Chromium Deficiency The Predicted Underlying Cause for Up-regulation of Calcium8 Signaling in Non-Excitable Cells

Based on the Author's US Provisional Patent:  60/496, 397 

Atef  M.  Elayyan
Amman  -   Jordan
atefesmail65@gmail.com

https://orcid.org/0000-0002-3198-5418

ABSTRACT

BACKGROUND:
Calcium signaling controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, generated by various plasma membrane calcium channels.  The up-regulation of calcium signaling in non-excitable cells is implicated in many disorders like diabetes complications, asthma, chronic allergy, and cancer.

PURPOSE:
The purpose of this research was to discover the mechanisms underlying the upregulation  of calcium signaling in non-excitable cells, and how to reverse them.  This may present a therapeutic approach for many disorders associated with the up-regulation of calcium signaling in non-excitable cells as mentioned above.

RESULTS:
We propose firstly, that chromium chloride (CrCl₃)   is the positively charged molecule attached to the nitrogen atoms on the amino acids lysine or arginine side chains in  voltage sensing segments S₄ in L-type calcium channels situated in the cell membrane of some non-excitable cells with covalent bonds.  

Secondly, we propose   that  when it totally releases  from  these segments   due  to a combination of genetic vulnerability, environmental risk factors, oxidative stress, and aging, we predict that a continuous  extracellular calcium ion influx  diffuses into  the cytosol leading to amplification of  oscillation frequency of  cytosolic calcium currents thus  predicted to   up-regulate  calcium signaling which in turn over- activates calcium-dependent signaling pathways. These over-activated signaling pathways are also predicted to cause specific  over-activated and impaired  biological processes leading to  many conditions including but not limited to diabetes complications like ( retinopathy, cataracts, open-angle glaucoma, nephropathy, essential hypertension, ED ), cancer, asthma, and chronic allergy. 

Finally, we propose that when  this molecule totally releases from voltage sensing segments (S4) in voltage-gated ion channels in some peripheral, autonomic or proximal neurons, it is predicted to lead to altered ion concentration and impaired neuronal function, thus, diabetic neuropathy and ED  are predicted to develop.

CONCLUSION:
Based on our preliminary results – which are consistent with well established scientific literature -  we predict that chromium supplements serve as a long-term  and intermittent  adjuvant approach that may alleviate symptoms of essential hypertension and  ED, or even  cure neuropathy, cataract, asthma, and chronic allergy effectively. Also, may retard the progression of other diabetes complications like ( retinopathy, nephropathy, open-angle glaucoma), and cancer effectively.  Daily dose 200 mcg orally for a limited period of 12 weeks for adults along with standard treatment, preferably under medical supervision. 

Keywords:  Calcium signaling, asthma, chronic allergy, cancer, diabetes complications, diabetic retinopathy, cataracts, diabetic neuropathy, diabetic nephropathy, glaucoma, essential hypertension & ED.

MATERIALS AND METHODS:

We depended on this research on a published medical study showing that diclofenac sodium drug may affect the male ejaculation process.  In order to study this effect on the modulation of the activity of voltage-gated ion channels located in chromaffin cells at adrenals that could be involved in the ejaculation process, 100 mg of diclofenac sodium was administered daily for ten days to a normal  35 years old male, during which frequent ejaculations occurred.  At the end of the ten-day period, results were recorded. Then this subject was administered   120 mcg of chromium chloride daily for 12 weeks.  At the end of the 12 week period, these results were also recorded. We  have proposed  the following hypothesis:

HYPOTHESIS No. 1:

We propose that nerve impulse resulting from sexual stimulation travels from pelvic plexuses throughout sympathetic trunk to suprarenal plexuses and is blocked temporarily in voltage-gated ion channels in chromaffin cells at adrenals depolarizing ion channels.  When depolarization reaches the threshold of channels, these channels open and electrolyte discharge (orgasm) occurs and nerve impulse is released throughout suprarenal plexuses to the sympathetic trunk then through pelvic plexuses to pelvic muscles causing contraction and squeezing seminal vesicles thus, ejaculation process occurs. 

This hypothesis is based on a fact that nerves are connected to voltage-gated ion channels located in chromaffin cells at adrenals, so there should be a function for these nerves. Also, it is based on an in vitro experiment stating that: ( By using fluorescent dye to detect Calcium ions influx it was noticed that there is a blocking of calcium ions influx in Adrenal chromaffin cells).

Also, this evidence comes from anatomy:  " The suprarenal plexus supplies the suprarenal gland, being distributed chiefly to its medullary portion;  its branches are remarkable for their large size in comparison with that of the organ they supply "  (Gray, Henry.  1918. Anatomy of the human body). 

·       So, if nerve impulse is not blocked temporarily in voltage-gated ion channels located in chromaffin cells at adrenals, due to voltage-gated ion channels gating mechanism malfunction, then premature ejaculation will occur.

In order to test this hypothesis, we conducted the following case study:

CASE STUDY No. 1:

100 mg of diclofenac sodium was administered daily for ten days to a normal 35 years old male, during which frequent ejaculations occurred.  At the end of  the ten days, results were recorded.

RESULTS OF CASE STUDY NO. 1:

·         It was observed that the subject suffered from premature ejaculation followed by administering 100 mg of diclofenac sodium daily for ten days during which frequent ejaculations occurred.

HYPOTHESIS No. 2:

Also, we propose that: “ There should be molecules in the structure of voltage-gated ion channels located in chromaffin cells at adrenals vital for the function of these channels.  The release of these molecules leads to a failure in these channels thus they no more can block nerve impulse resulting from sexual stimulation leading to premature ejaculation.  And valency of these molecules should be higher than sodium valency (i.e higher than1⁺) because diclofenac sodium drug absorbed it causing failure in ion channels leading to premature ejaculation. 

Since sodium atom lies at the end of the molecular structure of this drug, so the sodium atom could be substituted by another atom with a valency higher than 1⁺. The molecular structure of diclofenac sodium is C₁₄H₁₀Cl₂No₂Na.

Since chromaffin cells are stained with chromium chloride  it gives them brownish color due to  oxidation of catecholamines so,  (CHROMIUM CHLORIDE) could be  the molecules vital for the function of voltage-gated ion channels  in chromaffin cells in adrenals, since valency of chromium  is  Cr ⁺³  and it's higher than valency of  Sodium (Na ¹⁺ ) .

·         So, if male suffering from premature ejaculation is given chromium chloride for a limited period of time,  which is implicated in voltage-gated ion channels gating mechanism located in chromaffin cells, then he will be free from premature ejaculation.

To test this hypothesis, we have conducted the following case study:

CASE STUDY No. 2:

In a case study in the same subject who had been given diclofenac sodium and caused him premature ejaculation, the subject was given  120 mcg of chromium chloride daily for 12 weeks. At the end of the 12 weeks, results were recorded.

RESULTS OF CASE STUDY No. 2

·         It was observed that the subject was free from premature ejaculation followed by administering 120 mcg chromium chloride daily for 12 weeks:

ANALYSIS OF CASE STUDIES:

In the first case study: 

It was observed that the subject suffered from premature ejaculation followed by administering 100 mg of diclofenac sodium daily for ten days during which frequent ejaculations occurred. Because diclofenac sodium absorbed chromium chloride from voltage-gated ion channels located in a chromaffin cell at adrenals which are involved in the ejaculation process thus, gating mechanism in these ion channels malfunctioned and could not block temporarily nerve impulse resulting from sexual stimulation, thus, the subject suffered from premature ejaculation.

Hence, providing an answer to hypothesis No. 1.  ( Mutations   introduced in the positive residues of S₄ segments alter the voltage dependence of channel  activation) N.   Davidson, 1989.

Strong evidence supporting these case studies  and proves that chromium chloride is a modulator of activity of voltage-gated ion channels and supporting that diclofenac sodium drug absorbs chromium chloride from voltage-gated ion channels in chromaffin cells leading to malfunction of gating mechanism, thus permanent premature ejaculation will occur according to  the following formula:

C₁₄H₁₀Cl₂No₂Na+ CrCl₃            C₁₄H₁₀Cl₂(No₂)₃Cr + NaCl

n the second case study: 

It was observed that the subject was free from premature ejaculation followed by administering 120 mcg chromium chloride daily for 12 weeks.  Because of chromium chloride attached with S₄ ( voltage sensing segments) in the same voltage-gated ion channels located in chromaffin cells at adrenals and restored totally released chromium chloride in first case study so,  gating mechanism in these ion channels restored  its proper functionality and could block  temporarily nerve impulse resulting from sexual stimulation thus, the subject recovered from premature ejaculation.  Hence, providing an answer to hypothesis No. 2.

Pathophysiology  of Some Disorders Associated with Up-regulation of Calcium Signaling:

ASTHMA:

Asthma is a chronic disease involving the airways in the lungs. These airways, or bronchial tubes, allow air to come in and out of the lungs.

If you have asthma your airways are always inflamed along with mucus overproduction. They become even more swollen and the muscles around the airways can tighten when something triggers your symptoms. This makes it difficult for air to move in and out of the lungs, causing symptoms such as coughing, wheezing, shortness of breath and/or chest tightness.

In asthmatics, chromium chloride is proposed to release totally from some L-type calcium channels situated on airway smooth muscle cell membranes.  These channels mediate intracellular and extracellular calcium release,  thus,  a continuous extracellular calcium ions influx diffuses into the cytosol.  This influx does not initiate any action unless these patients are exposed to allergens, air pollution or certain medications.

Upon exposure of asthmatics to  the above-mentioned antigens,  these antigens bind to the antigen-binding sites, which are situated on the variable regions of the IgE molecules bound to the mast cell surface.  Mast cells can be stimulated to degranulate by antigens through cross-linking with immunoglobulin E (IgE) receptors leading to histamine production.

Binding of histamine to histamine H1 receptor in airway smooth muscles results in phospholipase C (PLC) activation leading to the generation of the second messenger's inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃stimulates intracellular Ca²⁺ release from ER and DAG causes protein kinase C (PKC) activation. 

Another pathway involved in ASM contraction is  SOCE ( Store-operated calcium entry) channels pathway. When ER compartments deplete, as mentioned above, SOCE  ( Store-operated calcium entry) channels are activated to replenish the depleted ER compartments. SOCE entry channels provide Ca ions for the  ER  necessary for ASM contraction.  

Also, another pathway involved in airway smooth muscle contraction is via leukotrienes, which are pro-inflammatory mediators more potent than histamine,  released by mast cells in response to allergens.  They activate  Cysteinyl-LTs receptors which induce calcium ions release from ER to the cytosol.  This process provides Ca ions for  the cytosol necessary for ASM contraction.
    

The oscillation frequency of calcium ion currents generated via the above-mentioned pathways is predicted to be amplified by the extracellular continuous calcium, ions influx resulted from the proposed release of chromium chloride from L-type calcium channels.  The information transmitted by these Ca²⁺ waves can arrive as a stimulus at the plasma membrane and is translated into intracellular Ca²⁺ oscillations (Falcke 2004).High-frequency calcium ions oscillations give a strong contractile response ( Inga Wang, et al -  2008).

These  high-frequency calcium ion  oscillations  are  predicted  to up-regulate the above mentioned calcium signaling pathways  which in turn over-activate the PKC pathway leading to prolonged activation of calmodulin interaction stimulating myosin light chain (MLC) phosphorylation causing ASM  hyper-responsiveness characterized by severe and prolonged ASM contraction,  thus,  an asthmatic attack develops.

Rho-associated kinase, also known as ROCK, belongs to the AGC (PKA/PKG/PKC) family of serine/threonine kinases.The Rho signaling pathway, mediated through ROCK, plays a major role in the of smooth muscle contraction, through the regulation of the actin-myosin filament bundles (   regulation A. J. Lee and I. Goldberg, - 2011), ( B. Colligris, et al, 2012).

 The up-regulated  RhoA/Rock pathway leads to continuous phosphorylation of myosin light chain ( MLC).  Activated RhoA/ROCK  pathway leads to the phosphorylation of myosin light chain (MLC) (E. Nakajima, et al - 2005), in which combined with actin induces a calcium-independent smooth muscle contraction.

In summary, up-regulation of calcium signaling and over-activation of calcium-dependent and independent signaling pathways, via the above-mentioned mechanisms are proposed to be implicated in the pathogenesis of asthma. 

CASE STUDY:

In a case study in one 35 years old male diagnosed with asthma, the subject was given  120 mcg of chromium picolinate daily for 12 weeks orally.  At the end of the 12 weeks, the subject showed a significant improvement in the symptoms of this condition.

CHRONIC ALLERGY:

Allergies, also known as allergic diseases, are a number of conditions caused by hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic dermatitis, and anaphylaxis. Symptoms may include red eyes, an itchy rash, sneezing, a runny nose, shortness of breath, or swelling. Food intolerances and food poisoning are separate conditions. Common allergens include pollen and certain food. Metals and other substances may also cause problems.

In allergics, chromium chloride is proposed to totally release from some L-type calcium channels situated in mast cells membrane.  These channels mediate intracellular and extracellular calcium release,  thus a continuous extracellular calcium ions influx diffuses into the cytosol.  This influx does not initiate any action unless these patients are exposed to allergens.

Upon exposure of allergics to airborne allergens, certain medications, certain food, insect stings or a touch of a certain substance,  these antigens bind to the antigen-binding sites, which are situated on the variable regions of the IgE molecules bound to the mast cell surface.  Mast cells can be stimulated to degranulate by allergens through cross-linking with immunoglobulin E (IgE) receptors leading to overproduction of histamine and other mediators through the following process: 

Binging of IgE antibodies to their receptors  on mast cells result in phospholipase C (PLC) activation leading to the generation of the second messenger's inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃stimulates intracellular Ca²⁺ release from the ER and DAG causes protein kinase C (PKC) activation.  This process  provides Ca ions for  the cytosol necessary for the degranulation process.

Another pathway involved in degranulation is  SOCE ( Store-operated calcium entry) channels pathway. When ER compartments deplete, as mentioned above, SOCE  ( Store-operated calcium entry) channels are activated to replenish the depleted ER compartments. SOCE entry channels  provide Ca ions  for the  ER  necessary for the deregulation process.

Also, another pathway involved  in the immune response is via leukotrienes, which are pro-inflammatory mediators more potent than histamine,  released by mast cells in response to allergens.  They activate  Cysteinyl-LTs receptors which induce calcium ions release from ER to the cytosol, contributing to immune mediators release.

The oscillation frequency of calcium ion currents generated via the above-mentioned pathways is predicted  to be amplified by the extracellular continuous calcium ions influx resulted from the proposed release of chromium chloride from L-type calcium channels.  The information transmitted by these Ca²⁺ waves can arrive as a stimulus at the plasma membrane and is translated into intracellular Ca²⁺ oscillations (Falcke 2004). High-frequency calcium ions oscillations are predicted to enhance the degranulation process.

These high-frequency calcium ion  oscillations  are  predicted  to cause continuous immunological hyperactivity characterized by prolonged degranulation and overproduction of histamine and other meditators from mast cells, thus,  the allergic attack develops.

In summary, up-regulation of calcium signaling and over-activation of calcium-dependent signaling pathways, via the above-mentioned mechanisms are proposed to be implicated in the pathogenesis of chronic allergy.

CASE STUDY:

In a case study in one 26 years, old female diagnosed with allergic rhinitis, the subject was given  120 mcg of chromium picolinate daily for 4 weeks orally.  At the end of the 4 weeks, the subject showed a significant improvement in the symptoms of this condition.

CANCER:
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.  These contrast with benign tumors, which do not spread to other parts of the body.  Possible signs and symptoms include a lump, abnormal bleeding, prolonged cough, unexplained weight loss and a change in bowel movements.  While these symptoms may indicate cancer, they may have other causes.  Over 200 types of cancers affect humans.

We propose that when chromium chloride  totally releases from  L-type calcium channels in the cell nucleus, a continuous  calcium ions  influx diffuses  into the cytosol  which is predicted to amplify oscillation frequency of cytosolic calcium currents which up-regulate calcium signaling which in turn over-activates calcium-dependent multiple signaling pathways such as PKC isoforms, TGF-β isoforms, TNF, VEGF, PI3K-Akt,  Ras-ERK, STAT,  MAPK, APC, NOTCH, HH,  and other pathways, while down-regulation of some pathways. 

These over-activated multiple pathways are predicted to initiate a cascade of uncontrolled biological processes like cell proliferation, cell growth, angiogenesis, apoptosis evasion, immune evasion,  and metastasis, thus, cancer is predicted to develop. 

Calcium signaling pathways are remodeled or deregulated in cancer that results in changes in their physiology and distinguish them from non-malignant cells ( Hanahan and Weinberg 2000; Wang et al. 2010).

Deregulation of the calcium signal is often deleterious and has been linked to each of the 'cancer hallmarks'.  Stewart TA^, et al (2015).

Studies show that there is a critical role for RhoA/ROCK activation in the pathogenesis of cancer. 

In summary, up-regulation of calcium signaling and over-activation of calcium-dependent and independent signaling pathways, via the above-mentioned mechanisms are  proposed  to be implicated in   the  pathogenesis of cancer.

DIABETES COMPLICATIONS:

Prolonged hyperglycemia leads to the development of long-term microvascular and macrovascular complications which cause morbidity and mortality. Microvascular complications (involving small vessels such as capillaries) and macrovascular complications (involving large vessels, such as arteries and veins). Microvascular complications include diabetic retinopathy, macular edema, cataract, glaucoma, and nephropathy, while macrovascular complications include coronary artery disease, peripheral arterial disease, stroke, and ED.  Also, neuropathy due to the  impairment  of  some nerve cells.

Hyperglycemia-induced oxidative stress is now regarded as the driving force for the development of diabetic complications. Oxidative stress in diabetes results from stimulation of the polyol pathway and the formation of advanced glycation end products (AGE).  Hyperglycemia generates reactive oxygen species (ROS) and weakens antioxidative mechanisms by scavenging enzymes and substances.  ( Omolola R. Ayepola et al).

Many studies indicate that oxidative stress contributes to increased signaling transduction leading to VEGF and cytokines overproduction, leading to alterations in tight junctions and vascular hyperpermeability,  thus some diabetic complications develop.  ( Tiffany Frey1 and David A. Antonetti - 2011).

Therefore, intermittent antioxidant supplementation is highly recommended to be included in the diabetics standard treatment plan as long-term adjuvant therapy. To be followed by intermittent chromium supplementation. (Author).

Another point to state is long-term hyperglycemic state results in mitochondrial dysfunction.  Thus,  mitochondria continue to generate ROS and RNS even though glucose levels are controlled and conventional antioxidants are supplemented.  In this case, alpha-lipoic acid or coq10 is highly recommended that may alleviate mitochondrial dysfunction,  scavenge ROS, and RNS, improve cognitive functions and arterial stiffness in the elderly diabetics.

Finally, postprandial hyperglycemia is associated with diabetes complications via two pathways: 1) It triggers oxidative stress. 2) Via direct activation of PKC receptors through the DAG-PKC pathway.  DAG   is derived from glucose via De novo synthesis of glucose.  As it is widely accepted, PKC activation is associated with the development and progression of diabetes complications.  We shall discuss this in detail later in this article.

In summary, many studies conclude that antioxidants are an important long-term adjuvant approach that may prevent the development of diabetes complications.
DIABETIC RETINOPATHY:
People with diabetes can have an eye disease called diabetic retinopathy. This is when high blood sugar levels cause damage to blood vessels in the retina. These blood vessels can swell and leak. Or they can close, stopping blood from passing through. Sometimes abnormal new blood vessels grow on the retina. All of these changes can steal your vision.

Increased glucose levels promote the formation of  ROS  (oxidative stress) as discussed above.  ROS ( reactive oxygen species )  are proposed to  totally release chromium chloride from L-type calcium channels in  support cells such as glia, microglia, and pericytes, a continuous  calcium ions  influx diffuses into the cytosol,   which is predicted to amplify oscillation frequency of cytosolic calcium currents and up-regulates calcium signaling which in turn over-activates the multiple PKC isoforms, leading to overproduction of cytokines (TNF-α, IL-1β) and VEGF, thus diabetic retinopathy is predicted to develop. 

A number of studies support a role for ROS in the activation of signal transduction pathways that result in increased production of growth factors and cytokines. (Tiffany Frey1 and David A. Antonetti -2011). Cytokines (TNF-α, IL-1β) and VEGF  overproduction results in cellular modifications in adhesion cells and tight junctions leading to blood-retinal barrier breaks down and vascular hyperpermeability, which are hallmarks of diabetic retinopathy.

CASE STUDY:

In a case study in one 50 years old male diagnosed with diabetic retinopathy, characterized by fluid leakage, macular edema, and blurred vision. The subject was given several anti-VEGF injections and laser sessions.  We started a conventional antioxidant course for 12 weeks, followed by a course of chromium picolinate 120 mcg daily for 4 weeks orally.  During which anti-VEGF injections were given to the subject.  At the end of the 4 weeks, the subject showed limited improvement in symptoms of this condition even with controlled glucose levels.  Thus, he continued to take anti-VEGF injections. 
  
We doubted that there might be mitochondrial dysfunction in retinal capillaries, in this case, the mitochondria continue to generate ROS even though with controlled glucose levels and conventional antioxidant administration.  So, we decided to give the subject a course of 300 mg alpha-lipoic acid daily for 12 months, to be followed by a course of 120 mcg daily of chromium picolinate for 4 weeks.  During the past period of time, he was given anti-VEGF injections.  Glucose levels were also under control. 

After 6 months of the  end of 4 weeks, the subject showed a significant improvement in the symptoms of diabetic retinopathy.  There was no leakage in the retinal capillaries, no macular edema as OCT images show, visual acuity has improved significantly and blurred vision has been improved. Also, this intervention retarded the progression of this condition.  The subject discounted anti-VEGF injections, but still under conventional antioxidant administration on a regular basis and chromium picolinate on intervals as maintenance intervention to protect him against re-development of diabetic retinopathy and other complications.

CATARACTS:

A cataract is a clouding of the lens in the eye which leads to a decrease in vision.^[ Cataracts often develop slowly and can affect one or both eyes. Symptoms may include faded colors, blurry vision, halos around light, trouble with bright lights, and trouble seeing at night.  This may result in trouble driving, reading, or recognizing faces.  Poor vision caused by cataracts may also result in an increased risk of falling and depression.  Cataracts cause half of all cases of blindness and 33% of visual impairment worldwide. In lens cells, proteins are lined up in a way to permit light to pass through the lens. 

The theory of light scattering indicates that aggregation of the crystalline lens proteins can produce opacity. Furthermore, it is known that calcium increases in the cataractous lens.  without any obstacles.  Cataracts are proposed to associate with increased levels of calcium ions in lens cells.

Marko Gosak et al, have demonstrated that their findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered  Ca²⁺ signaling of LECs in cataractogenesis. ( MarKo Gosak et al, 2015).

We propose  that, oxidative stress  releases  chromium chloride from lens cell proteins, which are predicted to up-regulate calcium signaling and increases the levels of calcium ions in these cells.  High calcium  ions levels are  predicted to cause miss folding and aggregation of a2 crystallin in lens cells which are apparently associated with disulfide bond formation resulting in the development of  insoluble light scattering aggregates, thus cataracts  are predicted to develop.

J. A. Jedziniak et al,  have investigated the effectiveness of calcium ion in producing aggregates of the soluble proteins of the bovine lens. The experimental results show 1)  CaCa₂ produces aggregates of the soluble protein of the lens;  2)  The aggregation can be reversed and transparency restored when calcium is removed either by dialysis or chelation. (www.iovs.arvojournals.org ).

CASE STUDY:
In a case study in one 52 years old male diagnosed with cataract, the subject was given  120 mcg of chromium picolinate daily for 12 weeks orally after a course of conventional antioxidants.  At the end of the 12 weeks, the subject showed a significant improvement in the symptoms of this condition.  The subject continued  intermittent antioxidant  supplementation  after the course of chromium picolinate supplementation.

OPEN ANGLE  GLAUCOMA:
Open-angle glaucoma, the most common form of glaucoma, accounting for at least 90% of all glaucoma cases:  Is caused by the slow clogging of the drainage canals, resulting in increased eye pressure.   It has a wide and open-angle between the iris and cornea develops slowly and is a lifelong condition, and has symptoms and damage that are not noticed.

OA glaucoma is the most common cause of irreversible blindness. This neurodegenerative disease becomes more prevalent with aging and oxidative stress.

Several signaling pathways are implicated in OA glaucoma pathogenesis including TGF-, MAP kinase, Rho kinase, BDNF, JNK, PI-3/Akt, PTEN, Bcl-2, Caspase, and Calcium-Calpain.  We shall discuss the two main pathways:  TGF and Rho pathways:

TGF Pathway:

We propose that when chromium chloride totally releases from L-type calcium channels in trabecular meshwork smooth muscle cells due to aging and diabetic induced oxidative stress, a continuous influx of calcium ions diffuses into the cytosol which is predicted to up-regulate calcium signaling. 

Up-regulated calcium signaling, in turn, over-activates  the PKC pathways, leading to overexpression of TGF-B1  and TGF-B2 causing fibrosis by increasing the production and deposition of extracellular matrix proteins in trabecular meshwork cells, thereby blocking the outflow of aqueous humor,  leading to elevated IOP, thus,  OA glaucoma is predicted to develop.

Patients with OAG have significantly increased levels of TGF-2 in the aqueous humor compared to people with other types of glaucoma and healthy controls.  (A. Ozcan et al. 2004).

Rho/ROCK Pathway:
The up-regulated  RhoA/Rock pathway leads to continuous phosphorylation of myosin light chain ( MLC).  Activated RhoA/ROCK  pathway leads to the phosphorylation of myosin light chain (MLC) (E. Nakajima, et al - 2005), in which combined with actin induces a calcium-independent smooth muscle contraction, which contributes to outflow obstruction in trabecular meshwork leading to elevated IOP, which is a hallmark of OA glaucoma, thus  OA glaucoma is predicted to develop.

When elevated IOP  is not treated, it causes biomechanical stress to the optic nerve. Two hypotheses have emerged to explain the development of glaucomatous optic neuropathy, the mechanical and ischemic theories. The mechanical theory stresses the importance of direct compression of the axonal fibers and support structures of the anterior optic nerve, with distortion of the lamina cribrosa plates and interruption of axoplasmic flow, resulting in the death of the RGCs. The ischemic theory focuses on the potential development of intraneural ischemia resulting from decreased optic nerve perfusion. This perfusion may result from the stress of IOP on the blood supply to the nerve or from processes intrinsic to the optic nerve.

Also, we propose that biomechanical stress causes the total release of chromium chloride from the optic nerve voltage-gated ion channels, which is predicted to cause degeneration of the optic nerve.

Apoptosis:
Apoptosis refers to a common mode of cell death. It is a subtle process where the cell initiates a death program and commits suicide.  There are many triggering factors for apoptosis, be it extracellular or intracellular events. These include trophic factor deprivation or oxidative damage, both of which have been postulated to induce RGC apoptosis in glaucoma. 

Caspases are proteases that execute the dismantling and demolition of apoptotic cells. Caspases are categorized into two broad groups: initiators (e.g. caspases 8 and 9) which activate other caspases and effectors (caspase 3) which cleave specific substrates involved in cellular disassembly.    Some experimental glaucoma models have shown that the initiator caspases are activated, while inhibition of the effector caspases can be neuroprotective. 
Neurotrophin withdrawal:
Neurons require neurotrophic growth factors, which are acquired by retrograde axoplasmic transport. These growth factors, known as neurotrophins, regulate cellular metabolism hence maintaining the normal cellular milieu. Thus, where neurotrophic support is absent due to retrograde axonal transport block, RGCs die.  This group of small growth peptides comprises brain-derived neurotrophic factors (BDNF), nerve growth factors (NGF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). Lack of BDNF and NGF secreted by RGC targets results in apoptosis of developing RGC.
Calcium-dependent pathways:
Calcium overload is also responsible for the activation of calpain and caspase cascades, leading to apoptosis.  Finally, Calcium-induced neuronal apoptosis is dependent on calcineurin which is a calcium-dependent phosphatase and facilitates the dephosphorylation of the pro-apoptotic mitochondrial membrane-bound proteins i.e. Bad. The dephosphorylation, in turn, results in its translocation from the cytoplasm to the mitochondria, where it binds to Bcl-2 or Bcl-X_L, forming protein complexes, which raise mitochondrial membrane permeability, release cytochrome C and cause neuronal suicide.
Vascular insufficiency
Impaired blood flow and disruption of autoregulation of the optic nerve blood flow are thought to contribute to the pathogenesis of glaucoma. Vascular insufficiency is also associated with elevated endothelin-1 levels in the aqueous humor and plasma. Endothelin-1 is a potent vasoconstrictor that further compromises blood supply to retinal tissues.

Proposed pathophysiology of elevated endothelin-1 and tumor necrosis factor (TNF) is due to the over-activation of  the PKC pathways resulted from up-regulation of calcium signaling due to the proposed release of chromium chloride from L-type calcium channels.   Another pathway implicated in vascular insufficiency is impairment of NO synthesis, which results in impairment of vascular vasodilation which contributes to vascular insufficiently.  

In summary, while some diabetics may develop OA glaucoma especially with aging, long-term diabetes and family history, it is not necessarily that all diabetics develop OA glaucoma throughout their lifespan.

DIABETIC NEUROPATHY:
It is nerve impairment and one of the most common complications of diabetes.  More than 70 % of diabetics will develop diabetic neuropathy at some point.  There is no known cure for diabetic neuropathy globally.

There are four types of diabetic neuropathy:  Peripheral, autonomic, proximal and focal neuropathy. 

Proposed Pathophysiology:
Proposed pathophysiology of peripheral neuropathy is that when chromium chloride is proposed to totally release from voltage sensing segments (S₄) in voltage-gated ion channels like sodium, potassium, calcium and chloride channels from some peripheral neurons due to diabetic induced oxidative stress,  thus leading to altered ion concentration which impairs neuronal function. 

For example, in voltage-gated sodium channels in some peripheral neurons,  this proposed total release is predicted to inhibit the inactivation of these channels leading to continuous sodium ions influx into these neurons, causing recurrent action potentials and hyperexcitability.  The brain interprets this hyperexcitability as pain.  Also, in these neurons, voltage-gated potassium channels activity is reduced due to increased  (BDNF)  protein activity likely through tyrosine receptor kinase B (TrkB ) stimulation which contributes to further hyperexcitability. 

Finally, altered ion concentration due to proposed chromium chloride release is also predicted to inhibit the generation of action potentials and propagation of nerve impulse, thus, leading to numbness, tingling, and loss of sensation in feet, hands, and loss of balance and coordination.

Proposed pathophysiology of autonomic  neuropathy is that  chromium chloride release   from voltage sensing segments (S₄) in  voltage-gated  ion channels   in  some autonomic nerves  is predicted  to alter ion concentration, inhibits  generation of action potentials and propagation of nerve impulse, thus leading to changes in the function of  internal organs controlled by the autonomic system, thus, autonomic neuropathy  is predicted to develop.

Proposed pathophysiology of proximal   neuropathy is that chromium chloride release from voltage sensing segments (S₄) in  voltage-gated  ion channels   in  some motor  nerves  is predicted  to alter ion concentration, inhibits the generation of action potentials and propagation of nerve impulse, thus leading to  muscle weaknesses, and trouble getting up from a seated position,  thus, proximal neuropathy  is predicted to develop.  As for pain pathophysiology, it is the same as mentioned in peripheral pathophysiology above.

As for focal neuropathy, it is temporary due to nerve compression leading to blocking of nerve impulse, rather than nerve impairment resulted from diabetes. Elderly diabetics are three times more likely to develop nerve compression and this type of diabetic neuropathy.  So, chromium picolinate has no effect on this type of diabetic neuropathy.

CASE STUDY:

In a case study in one 52 years old male diagnosed with peripheral and autonomic diabetic neuropathy, the subject was given  120 mcg of chromium picolinate daily for 12 weeks orally after a course of antioxidants.  At the end of the 12 weeks, the subject showed a significant improvement in symptoms of these two conditions.  The subject continued  intermittent antioxidant  supplementation  after the course of chromium picolinate supplementation.

DIABETIC NEPHROPATHY:
Diabetic nephropathy (diabetic kidney disease) is kidney damage that results from having diabetes.  Having high blood glucose levels due to diabetes can damage the part of the kidneys that filter your blood. The damaged filter becomes 'leaky' and lets protein into your urine.

For some people, diabetic nephropathy can progress to kidney failure. However, most people with diabetes do not develop kidney disease that progresses to kidney failure.

Diabetic nephropathy is characterized by initial glomerular hyperfiltration, progressive accumulation of ECM in glomerular  mesangium and tubulointerstitium, and progressive renal insufficiency.

Glomerular hyperfiltration:
The increased glomerular filtration rate is described in the kidney of diabetic patients and animal models. This alteration is likely to be the result of hyperglycemia-induced decreases in arteriolar resistance, leading to an elevation of glomerular filtration pressure. Multiple mechanisms have been proposed to explain the increases in glomerular filtration rate and filtration pressure, including an enhanced activity of angiotensin II and prostaglandin productions.  

Activation of the DAG-PKC pathway may play a role in both the enhancement of angiotensin II actions29 and increases in vasodilatory prostaglandins. The enhanced production of prostaglandin E2 induced by diabetes and hyperglycemia could be the result of sequential activation of PKC and cytosolic phospholipase A2, a key regulator of arachidonic acid synthesis.

Increases in the activities of nitric oxide (NO) may also enhance the glomerular filtration rate.  Urinary excretion of NO2 and NO3, metabolites of NO, has been reported to be increased in diabetes of short duration, NO production can be mimicked by PKC agonists and inhibited by PKC inhibitors when induced by hyperglycemia,  suggesting that NO production might be increased in diabetes through PKC-induced up-regulation of inducible NO synthase.
Accumulation of ECM:
Thickening of glomerular basement membrane and accumulation of ECM in the glomerular mesangium and tubulointerstitium are the hallmark of diabetic nephropathy. It has been reported that high glucose increased the production of type IV collagen and fibronectin in mesangial cells. This finding could be mimicked by phorbol ester, PKC agonist, and reversed by general PKC inhibitors.

Many studies have suggested that transforming growth factor (TGF)-b1 plays a key role in the accumulation of ECM. It has been reported that PKC activation can increase the production of ECM and TGF-β1 expression and that PKC inhibitors can prevent hyperglycemia or diabetes-induced increases in ECM accumulation and TGF-β1 production in mesangial cells or renal glomeruli.

Vascular permeability and albuminuria
Increased vascular permeability is another characteristic of systemic vascular abnormality in diabetic animals, suggesting endothelial cell dysfunction. PKC activation can directly increase the permeability of albumin and other macromolecules through barriers formed by endothelial cells.

A number of studies support a role for ROS in the activation of signal transduction pathways that result in increased production of growth factors and cytokines. (Tiffany Frey1 and David A. Antonetti -2011). Cytokines (TNF-α, IL-1β) and VEGF  overproduction are predicted to results in cellular modifications in adhesion cells and tight junctions contributing to diabetic nephropathy development.

Rho/Rock Pathway:
Studies show that there is a critical role for RhoA/ROCK activation in the pathogenesis of diabetic nephropathy.

In summary, we propose  that, when chromium chloride releases  from L-type calcium channels in some renal tissues due to diabetic induced oxidative stress,  it is predicted to up-regulate  calcium signaling which in turn  over-activates  the PKC pathway  leading  to overproduction of VEGF and cytokines  (TNF-α, IL-1β)  which leads to a/m dysfunctions, thus diabetic nephropathy  is predicted to develop.

Ruboxistaurin, a specif PKC-β isoform inhibitor, has been shown to ameliorate many functional features of experimental diabetes.  In experimental diabetic animals, administration of RBX prevented elevated glomerular filtration rate, albuminuria, mesangial expansion, production of ECM proteins, including collagen IV and fibronectin, expression of  TGF-β and CTGF, and glomerulosclerosis ( despite continued hyperglycemia).  Koya D. Jirousek MR, Lin YW, et al.  1997).

ESSENTIAL HYPERTENSION:

Essential hypertension is common among patients with diabetes, with the prevalence depending on the type and duration of diabetes, age, sex, race/ethnicity, BMI, history of glycemic control, and the presence of kidney disease, among other factors. Furthermore, hypertension is a strong risk factor for atherosclerotic cardiovascular disease (ASCVD), heart failure, and microvascular complications.

In normal conditions, attachment of vasoconstriction peptides endothelin-1 and angiotensin II  to their receptors leads to activation of these receptors in smooth muscle cells results in phospholipase C (PLC) activation leading to the generation of the second messenger's inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃stimulates intracellular Ca²⁺ release from the sarcoplasmic reticulum and DAG causes protein kinase C (PKC) activation.

Additionally, different Ca²⁺ entry channels, such as voltage-operated (VOC), receptor-operated (ROC), and store-operated (SOC) Ca²⁺ channels, TRPC3, TRPC6 channels,  as well as Ca²⁺-permeable nonselective cation channels (NSCC), are involved in the elevation of intracellular Ca²⁺concentration.

In essential hypertensive patients, chromium chloride is proposed to release totally from some L-type calcium channels situated in arterial vascular smooth muscle cell membranes due to diabetic induced oxidative stress. Thus,  a continuous extracellular calcium ions influx diffuses into the cytosol. 

This continuous influx is translated into cytosolic high-frequency calcium oscillations. The information transmitted by these Ca²⁺ waves can arrive as a stimulus at the plasma membrane and is translated into intracellular Ca²⁺ oscillations (Falcke 2004).

These high-frequency calcium ion  oscillations  are proposed to up-regulate the above mentioned calcium signaling  pathways which in turn over-activate the PKC pathway leading to prolonged activation of calmodulin interaction stimulating myosin light chain (MLC) phosphorylation causing ASM  hyper-responsiveness characterized by severe and prolonged ASM contraction,  thus,  essential hypertension  is predicted to develop.

Finally, the up-regulated  RhoA/Rock pathway leads to continuous phosphorylation of myosin light chain ( MLC).  Activated RhoA/ROCK  pathway leads to the phosphorylation of myosin light chain (MLC) (E. Nakajima, et al - 2005), in which combined with actin induces a calcium-independent smooth muscle contraction.

In summary, up-regulation of calcium signaling and over-activation of calcium-dependent  and independent signaling pathways, via the above-mentioned mechanisms, are  proposed  to be implicated in  the  pathogenesis of essential hypertension.
DIABETIC  ERECTILE  DYSFUNCTION:

Erectile dysfunction is a common problem for men who have diabetes — but it's not inevitable. Erectile dysfunction — the inability to get or maintain an erection firm enough for sex — is common in men who have diabetes, especially those with type 2 diabetes. It can stem from damage to nerves and blood vessels caused by poor long-term blood sugar control.

Experimental in vivo studies have implicated central and peripheral neuropathy, impaired neurotransmission, and endothelial dysfunction in the pathogenesis of diabetic ED.

Relaxation of erectile tissue requires nitric oxide from nonadrenergic-noncholinergic neurons and the endothelium.  We propose when chromium chloride totally releases from these neurons due to diabetic induced oxidative stress, these neurons are predicted to become unable to synthesize nitric oxide necessary for cavernosal smooth muscle relaxation.

Penile tissue from diabetic men with ED demonstrates impaired neurogenic and endothelium-mediated relaxation of smooth muscle.  ( Neelima V. Chu, MD and  Steven V. Edelman, MD, 2001).

We propose that when chromium chloride releases from L-type calcium channels in smooth muscles in cavernosa corpora due to diabetic induced oxidative stress, a continuous influx of calcium ions is predicted to diffuse into the cytosol of these cells. This continuous influx is translated into cytosolic high-frequency calcium oscillations, which is predicted to up-regulate calcium signaling pathways.

These up-regulated  calcium signaling pathways over-activate the  PKC pathway leading to prolonged activation of calmodulin interaction stimulating myosin light chain (MLC) phosphorylation causing vascular smooth muscles prolonged contraction, thus they  do not respond to nitric oxide relaxation thus,  restricting  arterial blood flow  to  the cavernosa corpora, thus, predicted  to diabetic ED development.

Another  impairment implicated in diabetic induced ED, is venous leakage characterized by valve impairment in deep dorsal vein due to cytokines overproduction resulted from  over-activation  of the PKC pathway due to upregulation of calcium signaling from chromium chloride release, thus cavernosal corpora fail to hold the blood inside which also contributes to diabetic ED.  In this case, flavonoids the administration is recommended in addition to using penile restriction bands.

Also, another impairment implicated in diabetic ED in deep and dorsal arteries stiffness leading to restricted blood flow to the penis.  In this case, antioxidants are recommended to alleviate arterial stiffness in addition to administration medications like sildenafil citrate which increases blood flows into the penis.

In late-stage  diabetic ED, cavernosa corpora become leaky, due to  prolonged diabetic induced oxidative stress which is proposed to release chromium chloride from L-type calcium channels in cavernosal tissue,  calcium signaling upregulation  is predicted to activate PKC pathways, leading to overproduction of VEGF and cytokines which leads  to hyper permeability and dysfunctional tight junctions, thus irreversible  diabetic ED  is predicted to develop.

In summary, up-regulation of calcium signaling, over-activation of calcium-dependent signaling pathways, via the above-mentioned mechanisms, and other above-mentioned impairments are  proposed to  be implicated in the  pathogenesis of diabetic erectile dysfunction.

DISCUSSION:

It is well documented that the up-regulation of calcium signaling is associated with VEGF & cytokines overexpression in retinopathy, nephropathy, and glaucoma.  Increased lens light scattering in cataracts, smooth muscle hypercontractility in  asthma and essential hypertension,  mediators overexpression and hyper immunological response in allergy, and initiation of a cascade of uncontrolled biological processes in cancer.

The predicted mode of  the action of chromium supplements  in non-excitable cells is via restoring  L-type calcium channels proper functionality, calcium ions homeostasis. And reversing altered ion concentration and nerve cells impaired function (excitable-cells) in diabetic neuropathy and ED.  

CONCLUSION:

Based on our preliminary results – which are consistent with well established scientific literature -  we predict that chromium supplements serve as a long-term and intermittent adjuvant approach that may alleviate symptoms of essential hypertension and ED, or even cure neuropathy, cataract, asthma, and chronic allergy effectively. Also, may retard the progression of other diabetes complications like ( retinopathy, nephropathy, open-angle glaucoma), and cancer effectively.  Daily dose 200 mcg orally for a limited period of 12 weeks for adults along with standard treatment, preferably under medical supervision.  As for diabetes complications, chromium should be co-supplemented with antioxidants.  As for diabetes complications, chromium should be co-supplemented with antioxidants, and glucose levels are controlled.  Also, for diabetic neuropathy and ED, only chromium picolinate supplementation is predicted to be effective, due to its high bioavailability.

LIMITATIONS:

We would like to point out that there are limitations in this research since it needs further in vitro empirical validation.

ACKNOWLEDGMENT:
We would like to extend our grateful gratitude to Dr. Mustafa Naka, and  Dr. Maram Elayyan for their valuable assistance.
FOOTNOTES:
Chromium supplementation  should be for limited periods of time for kidney and liver patients.

This research is based on trivalent chromium which is safe and beneficial to humans.  While Hexavalent chromium is harmful to humans and may cause lung cancer.

This research does not endorse any product; it is for the sake of patients all over the world.

Written consents had been taken from all participants enrolled in case studies mentioned in this research.

No potential conflict of interest exists.

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