC-MS for exploring its chemical composition.

The present study reports on an artificially intelligent nanoarray based on molecularly modified gold nanoparticles and random network of single-wall carbon nanotubes for noninvasive diagnosis and classification of 17 different diseases from exhaled breath. The nanoarray was used for the practical evaluation of 1404 subjects in nine clinical settings worldwide. Blind experiments with the artificially intelligent nanoarray showed that 86% accuracy could be achieved, allowing discrimination between each pair of the diseases, and that each disease has its own unique volatile molecular print compared to both healthy controls and other diseases.

The artificially intelligent nanoarray had a low or no vulnerability to clinical and demographical confounding factors. The findings by nanoarray were examined by an independent analytical technique, GC-MS. This analysis found 13 exhaled VOCs associated with various diseases, and their composition differs from one disease to another, thereby validating the nanoarray results. While further and larger translational studies are required to validate these findings, this work provides a shuttling pad for in statu nascendi “volatolomics” field (the omics of volatile biomarkers), as well as a method for obtaining affordable, easy-to-use, inexpensive, and miniaturized tools for personalized screening, diagnosis, and follow-up of a range of diseases.

Control samples ruled out the possibility of coincidence and/or external biases. Of special importance, results from the artificially intelligent nanoarrays support the hypothesis that similarities in pathophysiological processes are expressed in quite similar breath patterns. The results also indicated that the adjustment for confounding factors was successful. The subgroups were not clustered according to similarities in demographic features or geographical location, which also stresses that the artificially intelligent nanoarray analysis is less sensitive to possible confounding factors since we have seen in some cases trends in the control groups that were like those seen among the diseases.

In some cases, two or more diseases shared the same control group, as in (1) Crohn’s disease, ulcerative colitis, and irritable bowel syndrome; (2) kidney and bladder cancer; and (3) idiopathic and atypical Parkinsonism. Therefore, the last analysis was not applicable in these cases (Figure 3, hatched boxes). In contrast to the high accuracy achieved among diseases (86%), the classification of the control samples resulted in random results with a total accuracy of 58%, ruling out the possibility of coincidence. In certain comparisons, the results were higher than the arbitrary classification of the control subjects.

In some cases, two or more diseases shared the same control group, as in (1) Crohn’s disease, ulcerative colitis, and irritable bowel syndrome; (2) kidney and bladder cancer; and (3) idiopathic and atypical Parkinsonism. Therefore, the last analysis was not applicable in these cases (Figure 3, hatched boxes). In contrast to the high accuracy achieved among diseases (86%), the classification of the control samples resulted in random results with a total accuracy of 58%, ruling out the possibility of coincidence. In certain comparisons, the results were higher than the arbitrary classification of the control subjects.

The artificially intelligent nanoarray analyzes the collective breath VOC patterns in a black-box approach. To identify and quantify the specific VOCs associated with each disease state, a second breath sample obtained from all participants was analyzed by GC-MS. This identified over 150 different VOCs in the different cohorts, but only 35 VOCs were selected for further investigation. The choice was made on the following criteria: (i) they were common to >70% of the total population (patients and controls); (ii) they were easily identified and verified by the analysis of pure standards; and (iii) they had concentrations in ambient air samples at least 10-fold lower (on average) than in the equivalent breath samples. Owing to the demographic differences between the groups, multiple linear regression for the abundance of each VOCs was first carried out to explore any possible correlation between abundance and the covariates (age, sex, location, and smoking status). The results indicate that the abundances of 15 VOCs were negatively correlated with age and/or smoking; three of them were also correlated with gender. However, there was no significant correlation between the abundance of those VOCs and the sampling site. Therefore, each VOC with significant correlations (p < 0.05) was adjusted according to the calculated coefficient corresponding to the confounding element (see SI, Table S16).

Regression models applied to the raw GC-MS data showed that the abundance of exhaled VOCs was affected by some common confounding factors. A number of the VOCs was affected by age and/or smoking habits (e.g., 2-ethylhexanol, 3-methylhexane, 5-ethyl-3-methyloctane, acetone, ethanol, ethyl acetate, ethylbenzene, isononane, isoprene, nonanal, styrene, toluene, and undecane), whereas three of them were also affected by the gender of the subject (isononane, nonanal, and undecane). This effect stemming from the first part of the VOCs could be explained by the relationship between the anatomical and physiological changes in the respiratory system and circulation associated with aging and/or smoking injury.(65) It includes stiffness and degeneration of the elastic fibers, fibrosis, aging-associated destruction of lung parenchyma, emphysema, and chronic bronchitis, mainly among smokers.(66) These alterations could easily affect the diffusion of VOCs through the blood–air barrier by altering the thickness or permeability of the epithelium (the so-called membrane conductance) or by reducing the total surface area of the membrane.(66) These factors could easily alter the flux, according to Fick’s first law, affecting the diffusion of gases in the exhaled air, eventually reducing/stressing the expression and/or concentrations of a wide range of the exhaled VOC components.(5) The effect stemming from the second part of the VOCs might be attributed to hormonal or structural gender-related differences.(67)

Brain to immune system communication


This review outlines the mechanisms underlying the interaction between the nervous and immune systems of the host in response to an immune challenge. The main focus is the cholinergic anti-inflammatory pathway, which we recently described as a novel function of the efferent vagus nerve. This pathway plays a critical role in controlling the inflammatory response through interaction with peripheral α7 subunit–containing nicotinic acetylcholine receptors expressed on macrophages. We describe the modulation of systemic and local inflammation by the cholinergic anti-inflammatory pathway and its function as an interface between the brain and the immune system. The clinical implications of this novel mechanism also are discussed.


Inflammation is a normal response to disturbed homeostasis caused by infection, injury, and trauma. The host responds with a complex series of immune reactions to neutralize invading pathogens, repair injured tissues, and promote wound healing (1,2). The onset of inflammation is characterized by release of pro-inflammatory mediators including tumor necrosis factor (TNF), interleukin (IL)-1, adhesion molecules, vasoactive mediators, and reactive oxygen species (13). The early release of pro-inflammatory cytokines by activated macrophages has a pivotal role in triggering the local inflammatory response (2). Excessive production of cytokines, such as TNF, IL-1β, and high mobility group B1 (HMGB1), however, can be more injurious than the inciting event, initiating diffuse coagulation, tissue injury, hypotension, and death (2,46). The inflammatory response is balanced by anti-inflammatory factors including the cytokines IL-10 and IL-4, soluble TNF receptors, IL-1 receptor antagonists, and transforming growth factor (TGF)β. Although simplistic (7,8), the pro-/anti- terminology is widely used in the discussion of the complex cytokine network. Apart from their involvement in local inflammation, TNF and IL-1β are signal molecules for activation of brain-derived neuroendocrine immunomodulatory responses. Neuroendocrine pathways, such as the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic division of the autonomic nervous system (SNS) (915), control inflammation as an anti-inflammatory balancing mechanism. The host thereby mobilizes the immunomodulatory resources of the nervous and endocrine systems to regulate inflammation.

Restoration of homeostasis as a logical resolution of inflammation does not always occur. Insufficient inflammatory responses may result in increased susceptibility to infections and cancer. On the other hand, excessive responses are associated with autoimmune diseases, diabetes, sepsis, and other debilitating conditions. When control of local inflammatory responses is lost, pro-inflammatory mediators can spill into the circulation, resulting in systemic inflammation that may progress to shock, multiple organ failure, and death. Effective therapies for diseases of excessive inflammation are needed.

We recently discovered the anti-inflammatory role of the vagus nerve (16,17) in an animal model of endotoxemia and shock. This previously unrecognized immunomodulatory circuit termed the “cholinergic anti-inflammatory pathway” is a mechanism for neural inhibition of inflammation (18,19), and interfaces the brain with the immune system. Can it be a “missing link” in neuroimmunomodulation that will validate the notion of a mind-body connection?

This review outlines brain-derived control mechanisms of immune function and specifically the role of cholinergic anti-inflammatory pathway in the regulation of inflammation.


Communication between the immune, nervous, and endocrine systems is essential for host defense and involves a variety of mediators including cytokines, neurotransmitters, hormones, and humoral factors. The influence of the brain on immune function and the mechanisms involved in these interactions have been elucidated over the past 3 decades (1719). Two important questions arise when describing the brain-derived immunomodulation: 1) How is the brain initially signaled by cytokines to trigger corresponding neural and neuroendocrine responses; and 2) how is immunomodulation achieved through these mechanisms?

Immune-To-Brain Communication

The brain can monitor immune status and sense peripheral inflammation through 2 main pathways: neural and humoral (Figure 1; for a review, see 20, 21).


Neural and humoral pathways in immunomodulation. During immune challenge activated macrophages and other immune and nonimmune cells release cytokines that signal the brain for activation of immunomodulatory mechanisms. Central immunomodulation is achieved

Neural pathway

The neural mechanism relies upon activation of vagus nerve afferent sensory fibers that signal the brain that inflammation is occurring. Immunogenic stimuli activate vagal afferents either directly by cytokines released from dendritic cells, macrophages, and other vagal-associated immune cells, or indirectly through the chemoreceptive cells located in vagal paraganglia (22). For instance, intraperitoneal administration of endotoxin can induce IL-1β immunoreactivity in dendritic cells and macrophages within connective tissues associated with the abdominal vagus nerve and subsequently in vagal paraganglia and afferent fibers (23). Visceral vagus afferent fibers, residing in the nodose ganglion, terminate primarily within the dorsal vagal complex (DVC) of the medulla oblongata. The DVC consists of the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMN), and the area postrema (AP) (24). The DMN is the major site of origin of preganglionic vagus efferent fibers; cardiovascular vagal efferents also originate within the medullar nucleus ambiguous. The AP, which lacks a blood-brain barrier, is an important circumventricular organ and site for humoral immune-to-brain communication, as described below. The main portion of vagal sensory input is received by neurons in the NTS that coordinate autonomic function and interaction with the endocrine system (25). Ascending projections from the NTS reach forebrain sites including hypothalamic nuclei, amygdala, and insular cortex. One of the hypothalamic nuclei receiving input from the NTS is the paraventricular nucleus (PVN). The PVN is associated with the synthesis and release of corticotropin releasing hormone (CRH), an important substance in the HPA axis.

This ascending link between the NTS and PVN provides a pathway that can modulate neurohormonal anti-inflammatory responses. Synaptic contacts also exist between the neurons in the NTS and C1 neurons in the rostral ventrolateral medulla (RVM), which occupies an important role in control of cardiovascular homeostasis. The RVM neurons in turn project to the locus coeruleus (LC), which is the main source of noradrenergic innervations of higher brain sites, including the hypothalamus and PVN. Projections emanate from the RVM and LC to sympathetic preganglionic neurons in the spinal cord. There are also descending pathways from the PVN to the RVM and NTS. These ascending and descending connections provide a neuronal substrate for interaction between HPA axis and SNS as an immunomodulatory mechanism.

The transmission of cytokine signals to the brain through the vagal sensory neurons depends upon the magnitude of the immune challenge. Subdiaphragmatic vagotomy inhibits the stimulation of the HPA axis (26) and norepinephrine (NE) release in hypothalamic nuclei (27) in response to intraperitoneal administration of endotoxin or IL-1β. Intravenous endotoxin administration induces expression of the neural activation marker c-Fos in the brainstem medulla, regardless of the integrity of the vagus nerve (28). Vagotomy fails to suppress high dose endotoxin-induced IL-1β immunoreactivity in the brain (29) and increases blood corticosterone levels (30). It is likely that the vagal afferent neural pathway plays a dominant role in mild to moderate peripheral inflammatory responses, whereas acute, robust inflammatory responses signal the brain primarily via humoral mechanisms.

Humoral pathway

A large body of evidence supports the involvement of humoral mechanisms in the immune-to-brain communication, especially in cases of systemic immune challenge (21, 3133). The question remains, however, of how the circulating cytokines interact with brain structures involved in the anti-inflammatory response, and how circulating cytokines induce central cytokine production associated with fever and sickness. Blood-borne IL-1β and TNF can cross the blood-brain barrier and enter cerebrospinal fluid and the interstitial fluid spaces of the brain and spinal cord by a saturable carrier-mediated mechanism (34) that may function only at very high plasma cytokine concentrations. Cytokines also can bind to receptors at the surface of the endothelium of the brain capillaries and can enhance the synthesis and release of soluble mediators such as prostaglandins and nitric oxide, which diffuse into the brain parenchyma and modulate the activity of specific groups of neurons (21,35,36). It has been suggested that prostaglandins mediate fever and HPA axis activation (13).

Cytokine-to-brain communication also may occur via circum-ventricular organs that lack normal blood-brain barrier function. Among the circumventricular organs, the AP appears to represent the best candidate for such a transduction site (for a review, see 37). The AP is located in the floor of the caudal 4th ventricle (38) and dendrites of neurons in the NTS and DMN penetrate both the AP and floor of the 4th ventricle (39,40). The close proximity of AP to NTS and RVM and the existing neural connections provide a way of signaling the SNS and HPA axis. Cytokine-induced production of prostaglandins within the AP, NTS, and RVM may activate the catecholamine projections to the PVN, resulting in subsequent HPA axis activation (37). This is one possible interaction between the neural and humoral mechanisms of immune-to-brain communication through which the brain mediates anti-inflammatory responses.

Apart from their function in signaling the brain for immunomodulatory responses, cytokines play a multifunctional role in brain injury and neurodegenerative diseases (for review, see 4143).

Brain-To-Immune Communication

The brain exerts strong modulatory effects on immune function by activation of the HPA axis and the SNS, which results in increased synthesis and release of glucocorticoids and catecholamines (see Figure 1). The immunomodulating properties of α-melanocyte stimulating hormone (α-MSH) and estrogens also are known. The HPA axis is a neurohormonal pathway; its role in the regulation of the immune function has been widely studied (for reviews, see 14,44–46). The components of the HPA axis are the hypothalamic PVN, the anterior pituitary gland, and the adrenal cortex. Specialized neurons in PVN synthesize CRH, which is released into the pituitary portal blood system and stimulates the synthesis of adrenocorticotropin hormone (ACTH) from the anterior pituitary. ACTH is the main inducer of the synthesis and release of immunosuppressive glucocorticoids (cortisol in humans and corticosterone in rats) from the adrenal cortex. Pro-inflammatory cytokines trigger the HPA axis via the neural or humoral mechanisms described above. At both the hypothalamic and pituitary level, the HPA axis is subject to a classical negative feedback loop by the final product: glucocorticoids. ACTH also inhibits the synthesis of CRH from the PVN. The hypothalamo-pituitary circuit of the HPA axis is regulated by neural mechanisms including acetylcholine (ACh)-, catecholamine-, GABA-, serotonin-, and histamine-mediated modulation.

Glucocorticoids exert their effects by binding to intracellular receptors and subsequently triggering up-regulation or down-regulation of gene expression (47). Apart from triggering the activation of the HPA axis, cytokines such as IL-1 and IL-6 also can alter peripheral glucocorticoid effects by directly influencing the function of corresponding glucocorticoid receptors (45). Immunosuppressive glucocorticoid influence is mainly linked to suppression of nuclear factor-κB activity (14,48,49), which plays an important role in regulating cytokine synthesis (50). As summarized by Webster and others (14), glucocorticoids inhibit the synthesis of pro-inflammatory cytokines, such as TNF, IL-1, IL-8, IL-11, IL-12, and interferon-γ; and they activate the synthesis of the anti-inflammatory cytokines IL-4 and IL-10. Inhibition of neutrophil, eosinophil, monocyte, and macrophage infiltration, and adhesion molecule expression are attributed to glucocorticoid suppression of local inflammation (14,45). Glucocorticoids are also potent clinical anti-inflammatory agents (45).

The SNS plays a dual role in the regulation of inflammation, because it mediates both pro- and anti-inflammatory activities; it is thus an integral component of the host defense system against injury and infection (15,51). The locus coeruleus (LC) and RVM, brain functional components of the SNS, project to sympathetic preganglionic cholinergic neurons in the spinal cord. Sympathetic innervation of primary (thymus and bone marrow) and secondary (spleen, lymph nodes, and tissues) lymphoid organs is the anatomic basis for modulation of immune function by the SNS (15,51,52). Sympathetic postganglionic norepinephrine (NE)-ergic and neuropeptide Y-ergic neurons also innervate blood vessels, the heart, the liver, and the gastrointestinal tract (15,52). NE, released from sympathetic postganglionic nerve endings, exerts anti-inflammatory effects by interacting with adrenoceptors expressed on lymphocytes and macrophages.

Adrenoceptors belong to the G-protein coupled receptor superfamily and are divided into α and β subtypes, which can be further subdivided. Sympathetic control on cytokine production is achieved by 2 mechanisms: (1) synaptic-like junctions with corresponding adrenoceptors (as in the spleen) and (2) nonsynaptic, distant mechanisms, such as NE diffusion through the parenchyma before interaction with the receptor. The nonsynaptic mechanism plays a dominant physiological role (15). The release of NE is subject to complex presynaptic regulation, involving the effects of neuropeptide Y, acetylcholine, dopamine, prostaglandins, and other micro-environmental factors. Sympathetic immunomodulation also is mediated via epinephrine, and to a lesser extent, by NE released from the chromaffin cells of the adrenal medulla. The chromaffin cells represent homologs of the sympathetic ganglia. Activation of the preganglionic sympathetic neurons innervating these cells leads to an increase in the release of catecholamines in the bloodstream, which can act systematically as hormones. Thus, sympathetic neural regulation is converted into “hormonal regulation” within the adrenal glands. The adrenals are therefore an important peripheral component of the CNS-controlled immunoregulation responsible for the synthesis of glucocorticoids (from the cortical cells) and catecholamines (from the medullar chromaffin cells).

SNS activation protects the organism from the detrimental effects of pro-inflammatory cytokines. Activation of β-adrenoceptors leads to marked inhibition of endotoxin induced serum TNF, IL-1, IL-12, interferon-γ, and nitric oxide production, and elevation of IL-6 and IL-10. Stimulation of β-adrenoceptors also is accompanied by suppression of the TNF and IL-1 expression caused by hemorrhagic shock. NE and epinephrine inhibit production of pro-inflammatory cytokines through stimulation of β2-adrenoceptor-cAMP-protein kinase A pathway, and they stimulate the synthesis of anti-inflammatory cytokines. Healthy volunteers receiving epinephrine and subsequent endotoxin treatment developed significantly decreased TNF levels and elevated IL-10 levels, as compared with controls exposed to endotoxin alone (53), raising the possibility that pharmacological control of cytokine production in septic patients could be achieved by selective adrenoceptor agonists and antagonists. A catecholamine-based strategy for treating sepsis and its complications is difficult, however, because of the broad effects of these agents and the need to balance the cardiovascular and immunomodulatory drug effects (15).

During the early stages of some cases of inflammation, stimulation of the SNS can be associated with activation of the local inflammatory responses and neutrophil accumulation (1,54). NE stimulation of the α2–subtype adrenoceptor has been linked to an increase in endotoxin-induced production of TNF and other cytokines (15,55). This dual role of the SNS in immunomodulation agrees with the general mode of SNS function, depending on the peripheral receptors involved. A classic example of dual effects is the “fight-or-flight” response, leading to increased perfusion of the heart, skeletal muscles, and brain, and release of glucose from the liver. Gastrointestinal motility and the blood supply to the skin are simultaneously depressed.

Anti-inflammatory properties of α-MSH have been shown in animal models of inflammation including sepsis and rheumatoid arthritis (for a review, see 56, 57). In rodents α-MSH is mainly produced from the intermediate lobe of the pituitary gland. The cells of the pituitary pars distalis and various extrapituitary cells, such as monocytes, astrocytes, and keratinocytes, are the source of this hormone production in humans. The activity of α-MSH is mediated through G-protein coupled melanocortin receptors widely distributed in peripheral tissues and in the brain. Immunomodulatory effects of α-MSH are associated with stimulation of its receptors on peripheral immune target cells and on glial cells in brain; α-MSH may also exert indirect effects via a brain-spinal cord pathway and sympathetic neurons (58). Although the exact molecular mechanisms of α-MSH immunosuppression and pro-inflammatory cytokine downregulation are not well understood, hormonal inhibition of nuclear factor κB may play an important role. The administration of endotoxin to normal human subjects is accompanied by an increase in α-MSH blood concentrations (in addition to ACTH) and decreased TNF plasma levels, demonstrating the role of α-MSH in the inflammatory response (59). It is not clear how pro-inflammatory cytokines cause stimulation of pituitary α-MSH secretion, but the ascending pathways from NTS to the hypothalamus underlying the HPA axis activation may play a role.

Female sex hormones and estrogens, in particular, also exert immunomodulatory and anti-inflammatory effects. Because their synthesis and blood concentrations are under control of hypothalamo-pituitary hormones, estrogen-affected immune functions can be imputed to the brain. Female sex hormones play immunoregulatory roles during pregnancy and in diseases like rheumatoid arthritis and osteoporosis (60). The classic mechanism of steroid hormone action (as described above for glucocorticoids) may contribute to estrogen immunomodulating activity. Estrogens are neuroprotectors (61); they prevent cartilage degradation during inflammation associated with increased production of IL-1 (62); and they inhibit the production of pro-inflammatory cytokines at different stages of their synthesis (63,64).


The sympathetic and parasympathetic parts of the autonomic nervous system rarely operate alone; autonomic responses represent the interplay of both parts. A link between the parasympathetic part of the autonomic nervous system and immunoregulatory processes was suggested 30 years ago, when alleviation of T-lymphocyte cytotoxicity by muscarinic cholinergic stimulation was noted (65). Despite this observation, the role of the parasympathetic/vagal efferents in immunomodulation is not completely understood (6668).

We recently demonstrated the existence of a parasympathetic pathway of modulation of systemic and local inflammatory responses (16,69), which focuses attention on neural immunomodulatory mechanisms via the vagus nerve.

Evidence for Parasympathetic (Vagus Nerve) Control of Systemic And Local Inflammation

Acetylcholine is an important neurotransmitter and neuromodulator in the brain. It mediates neural transmission in the ganglion synapses of both sympathetic and parasympathetic neurons, and is the principle neurotransmitter in the postganglionic parasympathetic/vagal efferent neurons. Acetylcholine acts through 2 types of receptors: muscarinic (metabotropic) (70) and nicotinic (ionotropic) (71). In addition to the brain and “wire-innervated” peripheral structures, the RNA for these receptor subtypes (muscarinic) and subunits (nicotinic) has been detected on mixed populations of lymphocytes and other immune and non-immune cytokine-producing cells (7277). Most of these cells can also produce acetylcholine (78).

We recently discovered that the α7 subunit of the nicotinic acetylcholine receptor is expressed on macrophages (16). Acetylcholine significantly and concentration-dependently decreases TNF production by endotoxin-stimulated human macrophage cultures via a post-transcriptional mechanism. Using specific muscarinic and nicotinic agonists and antagonists, we demonstrated the importance of an α-bungarotoxin-sensitive nicotinic receptor in the inhibition of TNF synthesis in vitro by acetylcholine. Acetylcholine also is effective in suppressing other endotoxin-inducible pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-18, by a post-transcriptional mechanism; release of the anti-inflammatory cytokine IL-10 from endotoxin-stimulated macrophages is not affected by acetylcholine (16).

Because of the immunosuppressive effects of acetylcholine in vitro, we studied the possible immunonodulatory role of the parasympathetic division of the autonomic nervous system in vivo. In a rat model, vagotomy without electrical stimulation significantly increases serum and liver TNF levels in response to intravenously administered endotoxin (Figure 2A and 2B), suggesting a direct role of efferent vagus neurons in the regulation of TNF production in vivo. Augmentation of efferent vagus nerve by direct electrical stimulation significantly attenuates endotoxin-induced serum and hepatic TNF (see Figure 2A and 2B). TNF amplifies inflammation by activating the release of pro-inflammatory mediators such as IL-1, HMGB1, nitric oxide, and reactive oxygen species (3,6).

TNF also plays an essential role in endotoxin-induced shock by inhibiting cardiac output, activating microvascular thrombosis, and modulating capillary leakage syndrome (4,79). These activities of TNF are consistent with the finding that attenuation of serum TNF via cervical vagus nerve stimulation prevents hypotension and shock in animals exposed to lethal doses of endotoxin (see Figure 2C) (16). Animals subjected to vagotomy without vagus nerve stimulation develop profound shock more quickly than sham-operated animals (see Figure 2C), demonstrating a role for vagus nerve efferent signaling in maintaining immunological homeostasis. Importantly, the immunomodulatory effects of the efferent vagus nerve also play a role in localized peripheral inflammation, because electrical stimulation of the distal vagus nerve also inhibits the local inflammatory response in a standard rodent model of carrageenan-induced paw edema (69). Pretreatment with acetylcholine, muscarine, or nicotine localized within the site of inflammation also prevents the development of hind paw swelling (69). Vagal efferents are distributed throughout the reticuloendothelial system and other peripheral organs, and the brain-derived motor output through vagus efferent neurons is rapid. The cholinergic anti-inflammatory pathway is therefore uniquely positioned to modulate inflammation in real time.

The most abundant dietary sources of choline—a precursor to acetylcholine—are animal fats such as egg yolks, cream, fatty cheeses, fatty fish, fatty meats, and liver. Non-animal sources include avocadoes and almonds.

Aerobic exercises influence acetycholine production.

DNA study could shed light on how genetic faults trigger disease

A new technique that identifies how genes are controlled could help scientists spot errors in the genetic code which trigger disease.

The method focusses on those parts of DNA – known as enhancer regions – which regulate the activity of genes and direct the production of proteins that have key functions within the body.

Errors in protein production can result in a wide range of diseases in people.

The new method could help researchers pinpoint the source of disease-causing mutations in enhancers. Until now, these genetic errors have been difficult to interpret as the link between enhancers and the genes they control was not clear.

Researchers at the MRC Human Genetics Unit were part of an international collaboration that identified all the enhancers – and the genes they activate – on a single human chromosome.

The team then tested the technique in zebrafish and found that genes are controlled by enhancers in a similar way, suggesting that this type of regulation takes place in all animals.

Individual genes may be under the control of many enhancers, which allow gene activation to be carefully regulated. This allows precise control of gene activity, which is important during development and in maintaining normal brain function.

The study, published in the journal Nature Communications, was funded by the Seventh Framework Programme of the European Union. The study was carried in close collaboration with researchers based in other parts of the UK, France, Germany, Australia, and Norway.

Professor David FitzPatrick, of the MRC Human Genetics Unit, who took part in the study, said: “This work is an important step in identifying which enhancers control which genes, and this will help us in interpreting the genetic changes we see in the part of the genome that does not code for protein.”

Increase the body’s oxygen carrying capacity with exercise, EPO and whole foods

How does food and supplements effectively increase EPO namely the body’s oxygen carrying capacity?

There is a distinct difference between unethical, harmful EPO-blood doping interventions and safe nutrition that effectively increases individual oxygen carrying capacity without compromising the athlete’s health or integrity.

Stimulates the production of red blood cells (RBC) with EPO hormone

EPO levels up to 48% safely improve performance in males, however beyond this level, the risk of compromised health increases. What nutritional protocol safely increases natural production of EPO?


ERYTHROPOIETIN (EPO) is a naturally occurring hormone that stimulates the production of red blood cells (RBC). Erythropoietin is a glycoprotein hormone produced in the kidneys containing a 165-amino acids structure. Most erythropoietin is produced by the kidney’s renal cortex. But some is also produced in the liver (mainly in the fetus), the brain and uterus. Erythropoietin production is stimulated by low oxygen levels in interstitial cells of the peritubular capillaries in the kidneys. Following its production in the kidneys, EPO travels to the bone marrow where it stimulates production of red blood cells.[2] In the absence of erythropoietin, only a few RBC’s are formed by the bone marrow. EPO increases the blood-oxygen carrying capacity but only up to a point, but beyond, it may compromise health and hinder blood flow dynamics with performance-limiting implications.


The margin between effective and lethal quantities of EPO is very narrow. EPO use can be LETHAL (many athletes seeking to derive its performance-enhancing effects have died from incorrectly-administered EPO…Inappropriate use of exogenous Erythropoietin can cause elevated Hematocrit levels (i.e. thickened blood is difficult to pump). Elevated EPO increases the risk of heart attack (due to the increase in hematocrit). Exogenous EPO is totally cleared from the urine within 48 hours of its administration and is cleared from the blood within 72 hours of its administration (although its physiological effects prevail for several months).[3]

What is the dynamic natural process to increase our bodys production of EPO?

A look at EPO’s complex pathway further illustrates a complex physiological process below, see PATHWAYS[4] including diagram on page 23.

Erythropoietin mediated neuroprotection through NF-kB

Dietary deficiency of specific foods and micronutrients, hormone imbalance, and lack of specific hypoxic training stress inhibit the endogenous (natural) production.

In normal adults, the kidneys produce EPO, which initiates approximately 90% of natural erythropoietin production. Tissue oxygenation exposure regulates the production of erythropoietin. Less oxygen saturation in the air we inhale either by altitude or hypoxic interval training stimulates the kidneys to appropriate chemical messengers to instruct bone marrow to increase the production of EPO to resolve lack of oxygen exposure. The reduced oxygen delivery deficit is sensed by the kidneys to be low when hematocrit (Hct) fails to restore oxygen levels in tissues or as a result of changes in how hemoglobin (Hb) and oxygen interact. Hypoxia or Anemia stimulates the kidney production of erythropoietin to increase production red blood cells. Erythropoietin released from the kidneys increases the rate of red blood cell division and differentiation of specific cells in the bone marrow.

Endogenous production of erythropoietin is regulated by the level of tissue oxygenation. ERYTHROPOIETIN (EPO) is released primarily by the kidney response to hypoxia or anemia, sending a highly specific hormone signal prompting cells in the bone marrow to produce RBCs. An important effect of erythropoietin is increased production of “Proerythroblasts.” These cells to mature rapidly, further accelerating production of red blood cells. The regulation of red blood cell production resembles a complete feedback loop. As a result, total oxygen-carrying capacity of the blood increases, the stimulus from hypoxia is alleviated then the production of erythropoietin decreases. In normal subjects, plasma erythropoietin levels range from 0.01 to 0.03 Units/mL, but may increase from 100- to 1000-fold during hypoxic or anemic states.

Epoetin Alfa stimulates erythropoiesis in anemic patients with CRF who do not require regular dialysis. The first evidence of a response to 3 X Week (T.I.W.) dose administration of Epoetin Alfa is an increase in the reticulocyte count within 10 days, followed by increases in the red cell count, hemoglobin, and hematocrit, usually within 14-42 days. Because of the length of time required for erythropoiesis, several days are required for erythroid progenitors to mature and appear in circulation. A clinically significant increase in hematocrit is usually not observed in less than 14 days and may require up to 42 days. Once the hematocrit reaches the suggested target range (30-36%), that level can be sustained by the absence of concurrent illnesses, nutrition or iron deficiencies.

Interval training hypoxia enhances EPO levels by the same mechanism as the prescription drug Procrit induces. When Procrit is administered 1-3 times per week, subsequent increases in plasma erythropoietin levels increase 100- to 1000-fold.[5] Increased hematocrit enables more oxygen to flow to the skeletal muscles. It is well known that distance runners and cyclists have illegally used recombinant EPO to enhance performance. A model for the regulation of erythropoietin production has been examined. This model proposes that a primary O2-sensing reaction in the kidney is initiated by a decrease in ambient PO2, a rapid decrease in gas exchange in the lung, a diminished oxygen-carrying capacity of hemoglobin, a molecular deprivation of oxygen, or a decrease in renal blood flow. Some of the agents that are released during hypoxia, which trigger the EPO cascade, are adenosine (A2 activation), eicosanoids (PGE2, PGI2, and 6-keto PGE1), oxygen-free radicals (superoxide and H2O2), and catecholamines with beta-2 adrenergic receptor agonist properties. It is further proposed that an increase in intracellular calcium leads to the inhibition of erythropoietin biosynthesis and/or secretion and a decrease in intracellular calcium increases erythropoietin production. The specific mechanism by which calcium regulates erythropoietin biosynthesis and secretion is not well understood.

However, a good correlation is seen with several agents that decrease intracellular calcium and increase erythropoietin production as well as with other agents that increase intracellular calcium and decrease erythropoietin production. When INOSITOL TRIPHOSPHATE levels are increased, an increase in the mobilization of intracellular calcium from the endoplasmic reticulum or another intracellular pool occurs. The increased intracellular calcium presumably activates calcium calmodulin kinase and produces a phosphoprotein that inhibits erythropoietin production/secretion.[6] Although recombinant EPO has exactly the same sequence of amino acids as the natural hormone, the sugars attached by the cells used in the pharmaceutical industry differs from those attached by EPO from a human kidney. This difference is easily detected by testing the athlete’s urine.


Roberts et al. examined exercise-induced hypoxemia (EIH) and plasma volume contraction as modulators of serum Erythropoietin (EPO) production. Five athletes cycled for 3 min at supra-maximal power outputs, at each of two different elevations (1,000 and 2,100 meters). Five subjects were exposed to normobaric hypoxia (F(I)O(2)=0.159), seven subjects underwent plasmapheresis[7] to reduce plasma volume and eight subjects were time controls for EPO levels. Oxyhemoglobin saturation was significantly reduced during exercise and during normobaric hypoxia. The time period of haemoglobin oxygen saturation <91% was 24+/-29 s (mean+/-S.D., n=5) for exercise at 1000 m, 136+/-77 s (mean+/-S.D., n=5) for exercise at 2100 m and 178+/-255 s (mean+/-S.D., n=5) with resting hypoxic exposure. However, significantly increased serum EPO levels were observed only following exercise (21-27% at 1,000 m and 31-41% at 2,100 m). Volume contraction also resulted in increased serum EPO 29-41% in spite of a significant rise in hematocrit of +2.2%. Despite similar degrees of arterial desaturation, only the hypoxemia induced by exercise was associated with an increase in serum EPO. This finding indicates that other factors, in addition to hypoxemia, are important in modulating the production of EPO in response to exercise. Volume depletion in the absence of exercise resulted in increases in EPO levels that were comparable with those observed in response to exercise. The paradoxical responses of increased hematocrit and increase in EPO in subjects undergoing plasmapheresis suggests that plasma volume may also modulate the production of EPO.[8]


Roberts & Smith measured the effects of exercise-induced hypoxia on the physiological production of erythropoietin. Twenty athletes exercised for 3 min at 106-112% maximal oxygen consumption. Estimated oxyhemoglobin saturation was measured by reflective probe pulse oximetry (Nellcor N200) and was validated against arterial oxyhemoglobin saturation by CO-oximetry in eight athletes. Serum erythropoietin concentrations-as measured using the INCSTAR Epo-Trac radioimmunoassay-increased significantly by 19-37% at 24 h post-exercise in 11 participants, who also had an arterial oxyhemoglobin saturation < or = 91%. Decreased ferritin levels and increased reticulocyte counts were observed at 96 h post-exercise. However, no significant changes in erythropoietin levels were observed in nine non-desaturating athletes and eight non-exercise controls. Good agreement was shown between arterial oxyhemoglobin saturation and percent estimated oxyhaemoglobin saturation (limits of agreement = -3.9 to 3.7. They concluded that a short 3 minutes supramaximal exercise period could induce both hypoxemia and increased erythropoietin levels in well-trained individuals. The decline of arterial hypoxemia levels below 91% during exercise appears to be necessary for the exercise-induced elevation of serum erythropoietin levels. Furthermore, reflective probe pulse oximetry was found to be a valid predictor of percent arterial oxyhemoglobin saturation during supramaximal exercise when percent estimated oxyhemoglobin saturation > or = 86%.[9]

Fitness of these athletes provides a physiological environment for increasing EPO naturally from short 3-minute all-out intervals.



Red blood cells carry iron-rich hemoglobin for up to 120-days, then they die. Unless there is a continual supply of Iron, Vitamin B-12, Vitamin C and Folacin, anemia and reduced oxygen carrying capacity appear in two forms:

  1. Low red blood cell count
  2. Malformed red blood cells.

Iron deficiency anemia is the most common form of anemia. Approximately 20% of women, 50% of pregnant women, and 3% of men are iron deficient. Iron is an essential component of hemoglobin, the oxygen carrying pigment in the blood. Iron is normally obtained through the food in the diet and by the recycling of iron from dying “retired” red blood cells. The causes of iron deficiency are iron-poor food, lead poisoning, chemotherapy, dehydration, poor absorption of iron from food or supplements, and blood loss. Anemia develops slowly, after the normal stores of iron from the body tissues and bone marrow are reduced to low levels. Women, in general, have smaller stores of iron than men and experience increased blood loss through menstruation, placing them at higher risk for anemia than men. Runners are also reported to lose more blood during heel strike during gait motion than cyclists, whose impact riding is noticeably less than running. In men and postmenopausal women, anemia is usually due to gastrointestinal blood loss associated with ulcers, nonsteroidal anti-inflammatory medications (NSAIDS), or colon cancer.

High-risk groups include:

  1. Women of child-bearing age who have blood loss through menstruation
  2. Pregnant or lactating women who have an increased requirement for iron
  3. Infants, children, and adolescents in rapid growth phases
  4. People with poor dietary intake of iron through little or no eggs, meat for several years.
  5. Blood loss from peptic ulcer disease, long term aspirin/NSAIDS, colon cancer
  6. Cancer-related drug therapy & chemotherapy
  7. Athletes whose sport requires running


The paradox of hematocrit in exercise physiology is that artificially increasing it by autotransfusion or erythropoietin doping improves VO2 max and performance, while in normal conditions there is a strong negative correlation between hematocrit and fitness, due to a training-induced “Autohemodilution”. Brun et al. reported that in professional soccer footballers:

  •         Physiological values of hematocrit in athletes were comprised between 36 and 48%
  •         Low hematocrit (<40%) was associated with a higher aerobic capacity

Subjects with the higher hematocrits (>44.6%) were frequently overtrained and/or iron-deficient, and their blood viscosity (and red cell disaggregability) tended to be increased.

Over the past 9 years several endurance athletes have complained of low hematocrit levels. In 6 elite endurance athletes ranging from 31-67 years age, hair lab analysis showed iron deficiency, in spite of a calorie-sufficient dietary iron intake. Proper nutrition permits the body to set optimal natural (EPO) hematocrit levels. The diet should contain specific blood-iron building blocks, first from whole foods, second from supplements. However, an athlete should not take supplemental iron unless prescribed and monitored by a physician.


The rate hematocrit increases varying specific to the individual but it may be improved applying dietary interventions with hypoxic interval sessions. The same dietary intervention that relieves Anemia is the protocol for increasing blood oxygen capacity of any endurance athlete. The most common cause is iron-deficiency anemia in red blood cells, smaller than usual and pale in color due to improper amounts of hemoglobin (the molecule in red blood cells that binds to oxygen and carries it in the blood).

Lack of iron for hemoglobin CAUSES:

  1. Loss of iron from the body due to blood loss
  2. Poor absorption of iron from one’s diet
  3. Lack of dietary iron
  4. Radiotherapy or Chemotherapy
  5. Anti-cancer drugs
  6. Certain types of viral infections
  7. Genetic reasons
  8. A result of malaria
  9. AIDS
  10. A deficiency of Vitamin B-12
  11. A deficiency of folic acid
  12. An imbalanced ratio of B-12:Folate


  • Tiredness and weakness
  • Lethargy
  • Dizziness, shortness of breath, and palpitations(rapid heart rate)
  • Headaches
  • Pale complexion
  • Brittle nails (due to lack of iron)
  • Irritability
  • Sore tongue
  • Unusual food cravings (called pica)
  • Decreased appetite
  • Headache – frontal
  • Blue tinge to sclerae (whites of eyes)
  • There may be no symptoms if anemia is mild.


Protein adequacy is a factor in erythropoietin (EPO) production. Inadequate protein nutrition can reduce the EPO produced. The erythroid response to Erythropoietin (EPO) is highly dependent on dietary protein adequacy and quality. The mouse spleen is an erythropoietic organ, which contains an EPO-responsive cell population that can be easily amplified by administration of the hormone. Researchers determined the effect of a protein-free diet offered freely to mice up to two days after injection of r-Hu EPO (1000mU/200 ul) on the response of the above population. Splenic cell suspensions from control and experimental mice were prepared in microwells containing 400 mU r-Hu EPO and appropriate medium.

The response to EPO was evaluated in terms of 3H-thymidine uptake. The results obtained indicate that acutely induced protein restriction suppressed the response of the EPO-responsive splenic cell population to EPO when it was imposed on mice immediately after hormone injection, and suggest the appearance of deficient rates of differentiation of erythropoietic units by protein restriction.[11]

Adequate dietary protein intake is 1.4-1.7 grams/kilogram body weight per day for an endurance athlete.

Inadequate dietary iron; Food sources of iron are red meat, liver, and egg yolks. Most flour, bread, and cereals are iron-fortified. If the diet continues to be iron-deficient, only a physician should prescribe and supervise iron supplementation.

Calorie inadequacy is a secondary factor in EPO production including red blood cell quality and quantity. In order to test the hypothesis that the early cessation of erythropoietin (Ep) production during hypobaric hypoxia is induced by lowered food intake, researchers compared the plasma Ep titer of rats after exposure to continuous hypoxia (42.6 kPa = 7000 m altitude) for 4 days with that in fed or fasted rats after exposure to discontinuous hypoxia. They found that plasma Ep was rather low after 4 days of continuous hypoxia. However, the Ep titer significantly rose again, when rats were maintained normoxic for 18 h and then exposed to repeated hypoxia for 6 h. Because this was also found in rats, which were deprived of food during the normoxic interval and the second hypoxic period, they concluded that the fall of the Ep titer during continuous hypoxia is not primarily due to reduced food intake. In addition, these findings show that fasting per se lowers the EPO-response to hypoxia in normal rats but not exhypoxic rats.[12] Calorie sufficiency (in spite of exercise expense) is required for optimal EPO-release. If training is causing weight loss, then EPO loss may be occurring.


EPO production also has hormonal-dependant roots complexly related to glucose metabolism, and calorie adequacy. The effect of Thyroid-T3 replacement and glucose supplementation on erythropoietin production was investigated in fasted hypoxic rats. It was found that 48 hr of fasting significantly reduced the circulating levels of thyroid hormones and the production of renal and extrarenal erythropoietin in response to hypoxia. These effects of fasting were completely abolished when the animals had free access to 25% glucose solution as drinking water, despite their lack of protein intake. Replacement doses of T3 (0.5 micrograms/100 gm per day) restored erythropoietin production in the fasted animals but also increased the response of the fed controls. To avoid the effect of endogenous T3, the experiments were repeated in thyroidectomized rats.

Erythropoietin production in athyroid rats was found to be markedly decreased, with values equivalent to those found in normal fasted animals, and were not affected by fasting or glucose supplementation. Replacement doses of T3 increased erythropoietin production in all three groups, but the fasted animals needed five times as much T3 to obtain a response similar to that observed in the fed group. Glucose supplementation enhanced the effect of T3 in the fasted animals but did not completely restore These results indicate that caloric deprivation is primarily responsible for the decreased erythropoietin production induced by fasting and that this effect is probably mediated by both a decreased level of T3 and a decreased responsiveness to it.[13]

A calorie deficit therefore requires 500% more Thyroid Hormone (T3) to maintain EPO levels. This is a good reason for monitoring calorie intake during high training calorie expense.


Dietary interventions significantly advance nonheme iron absorption rate during EPO production. It is very important to include foods to enhance nonheme iron absorption, especially when an exercise-induced iron loss is high or when no heme iron is consumed, such as in a vegetarian diet. Absorption of heme iron is very efficient; the presence of red meat increases absorption of non-heme iron +400%. Only 1-7% of the nonheme iron in vegetable staples in rice, maize, black beans, soybeans, and wheat are absorbed consumed alone. Vitamin C improves the rate of absorption of nonheme iron from red meats. Diets that include a minimum of 5 servings of fruits and vegetables daily provide adequate vitamin C to boost nonheme iron absorption. Calcium, polyphenols, tannins from tea, and phytates (a component of plant foods), rice, and grains inhibit the absorption of nonheme iron. Some of the protein found in soybeans inhibits nonheme iron absorption. Most healthy individuals maintain normal iron stores when the diet provides a wide variety of foods. However, if the diet contains large amounts of oxalates and phytates from dark green leafy vegetables and whole cereal grains the absorption of iron decreases due to binding with iron in the gut. High absorption of heme iron is further advanced by foods containing vitamin C in an acid environment found of the stomach. The recommended for daily iron intake is between 10-18 milligrams for adult males and postmenopausal females. Most endurance athletes consume too much iron. Iron is added to breads, cereals, and most packaged foods.

From a computer-generated dietary analysis on 16 endurance athletes and 9 non-athletes, iron intake from their reported food intake was assessed. The results of this data collect follows:



Diet then likely provides enough iron, but how foods are combined may affect iron absorption rate. Excess iron overdose is unhealthy and should be avoided. Common side effects of acute iron overload are gastro-intestinal pain, constipation, nausea, and heartburn. Excess iron levels may generate a continuous low-grade infection. Foods are the best source of iron. The best food source of iron is liver and red meats. These foods contain heme iron, which is better absorbed than non-heme iron. Non-heme iron can be found in dark green, leafy vegetables (spinach, chard and kale) and whole cereal grains (bran and whole wheat bread). Include dark green, leafy vegetables and whole cereal grains in the daily diet. Oxalates and phytates found in dark green leafy vegetables and whole cereal grains decrease the absorption of iron because they bind with iron in the gastrointestinal tract. Iron fortified cereals increase iron from the diet. Anemia may develop on a meat-free diet and/or if the iron store or intake is low.

Red meat contains arachidonic acid, an EPO-precursor nutrient, but it also contains high levels of saturated fats and cholesterol suggesting a little (now and then) is good but too much will harmfully compromise cardiovascular lipid levels. Adding iron to the diet in supplemental form is not recommended except under the supervision of a physician who is monitoring blood serum levels for a specific outcome. It has been shown that eating red meat 1-2 per week may contribute to providing substrates known to regenerate EPO as shown in animal research. The ability of Arachidonic Acid (AA), the bisenoic prostaglandin precursor to stimulate erythropoiesis and Erythropoietin (EP) Production in exhypoxic polycythemic mice and the programmed isolated perfused canine kidney was found to stimulate erythropoiesis when administered to exhypoxic polycythemic mice in the lowest dose tested (50 microgram/kg i.p.). Endogenously synthesized prostaglandins, their intermediates and/or other products of AA metabolism, such as prostacyclin and prostaglandins play an important role in the control EPO production.[14] Hematocrit levels are restored through the supplying dietary or supplemental specific substrates to support the body’s natural EPO-producing mechanisms during endurance exercise stress.


  1. Acidophilus – 15-30 Billion Count Probiotics
  2. Coenzyme Q10 – 150-300 mg daily
  3. Garlic – 2 cloves or 2 capsules up to 3 x day
  4. Kelp – 100-225 micrograms
  5. Vitamin B6 – 50-100 mg
  6. Vitamin B12 – 200-1,000 mcg
  7. Folic Acid – 800 mcg
  8. Proteolytic enzymes – Bromelain & Papain
  9. Selenium – 200 mcg
  10. Vitamin A – 15,000 IU daily or Beta Carotene – 25,000 IU daily
  11. Vitamin B Complex – 50-100 mg
  12. Vitamin C plus Bioflavonoids – 1-3 grams (divided dose)
  13. Vitamin E – 400 IU daily
  14. Copper – 2 mg
  15. Zinc 40 mg daily —->(Do not take zinc in amounts over 40 mg daily as it may interfere with metabolism of iron and copper)


There is a method to improve iron uptake in the absence of oxalate or phytate rich foods previously mentioned above If hematocrit, hemoglobin, or ferritin blood lab measures are low, the athlete may add 1-gram vitamin C to a 3-4 ounce lean cut of red meat cooked in an iron skillet 1-2 each week.. A complete dietary protocol for cancer patients going through chemotherapy and radiation was published and is applicable to over trained endurance athletes who present low hematocrit levels.[16]


Nutritional imbalance from caloric restriction (or exercise related expense), dehydration, fluid intoxication, excess calcium, excess inositol, excess oxalates foods[17], excess phytic acid from cereal grains[18], or a lack of hypoxic (interval training) are factors that inhibit the natural production of Erythropoietin (EPO).

Manipulating diet for protein and total calorie adequacy, monitoring hydration, supplements, timing food combinations, adding weekly hypoxic exercise followed by easy or rest days increases the release of natural EPO for healthy maximal oxygen carrying capacity. When individual hematocrit exceeds 48%, the risk of insulin resistance syndrome and stroke exponentially increase. Men with hematocrits above 48 percent have a 400% increased risk of non-insulin-dependent-diabetes mellitus. This research followed over 7,000 middle-aged men for more than 12 years, and discovered that the risk of diabetes increases proportionate to hematocrit increase.[19] [20] The upper recommended levels for a female is slightly lower at 45%.

This nutritional intervention parallels exercise intensitys effect for increasing EPO. Nutritional and training interventions for resolving low EPO levels during iron supplementation (only prescribed by a physician who should monitored progress) should not be permitted above a reference range of 48% in males and 45% in females. Similar research confirms this report.[21] [22] [23]

Erythropoietin (EPO) is most commonly known as the cytokine secreted by the kidneys that stimulates red blood cell production and is used as a drug for the treatment of anemias.

Epo is also secreted in the brain in response to hypoxia, such as ischemic stroke. Epo production in the brain is stimulated by the hypoxia-inducible transcription factor HIF-1 (see HIF pathway). Administration of Epo to the brain in rodents before hypoxic stress or other neuronal stresses is neuroprotective, preventing neuronal apoptosis. The erythropoietin receptor (EpoR) is known to associate with JAK kinases that phosphorylate and activate the STAT family of transcription factors (See Epo pathway).

The neuroprotection by Epo involves cross-talk between Epo receptor and anti-apoptotic pathways through activation of NF-kB by the JAK2 kinase (see NF-kB pathway). Epo stimulates JAK2 phosphorylation of I-kB, releasing NF-kB to translocate into the nucleus and activate transcription of neuroprotective genes. Neuroprotective genes activated by NF-kB include the anti-oxidant enzyme manganese superoxide dismutase and calbindin-D(28k). The erythropoietin receptor is also essential for proper brain development in mice. The absence of EpoR causes high levels of neuronal apoptosis in the developing mouse brain, further confirming the important role of Epo as a neuroprotective agent.

  • [6] Fisher JW. Pharmacologic modulation of erythropoietin production. Annu Rev Pharmacol Toxicol. 1988;28:101-22.
  • [7] Plasmapheresis is the process of separating certain cells from the plasma in the blood by a machine; only the cells are returned to the person. Plasmapheresis can be used to remove excess antibodies from the blood.
  • [8] Roberts D, Smith DJ, Donnelly S, Simard S., Plasma-volume contraction and exercise-induced hypoxaemia modulate erythropoietin production in healthy humans. Clin Sci (Lond). 2000 Jan;98(1):39-45.