Principals of Hard Core Acid!

This is an article on the most important aspects of acid/base balance with a few notes on extra sources of hard core acids and their consequence.

The problem with maintaining the alkaline design of the body the blood and then tissues are accumulating more of an acid load every day.  FOR EVERY ACID MOLECULE EXCRETED BY THE KIDNEYS, THAT ACID MOLECULE MUST TAKE A BASE MINERAL WITH IT TO KEEP IT NEUTRAL - to keep the acid part from burning up or destroying the kidneys.

For every animal protein acid (uric, nitric, phosphoric, sulphuric acids) molecule ingested, a base mineral must leave, eventu­ally, through the kidneys.  Since the base molecule is lost through defecation or urination and not reabsorbed back into blood circulation that equates to a double loss of base and why eating animal protein is so dangerous to the health of the body.

In a normal situation, the hydrochloric acid produced by the cover cells of the stomach is matched by the production of an equivalent amount of sodium bicarbonate (each molecule of sodium bicarbonate the stomach cells produce is matched by the counter production of just one, hydrochloric acid molecule). This sodium bicarbonate goes the other direction (except when ingesting food or liquids then the sodium bicarbonate remains in the stomach to buffer the acids of the food or drink) into the blood stream and circulates all around, first flushing out the ex­cess acid in the tissues and especially, freeing the collagen fibers and the colloidal connective tissue organ from the adsorbed acids stored there.  The acids buffered by sodium bicarbonate secreted by the cover cells of the stomach come from metabolism, diet and environment.

Any bicarbonate that is left over is picked up by the alkaline glands, the liver, the pancreas, the salivary glands, the pylorus glands,  the duodenum, etc.

An acid/alkaline imbalance happens because the sodium bicarbonate gener­ated by the stomach's cover cells, does not all go to the alkaline glands (pancreas, liver, salivary glands and the alkaline glands in the duode-num). On the way through the body to those glands, some of it gets used up by neutralizing acid residues stored in the connective tissue organ.

If there are not enough bases left over after the ingestion of a meal or enough bases to neutralize and clear the acids stored in the connective tissues,  a relative base deficiency develops, the latent tissue "acidosis", and the salivary glands, pylorus glands, liver and pancreas do not end up with enough alkaline sodium bicarbonate juices to ensure proper alkalization of the food and drink ingested.

Alkalization of the food and drink ingested cannot proceed without enough of these alkaline sodium bicarbonate juices from the salivary glands, pylorus glands, duodeum glands, liver and pancreas, etc., so the stomach has to produce more acid, in or­der to make enough sodium bicarbonate base, leading to ad nauseum, of which one can develop stomach ulcers and cancer. The ulcer or cancerous condition is not the result of too much acid, on the contrary, it is the result of too little alkaline sodium bicarbonate base!

The 'excess' acid in the gastric pits of the stomach is there as a toxic byproduct of sodium bicarbonte production, as the organism has to generate it so more sodium bicarbonate can be made to satisfy the needs of the salivary glands, pylorus glands, duodeum glands, intestinal glands, liver and pancreas.

NEW BIOLOGY (R)

If alkaline minerals are lost because they are excreted with the excess acids from animal protein consumed, it will be important to cut down on such consumption of animal protein an replace or supplement the alkaline mineral salts lost through urination and defecation.

These lost base minerals salts can be replaced with alkaline green fruit, vegetables, alkaline mineral salt supplementation, polyunsaturated oils, and good clean alkaline water.  I strongly recommend to eat alkaline green fruit, vegetables, mineral salts and attend to your alkaline water everyday.

Alkaline minerals from plants, organic minerals and ionized minerals contained in fresh/clean ionized water are the very best ones the body can use. Sodium from a plant for example, is much dif­ferent from the sodium from table salt. You can eat all the processed table salt you want and the cells themselves can still be sodium or base deficient. The sodium used for building cells has to be organic, from green plants or from a source such as the colloidal mineral salts from the North Shore of the Great Salt Lake.  Salt is the main base mineral in our body for transporting electrons for energy to the cells and building alkaline mineral compounds such as sodium bicarbonate or magnesium, potassium and calcium.

We simply do not eat enough green electron rich alkaline fruit and vegetables to compensate for the alkaline minerals lost, because of our "rich", fast life acidic diets. If we are base defi­cient and most of us are, then our whole body is in a relatively acid con­dition. That is all there is to it. We are not acidotic as they say in a hospi­tal, when things have gone so bad that the blood pH itself begins to change, Code Blue. Rather, we are full of stored acid residues, residues stored in the Pishinger space waiting for a ride out of the body on base minerals, like sodium bicarbonate that are not present. This is "latent tissue acidosis". 

General Results of a Base Sodium Bicarbonate Deficiency

1.     Tissue acidosis leads to the pleomorphic or evolutionary changes in the blood and body cells.  Whenever there is organized matter in nature that is dying or begin­ning to decay bacteria, yeast and then mold is born. As the human body ages, becoming acidic and toxic, aging body and blood cells will evolve into bacteria, then yeast and finally mold.  In other words, the living indestructible anatomical elements that make up body and blood cells change form and stick to­gether becoming a new form of bacteria and so on. This is how microorganisms are born.  They are born in us and from us. These new biological forms called mircoorganisms are not bad - they are the evidence of the evolution or change of matter due to a pH change in the environment.

2.     Sodium becomes deficient first from the blood serum (most of the sodium in the body is in the serum whereas the potassium is inside the cells). The acids and even excess protein itself can be, as one of the last resorts, stored in the cells themselves. This causes the cells to swell and edema develops. Of course, one is already sympto­matic by this time. The cells swell (edema) in order to dilute the metabolic acids in them, the acidic wastes that shouldn't be there. High blood pressure to cerebral edema can develop because of this acidic waste retention.

3.     Potassium leaves the cells to buffer acidic wastes and weakness, tiredness, and wasting develops.  Low blood pressure can be a result of this.

4.     Calcium leaves the bones, causing osteoporosis. The bone calcium goes into the blood and this may cause muscle cramps. The blood has to get rid of the "extra" calcium very quickly or one develops tetany. Tetany is a Charley Horse-type cramp, of every muscle in the body. The body does not deal well with calcium in the blood. It gets rid of it by depositing it in the tissues or excreting it. So why should we "mess around" with calcium? It should only be used, as a medicine, in a hospital. All the organic, good calcium that our bodies can use is contained in the electron rich alkaline fruit and vegetables, especially the dark, green leafy ones. We do not need milk and we do not need extra calcium supplements.  The body produces the calcium it needs from blood plasma ionic sodium.  The formula for the creation of calcium is as follows: Na + H <=> Mg + O <=> Ca.

There are many studies from around the world that show that the more animal protein and eggs a society consumes, the more osteoporosis that society develops. Osteoporosis is definitely an acid caused disease. The body's store of calcium is leached out of the bones by these metabolic acids to protect the delicate alkaline pH of the blood and tissues.

This calcium has to be excreted by the kidneys or in the feces; otherwise it will be deposited somewhere in the glands or organs. It can be deposited in the lining of the arteries, kidney, breast, brain, thyroid or gallbladder and this is how stones can develop.

Calcium chelated metabolic acids or stones can be deposited in the brain causing dementia or arthritic deposits form, on and on...and then the microbes come out of the acidic, hurt, swollen cells to help get rid of the deposits. Inflammation develops, pain, more swell­ing, edema, and blocked arteries. The amount of calcification of the body also corre­lates directly with the onset of 'old age'.  I have said that you do not get old you mold.

The blood and everything outside of the bones is already full of leached out calcium, because of the acidic condition produced by the base sodium defi­ciency. If you take extra calcium by mouth, where does it go? It does not go into the bones.  It goes to chelate acid.

CHRONIC DEGENERATIVE DISEASE IS CAUSED BY DIETARY and METABOLIC ACID

The condition of  "latent tissue acidosis" is not acknowl­edged by current medical savants.  If the above nonspecific symptoms are not recognized and dealt with for what they are, then, SPECIFIC ACIDIC DISEASES will develop.

The above GENERIC SYMPTOMS 'localize' in the weakess parts of the body, the locus minoris resistentiae, and frank organ degeneration begins to take place.

THE ABOVE IS CHRONIC DEGENERATIVE DISEASE.

This "localization in the body's place of least resistance" can take the form of any of the SPECIFIC, NAMED, CHRONIC, DEGENERATIVE, DISEASE. CHRONIC 

DEGENERATIVE DISEASE IS WHOLISTIC: IT AFFECTS THE WHOLE BODY.

If It's NOT HIV That Causes AIDS Then What Causes AIDS?

Contrary to popular belief, HIV is not necessary to explain acquired immune deficiency and the illnesses associated with AIDS.

To understand why this is so, it is first necessary to understand what AIDS is. AIDS is not a new disease or illness; it is a new name or designation for 29 previously known diseases and conditions. As the NIH states in its comprehensive report on AIDS, "the designation 'AIDS' is a surveillance tool."191 Since 1981, the surveillance tool AIDS has been used to track and record familiar diseases when they appear in people who have tested positive for antibodies associated with HIV.

The AIDS virus hypothesis supposes that the health problems renamed AIDS develop as a result of infection with HIV; that the virus somehow disables the body's defense system that protects against opportunistic illness, allowing the development of one or more of 29 diseases, such as yeast infection, certain cancers, pneumonia, salmonella, diarrhea, or tuberculosis, which are then diagnosed as AIDS. However, every AIDS indicator disease occurs among people who test HIV negative, none are exclusive to those who test positive and all AIDS diseases existed before the adoption of the name "AIDS."

Prior to the designation AIDS, these 29 diseases were not thought to have a single, common cause. In fact, all have recognized causes and treatments that are unrelated to HIV. For example, yeast infection is a widespread problem due to an imbalance of natural bacteria. The yeast infections that occur in people who test HIV positive and in people who test HIV negative are caused by the same imbalance of natural bacteria. All the opportunistic illnesses called AIDS have various, medically proven causes that do not involve HIV.

Immune deficiency can be acquired by several risk factors that are not infectious or transmitted through blood or blood products. The following factors are widely recognized causes of immune suppression, compromised health, and opportunistic infections, as documented in the medical literature for more than 70 years. Chronic, habitual and multiple exposures to these risks can cause the group of symptoms called AIDS.192 In fact, there is no case of AIDS described in the medical literature without one or more of these health risk factors.193

Physical Risk Factors

These risks include malnutrition and chronic lack of sleep. In 1985, orthodox AIDS researcher and director of NIAID, Dr. Anthony Fauci declared that malnutrition was the most prevalent cause of immune deficiency diseases throughout the world, particularly in developing regions such as Africa where common illnesses like measles run rampant and take millions of lives.194
The medical literature notes that malnutrition and infection are invariably linked, as one condition aggravates the other. Hunger and endemic disease are familiar problems in those countries around the globe thought to be under siege from AIDS. Intrauterine malnutrition occurs when expectant mothers are improperly nourished, and can result in prolonged, sometimes lifelong, immune suppression.195

Poverty, crowded living conditions and unclean water promote endemic disease and compromised health. The populations in many developing regions of the world are devastated by rampant infections with common microbes that pose little or no health threat to people in industrialized nations.

Infections due to malnutrition immunodeficiency are the world's leading causes of infant and child death.195 Among citizens of industrialized nations, subclinical malnutrition, rather than starvation leads to compromised immune function, especially when combined with chronic lack of sleep.196 People who make habitual and prolonged use of certain drugs like methamphetamines, heroin and crack cocaine often suffer from malnutrition and chronic lack of sleep.

Chemical Risk Factors

Immune-compromising chemicals include pharmaceutical drugs such as AZT and other cancer chemotherapy compounds, protease inhibitors, antibiotics and steroids, and recreational drugs such as cocaine, crack, heroin, nitrites (poppers), and methamphetamines (crystal, speed).
Chemotherapy targets and destroys the bone marrow cells from which all immune cells derive. They also kill fully formed immune cells in addition to killing B cells and red blood cells.196,197 Chemotherapy destroys the digestive system by killing the cells that compose the inner lining of the digestive tract which interferes with the body's ability to absorb and digest nutrients, causing malnutrition. Even when used very briefly, chemotherapy suppresses normal immune function, increases susceptibility to a variety of opportunistic infections, and can cause life-threatening anemia and diarrhea. AZT, ddI, ddC, D4T and 3TC are all chemotherapy compounds used as antiviral AIDS treatments.

There are many pharmaceutical drugs known to suppress the immune system, particularly when used for prolonged periods of time. Protease inhibitors cause impaired liver function and liver failure (the liver removes disease-causing toxins from the body) in addition to kidney failure, dangerously high cholesterol levels, diarrhea and other health-compromising effects. Steroids are a known cause of immune deficiency often prescribed to AIDS patients to counteract the muscle wasting caused by AZT.198 Antibiotics, especially when used habitually, can cause yeast infection and diarrhea, two conditions that can lead to malnutrition.199 Septra and Bactrim are sulfonamide antibiotics commonly prescribed for continuous, prophylactic or preventative use by HIV positives. These drugs are leftover from the days before penicillin; they do not target invading microbes as narrowly as modern antibiotics and are notorious for their side effects.200 Both cause nausea, diarrhea, vomiting, anorexia, bone marrow destruction, rashes, fever, hepatitis, and anemia by interfering with the production of red blood cells.201

The immunosuppressive effects of recreational drug abuse are well-documented in medical literature dating back to the turn of the century. They include pneumonias, mouth sores, fevers, endocarditis, bacterial infections and night sweats, all conditions now associated with AIDS.202 Amphetamine drugs suppress the appetite, causing chronic users to suffer from malnutrition. Many habitual users of heroin and crack do not provide themselves with adequate food, sleep, shelter and healthcare.

Prolonged exposure to common chemical toxins such as insecticides and herbicides can also impair immune function.203

Biological Risk Factors

These risks include multiple exposures to and/or chronic infections with syphilis, gonorrhea, chlamydia and other venereal diseases, hepatitis, tuberculosis, malaria, fungal diseases, amoebas and parasites such as giardia, bacterial infections such as staph and E coli, chronic bowel infections, blood transfusions, and the use of blood products. In addition to the damaging effects of recurrent infections, many of the pharmaceuticals used as treatment have adverse effects on immune function.

Factor VIII (the blood clotting agent used by hemophiliacs) and blood transfusions are immune suppressive and leave patients vulnerable to infection.204 Due to the serious conditions for which transfusions are necessary and the deleterious effects they have on the immune system, half of all HIV negative transfusion recipients die within a year of receiving a transfusion.204

Psychological Risk Factors

Chronic anxiety, panic, stress and depression have been shown to compromise health, damage immune function, and result in symptoms identical to AIDS.205 Mental stress provokes production of the hormone cortisol; excessive cortisol causes rapid and dramatic reductions in T cells, a condition known as lymphocytopenia. Within minutes, stress induces cortisol levels to increase as much as 20-fold. High levels of cortisol can eventually cause what medical texts describe as "significant atrophy of all the lymphoid tissue throughout the body" which may lead to "fulminating infection and death from diseases that would otherwise not be lethal."206
A profound fear of AIDS is enough to cause even people who repeatedly test HIV negative to develop physical symptoms of AIDS.207 Termed "AIDS-phobia," this condition is characterized by weight loss, wasting, reduced T cell counts and other signs considered indicative of AIDS, and typically follows intimate contact with people who sufferers believe may be HIV positive.

Beliefs and expectations are well-known to manifest in the physical body. The life-altering influence of beliefs was detailed dramatically in 1942 by Dr. Walter B. Cannon in his accounts of a phenomenon he called "voodoo death," a form of capital punishment practiced among certain Aboriginal tribes. Cannon reported that shaman, tribal medical authorities thought to possess special powers, were able to kill errant tribe members by simply pointing at them with a bone. Convinced of the shaman's ability to invoke a lethal curse, the people pointed at died within a matter of hours or days.208

In modern medicine, the power of expectation is a commonly accepted fact known as the "placebo effect." Placebos are inert chemical substances disguised as active preparations and given to patients in place of drugs. The health benefits gained from a placebo occur because the person taking it expects a positive effect. Since the benefits of any drug may be due in part to this placebo effect, most new drugs are tested against a placebo preparation.209
A recent study conducted at the University of Toronto demonstrated the profound physiological effects of expectation with regard to placebos. Researchers found that cardiac patients who strictly adhered to a placebo treatment regimen lived longer than patients who did not take their placebo regularly. In summarizing the study, lead researcher Dr. Paul Dorian noted, "What you believe has an important influence on your outcome."210

How These Risk Factors Apply to All AIDS Groups

There is not one case of AIDS described in the medical literature that does not include one or more immune-destroying health risk factors. There is no case of AIDS documented in a person whose sole risk is exposure to HIV. Every case of AIDS involves factors known to damage the immune system and leave a person vulnerable to debilitating infection and deadly illness.211

Men Who Have Sex With Men

Well-documented causes of immune dysfunction can explain AIDS illnesses among men who have sex with men although none of these causes are unique to this risk group or can be generalized to include all gay men. In fact, focusing attention on certain sexual practices rather than recognized health risks obscures our understanding of immune suppression and limits approaches to preventing and resolving AIDS.

Nitrites, more commonly known as poppers, are immune-suppressive, carcinogenic drugs chronically used by some gay men. At one time, 95% of gay men in major urban areas like Los Angeles, New York and San Francisco reported using poppers.212 Nitrite use correlates with Kaposi's Sarcoma (KS) and non-Hodgkin's lymphoma, two AIDS-defining cancers found almost exclusively in this risk group.213 There are several studies that further strengthen the correlation between poppers and KS by documenting KS in HIV negative gay men who use poppers.213 KS is hardly ever found among members of any other CDC risk group or among women with AIDS, and is never diagnosed in children or infants with AIDS.213 In 1981 when AIDS was first identified, half of all AIDS diagnoses were for KS. As popper use has diminished, so has KS which since 1993 has accounted for less than 5% of all new AIDS cases.214

In the only studies that asked gay men with AIDS about recreational drugs, 93% to 100% of participants acknowledged using cocaine, crack cocaine, poppers, heroin, ecstasy, methamphetamines like speed and crystal, and/or Special K (an animal tranquilizer).215
Combinations of parasitic infections that include amebiasis and giardiasis along with rectal infections, syphilis, and gonorrhea can result in acute diarrhea which in turn causes malabsorption and malnutrition, or wasting.216 This collection of infections and resultant problems was commonly known as Gay Bowel Syndrome in the years before AIDS.216 The CDC reports that 20% to 50% of all gay men in major US cities have been treated, often repeatedly, for intestinal parasites using immune suppressive pharmaceutical drugs.217 Antibiotic treatments for recurrent venereal infections are immune suppressive, as is the practice of using these antibiotics on a regular basis as a prevention. Steroids are another immune damaging drug frequently prescribed to offset the wasting caused by diarrhea and malabsorption.217
Campaigns that encourage HIV testing, the consuming of toxic AIDS drugs, and living in fear of AIDS are primarily directed at the gay community. Many gay magazines may have up to half of their commercial advertising devoted to AIDS-related promotions.218 Such constant emphasis on AIDS gives rise to the notion of the inevitability of AIDS, a belief which can evoke chronic terror, despair and hopelessness, psychological risk factors known to impair immunity and compromise health.

The chance of registering false positive on an HIV test is greater for people with high levels of non-HIV antibodies and microbes in their blood. Antibodies produced in response to the particular microbial and viral infections frequently found in some gay men are documented causes of false positive HIV test results.218

For people who test HIV positive, the drugs prescribed as preventative treatments for opportunistic AIDS-defining infections become harmful and even deadly when used on a daily, continuous basis. Bactrim and Septra, for example, are powerful sulfonamide antibiotics that kill digestive flora and cause anemia and bone marrow destruction. The anti-HIV drugs AZT, ddI, D4T, ddC and 3TC are all highly toxic chemotherapies that destroy the immune and digestive systems, in addition to causing five of the 29 official AIDS-defining illnesses.219 Two 1993 studies conducted in the US and Canada found that every one of several hundred gay men with AIDS had a history of significant recreational drug and/or AIDS drug use.220

Identifying this risk group as people who engage in habitual, prolonged use of recreational and/or pharmaceutical drugs, have chronic exposure to a multitude of infectious microbes, who suffer from chronic malnourishment and/or chronic fear of HIV and AIDS provides a more appropriate and comprehensive explanation of immune suppression that invites many possibilities for prevention and resolution.

Injection Drug Users

Members of this risk group account for 35% of all diagnosed AIDS cases, while another 4% of people diagnosed with AIDS cite heterosexual contact with injection drug users as their sole risk. However, the majority of people who initially claim intimate contact with IV drug users as their only risk later acknowledge taking drugs themselves.221

Considering only injection drug use as a high risk activity for AIDS disregards the immune suppressive effects brought about by habitual use of non-injected street drugs as well as the many health-compromising factors that can accompany the regular, long-term use of illicit chemicals. The emphasis on sharing needles over the damaging effects of the narcotics injected with the needles distorts our view of immune dysfunction and prevents application of practical solutions to the health problems common to this risk group.

Prolonged, habitual consumption of drugs such as heroin, crack, speed, and cocaine, whether taken by injection or other means, is well-known to disable immune function. Chronic use of these drugs is documented to bring about many conditions synonymous with AIDS including pneumonias, tuberculosis, mouth sores, fevers, night sweats, bacterial infections, and endocarditis. Malnutrition, the number one cause of immune deficiency diseases worldwide, and multiple infections are frequent side effects of habitual injection drug use, and are factors that suppress immunity.

Antibodies generated in response to the multiple infections and chemical toxins typical of chronic drug use can cause false positive readings on HIV tests. Positive test results most frequently lead to ongoing treatment with various immune suppressive antibiotics and chemotherapy drugs, and to a sense of hopelessness and profound despair.

A more compassionate and inclusive way to portray this diverse group is as people who engage in habitual, prolonged use of recreational drugs, have chronic exposure to a multitude of infectious microbes and toxins through septic syringes or septic living conditions; who suffer from chronic malnourishment, lack of adequate sleep, the immune suppressive effects of AIDS drugs, and/or the chronic despair that follows an HIV positive or AIDS diagnosis. The immune deficiency diseases caused by these multiple and variant factors can be resolved with treatments that do not involve toxic anti-HIV drugs and long-term use of powerful antibiotics.

Transfusion Recipients and Hemophiliacs

Hemophiliacs and blood transfusion recipients together make up 2% of adult AIDS cases in the US. As noted previously, Factor VIII, the blood clotting treatment used by hemophiliacs, is itself immune suppressive. Hemophilia is a life-threatening condition in people with or without an HIV positive diagnosis. Ryan White, the young HIV positive hemophiliac who became famous as an AIDS victim, actually died of common complications attributed to hemophilia (internal bleeding and liver failure), not of illnesses that define AIDS.223

Blood transfusions suppress the immune system. Medical experts note that higher amounts of blood transfusions among hospitalized patients correlate with higher death rates. The authors of one recent study on transfusions specifically mention that the immune suppressive effects of transfusions leave recipients vulnerable to deadly opportunistic infection.224

Factor VIII and blood transfusions can cause positive results on HIV antibody tests in persons never exposed to HIV by triggering the production of antibodies that react with the nonspecific proteins used in the HIV antibody test. Once a person has tested positive, they are subject to immune suppressive drug treatment regimens, and the terror of developing AIDS.
Members of these risk groups can be more accurately described as people with serious preexisting health challenges, critical or chronic exposure to immune suppressive blood products and toxic AIDS drugs, and/or who are affected by the chronic despair of a fatal diagnosis. Based on this view, immune compromising anti-HIV chemotherapy and continuous antibiotic treatments would compound preexisting health problems, rather than resolve them.

Heterosexual Contact

Six percent of Americans diagnosed with AIDS cite heterosexual contact as their sole AIDS risk. However, upon further investigation, 60% to 99% of these people are reclassified as injection drug users and/or men who have sex with men, groups with identifiable health risks documented to cause immune dysfunction.225 As previously noted, people diagnosed with AIDS voluntarily select a risk group from among six categories determined by the CDC which limits health risks to possible exposure to HIV through sex or blood.

The damage caused by AIDS chemotherapy and the acceptance of a fatal diagnosis are sufficient to bring about serious illness and even death in people with no other risk factors.
Members of this group may be better described as people with no health risk factors acknowledged by the CDC who, because of their positive HIV status, regularly consume chemotherapy and/or engage in continuous treatment with antibiotics and other immune suppressive pharmaceutical drugs, and/or suffer from the chronic panic and hopelessness of a fatal diagnosis.

Adolescents, Children and Infants

Although teenagers and children are not a specific AIDS risk group, cases of AIDS among young people, however rare, are a matter of great concern. The fact that babies are diagnosed with AIDS has been used as an argument against non-HIV explanations for AIDS illnesses. Despite widely held beliefs, the majority of AIDS cases that occur among children and adolescents can be explained by the same causes of immune suppression prevalent in adults with AIDS.
In 1998, new AIDS cases among this country's 26 million teens totaled 293; of these, 229 offered information which placed them in the two primary CDC defined AIDS risk groups for adults.226

Over 80% of the mothers of babies diagnosed with AIDS voluntarily acknowledge using injection drugs during pregnancy, a practice which almost universally results in intrauterine malnutrition. The remaining cases of AIDS in infants and children may be due to the immune suppressive medical treatments given in response to an HIV positive test result, or to the same factors that cause HIV negative babies to suffer from pneumonia, bacterial infections, and immune disorders. In 1998, new AIDS cases in children age 13 and under totaled 382.227

Residents of Developing Nations

In stark contrast to the US and Europe, AIDS cases in developing areas of the world are found almost exclusively among non-drug using heterosexuals.228 Mainstream AIDS experts offer no plausible reason why AIDS would spread primarily through drug-free heterosexual contact only outside the US and Europe.

A coherent explanation for AIDS cases in developing areas of the world is the well-known health risks shared by these countries, widespread poverty and malnutrition; lack of clean water, a regular food supply, and sanitary living conditions; limited access to medical care; endemic diseases such as tuberculosis, malaria, and parasitic infections that manifest in conditions identical to AIDS; and the practice of diagnosing AIDS based on a nonspecific set of clinical symptoms.

Although HIV tests are not required for an AIDS diagnosis in many parts of the world, widespread exposure to hepatitis, tuberculosis, leprosy, malaria and other conditions are more than sufficient to account for positive results on the nonspecific HIV antibody tests. 229
Resolving the immune suppressive conditions caused by poverty and malnutrition provides a means to alleviate the suffering of many people in developing nations who are currently counted and treated as victims of AIDS.

When considering non-HIV explanations for AIDS, consider that:
AIDS is a collection of familiar illnesses, not a disease.

Since 1993, more than half of all new AIDS diagnoses in the US are given to people who are not ill. In 1997, two-thirds of Americans diagnosed with AIDS had no symptoms or illness.*
Acquired immune deficiency predates the creation of the category "AIDS" and has numerous, well-documented causes.

There are no AIDS cases noted in the medical literature in which exposure to HIV has proved to be the sole health risk factor.

There are well-documented causes for every AIDS disease that do not involve HIV, and all illnesses now called AIDS occur in the absence of HIV.

HIV tests do not test for the actual virus, but for antiviral proteins or genetic material that are not specific to HIV.

The chance of a positive reaction on a nonspecific HIV antibody test increases proportionately with the level of other antibodies and microbes found in the blood.

Five of the six AIDS risk groups defined by the CDC have health risk factors that involve multiple, chronic exposure to viruses, bacteria and other antigens known to produce antibodies
identical to those associated with HIV.

Once a person has tested HIV antibody positive, chemotherapy and other immune suppressing chemicals are almost always prescribed for treatment or prevention of AIDS.

Alternative explanations for AIDS provide opportunities for effective AIDS prevention and for using practical, nontoxic approaches to resolving AIDS.

1997 was the last year that the CDC provided information on how many AIDS cases were diagnosed in people who are not sick.

Defined Terms:

Endemic: A medical term applied to a disease or disorder that is constantly present in a particular region or in a specific group of people.

Cancer Chemotherapy: Drugs used to treat cancer. Most anticancer drugs are cytotoxic (kill or damage cells). Others are synthetic forms of hormones. All anticancer drugs prevent cells from growing and dividing. Some work by damaging the cell's DNA; others block the chemical processes in the cell necessary for growth. Side effects of treatment include nausea, vomiting, and life-threatening diarrhea. By altering the rate at which cells grow and divide, anticancer drugs reduce the number of blood cells produced by the bone marrow, causing anemia and increased susceptibility to infection.

Endocarditis: Inflammation of the internal lining of the heart.

Incorrect Information about HIV and AIDS Costs Lives
Can you imagine receiving a fatal diagnosis without being told the diagnosis is based on an unproven idea and an uncertain test? Being instructed to take powerful, experimental drugs without being told these drugs compromise health, destroy functions necessary to sustain life, and were approved for use without adequate testing? Being informed that you have, or should expect, deadly illnesses without being told that these same illnesses are not considered fatal when they occur in "normal" people?

For anyone who tests HIV positive, getting all the facts is a matter of life and death. The important decisions a person makes should be based on thorough, verifiable data. All of us need and have the right to receive honest and complete information about HIV and AIDS.

Almost every AIDS organization in the country offers free instruction for people who test HIV positive. Standard information includes how to prepare a will, how to collect disability, health insurance, and public benefits, what drugs and tests to take, and which diseases to anticipate, all based on the assumption that HIV positives are or will be ill and do not have long to live.

Information on AIDS that is free from bias, that accurately describes tests and drugs, and offers facts that support a will to live, participate in society, and cultivate a healthy future are rarely, if ever mentioned. Some AIDS groups even lobby to limit public access to data that undermine their dire presentations of HIV and AIDS.

For many people handed an HIV positive diagnosis, these brief pages provide their first awareness that a normal, healthy life is not something they can only hope for, but something they can choose to achieve. Unfortunately for most people who test positive, the AIDS education they receive portrays their choices as being limited to toxic drug therapy or devastating illness, and encourages chronic fear, sadness, and resignation to an early death.

There are thousands of HIV positives who lead healthy lives without toxic AIDS drugs. What they have in common is not some unique, mysterious gene or a weakened strain of the virus, but an open-minded approach to information, an understanding of basic principles of medicine and science, and the knowledge that the responsibility for their well-being is ultimately their own. For more information on their lives, please see The Other Side of AIDS on page 94.

This book examines only a portion of the growing body of scientific, medical and epidemiological evidence that refutes popularly accepted ideas about HIV and AIDS. Readers are strongly encouraged to conduct further research and use the resources offered here.

To the degree that we allow unfounded ideas about HIV and AIDS to determine our actions, influence our choices, dictate our public policies, or define our world view, we are all victims of AIDS.

Since the 1984 announcement that HIV causes AIDS, all AIDS research has been based on the hypothesis that HIV, an inexplicably lethal new virus, is responsible for a group of previously known, disconnected diseases renamed AIDS. Setting the focus of all AIDS efforts on HIV, a virus that strains the rules of biology, epidemiology and logic, has rendered humankind few, if any, beneficial results.

The lives of over 400,000 Americans have been given to the notion that HIV is the only possible cause of AIDS, and that toxic drugs offer the only possible prevention, treatment, or hope for a cure. Many more lives have been forever altered by a positive result on a non-standardized test for harmless antibodies that may or may not be associated with HIV.

More than $50 billion in federal AIDS funding has provided no significant understanding of HIV, has produced no safe and effective therapies, and has not brought us any closer to ending AIDS. Instead, we have constructed a powerful AIDS establishment that regulates our news, limits our access to information, and demands an ever greater allocation of our resources and support. Rather than helping to resolve AIDS, we have funded the growth of multi-billion dollar industries, institutions and organizations that depend on AIDS and on our continued devotion to the narrow and unproductive HIV hypothesis.

Objective Examination of HIV and AIDS is Fundamental to Progress.

To understand and solve AIDS, it is necessary to investigate all legitimate scientific data, even when such information challenges our present understanding and perceptions. Progress in any area depends on the ability to engage in an unbiased evaluation of facts, to raise critical questions and to conduct an objective search for meaningful answers. Silence = Death...Of People, Ideas and Progress

" There is classical science, the way it's supposed to work, and then there's religion. I regained my sanity when I realized that AIDS science was a religious discourse. The one thing I will go to my grave not understanding is why everyone was so quick to accept everything the government said as truth. Especially the central myth: The cause of AIDS is known. What in the world made activists accept tha, ton the basis of a press conference, no less?

"My only theory is that AIDS requires the daily management of massive amounts of uncertainty, and people cling to any certainty they can find. Even if it's false."
Michael Callen, author, AIDS activist (deceased),Genre magazine, February/March, 1994
"Most HIV trials are useless rubbish. Research scientists [outside AIDS research] laugh at us. To them a good sample size is 30,000 people. We do studies with 1,500 people and think that's wonderful when the actual number of relevant patients is sometimes so small, you cannot rule out chance as the reason for the results you get. It is also unethical to run trials of drugs in places like Malaysia with only 30 people involved and then try to justify these flawed trials because some people got access to drugs who otherwise would have had nothing."

Kevin Frost, Manager of Research Programs for the American Foundation for AIDS Research (AmFAR), Positive Nation, September 1998

"The story of AIDS is deeply connected with the vicissitudes of the theory that viruses cause cancer and the failure of the cancer research program. Michael Verney-Elliot put it most acidly when he said: 'From the people who didn't bring you the virus that causes cancer, it's the virus that doesn't cause AIDS.'"

Jad Adams, Author, The HIV Myth, 1989

"AIDS is not another disease, it is the most metaphorical disease in history. It is the ultimate triumph of politics over science."

Michael Fumento, Author, The Myth of Heterosexual AIDS, 1990

"Perhaps I'd feel different about it if I thought people were dying from AIDS. But I don't. I think they're dying from bad medicine, bad drugs, bad attitudes. There is nothing I want from 'Big Daddy' I don't want his medicines, his laws, his approval."

Gavin Dillard, Author, In the Flesh, HIV positive since 1985, San Francisco Frontiers, May 20, 1999

"In the September 4 issue of the Journal of the American Medical Association, the CDC announced that a diagnosis of AIDS no longer requires an HIV test. The government now considers you an AIDS carrier if you suffer from any of the maladies on its new list of diseases indicative of AIDS, including such relatively common infections as herpes simplex, tuberculosis, Salmonellosis and the shockingly broad category 'other bacterial infections.' This broad definition will lead to countless new AIDS diagnoses, whether or not the person actually has AIDS. A major problem with the new AIDS definition is that it ignores the many environmental causes of immune suppression. Exposure to toxins, alcoholism, heavy drug use or heavy antibiotic use all can cause onset of the list of 'diseases' indicative of AIDS. The CDC itself conceded in a stunning remark near the end of the JAMA article that the new AIDS ground rules are highly suspect. 'The diagnostic criteria accepted by the AIDS surveillance case definition should not be interpreted as the standard of good medical practice,' warned the CDC."
Los Angeles Weekly, December 18, 1987

"The real trick is to get off the medication. I felt I was losing quality of life..."

Greg Louganis, HIV positive Olympic Gold Medalist,The State, April 15, 1997

"It's not even probable, let alone scientifically proven, that HIV causes AIDS. If there is evidence that HIV causes AIDS, there should be scientific documents which either singly or collectively demonstrate that fact, at least with a high probability. There are no such documents."

Dr. Kary Mullis, Nobel Laureate, HIV not Guilty, October 5, 1996

" If you think a virus is the cause of AIDS, do a control without it. To do a control is the first thing you teach undergraduates. But it hasn't been done. The epidemiology of AIDS is a pile of anecdotal stories selected to the virus-AIDS hypothesis. People don't bother to check the details of popular dogma or consensus views."

Dr. Peter Duesberg, Do You Think HIV Causes AIDS?,Scientists for Legitimacy in Science, 1995

"Beware the scientist who believes that mainstream research thinking on any public health issue is equivalent to truth. Or the scientist who bullies or ridicules other scientists because they oppose the prevailing view. This is a person who has become what I would call a propagandist and should not be trusted.

"I have worked as a medical science reporter for 30 years. I've interviewed thousands of scientists for newspaper and magazine stories, radio and television productions, and books. I've met scientists who at least try to keep an open and fair mind on scientific issues. I have also met many propagandists who think they're scientists. In all the time I've worked as a journalist, I've never come across a nastier group of people to interview than those propagandists who work in HIV research."

Nicholas Regush, Medical Science Reporter, Second Opinion, ABCNews.com, September 29, 1999

"As a scientist who has studied AIDS for 16 years, I have determined that AIDS has little to do with science and is not even primarily a medical issue. AIDS is a sociological phenomenon held together by fear, creating a kind of medical McCarthyism that has transgressed and collapsed all the rules of science, and has imposed a brew of belief and pseudoscience on a vulnerable public."

Dr. David Rasnick, Designer of Protease Inhibitors,SPIN magazine, June 1997

"Considering there is little scientific proof of the exact linkage of HIV and AIDS, is it ethical to prescribe AZT, a toxic chain terminator of DNA developed 30 years ago as cancer chemotherapy, to 150,000 Americans, among them pregnant women and newborn babies, as an anti-HIV drug?"

Rep. Gil Gutknecht (R-MN), US House of Representatives, Letter to NIAID Director Dr. Anthony Fauci, March 14, 1995

Bicarbonate and Dichloroacetate: Evaluating pH Altering Therapies in a Mouse Model for Metastic Breast Cancer

Ian F Robey^1* and Natasha K Martin^2,3

• *Corresponding author: Ian F Robey robeyi@email.arizona.edu

Author Affiliations

¹Arizona Respiratory Center, University of Arizona, Tucson Arizona, USA

²Centre for Mathematical Biology, Mathematical Institute, Oxford University, 24-29 St Giles', Oxford, OX1 3LB, UK

³Department of Social Medicine, University of Bristol, Canynge Hall, 29 Whatley Road, Bristol, BS8 2PS, UK

For all author emails, please log on.

BMC Cancer 2011, 11:235 doi:10.1186/1471-2407-11-235

The electronic version of this article is the complete one and can be found online at:http://www.biomedcentral.com/1471-2407/11/235

Received:	10 February 2011
Accepted:	10 June 2011
Published:	10 June 2011

© 2011 Robey and Martin; licensee BioMed Central Ltd. 

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.

Methods

We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.

Results

Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.

Conclusions

DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.

Keywords: 

Tumor; pH; Acidity; Dichloroacetate; Sodium bicarbonate

Background

The extracellular pH of malignant tumors is acidic (pH 6.5-6.9) compared to normal tissue (pH 7.2-7.4) [1-3]. Tumor acidity is thought to play a critical role in chemoresistance [4-6] and promotion of metastatic potential. For example, acidic culture conditions increase filopodia formations and expression of proteolytic enzymes involved in invasion [7]. Other reports have described enhanced invasiveness [8] and increased cathepsin B activity in acid-cultured tumor cells [9]. Acid-mediated metastatic potential has been demonstrated in vivo as well. Tumor cells pre-treated under acidic pH prior to tail vein injection formed a greater number of pulmonary metastases as well upregulated activity of metastatic effectors such as serine proteases and angiogenic factors[10].

Although decreased extracellular tumor pH is strongly associated with numerous cellular mechanisms including carbonic anhydrases [11], vacuolar ATPases [12], and sodium-ion exchangers [13], glycolysis is considered the major factor in promoting tumor acidity. Excessive tumor glycolysis, even in the presence of oxygen, is a hallmark of malignancy and leads to increased production of lactic acid [14,15]. The notion that acidic pH is sufficient for driving tumor cell invasion inspires the corollary prediction that neutralizing acidic tumor pH inhibits invasion and slows the spread of metastases. This question was tested in a survival study of tumor bearing mice. Female severe combined immunodeficient (SCID) mice with MDA-MB-231 breast tumor xenografts were chronically administered drinking water with sodium bicarbonate. This study reported that systemic bicarbonate buffered the extracellular pH in tumors to neutral levels (a pH of 7.2) and inhibited the spread of metastases which led to improved survival. Systemic bicarbonate was also effective against the spread of metastases in a model for prostate cancer[16].

Certain determinations are required before considering systemic buffering of tumor acidity as feasible approach to cancer treatment. Effective doses, susceptible tumor types, ideal buffers, safety approaches, and other factors in the tumor microenvironment would need to be identified and validated. To assist our understanding of these phenomena, mathematical models were developed to predict the safety and efficacy of systemic buffering of tumor acidity in humans. Our findings suggest that chronic use of oral bicarbonate as a cancer intervention is limited. Safe doses for consumption limit the amount of buffering to counteract tumor cell production of extracellular protons. In a previous study, it was reported that the saturating dose of oral bicarbonate in mice (which roughly translates to about 0.18 g/kg/day in humans) was only sufficient to counteract the acid load of a 15 mg tumor consisting of about 100,000 cells or 1.0 mm³ [16]. Moreover, chronic application of oral bicarbonate at doses higher than 0.5 g/kg/day is predicted to induce systemic alkalosis (Martin N, Robey I, Gaffney E, Gillies R, Gatenby R, Maini P: Predicting the Safety and Efficacy of Buffer Therapy to Raise Tumor pHe: An Integrative Modeling Study, submitted). Therefore, it is thought that safety and efficacy of oral bicarbonate could be enhanced by other reagents that directly or indirectly target the processes driving extracellular tumor acidity.

One possible method to enhance extracellular pH buffering is the orphan drug dichloroacetate (DCA). This analogue of acetic acid has been shown to selectively target tumor cell metabolism by indirectly activating pyruvate dehydrogenase (PDH) through inhibition of pyruvate dehydrogenase kinase (PDK) activity. Upregulated PDH activity shifts metabolism of pyruvate to lactate under the glycolytic cascade towards production of acetyl-CoA, which is fed into the Krebs Cycle to drive oxidative phosphorylation. This shift results in decreased cellular glycolysis with a subsequent increase in extracellular pH. It also triggers multiple apoptotic signaling events in the mitochondria[17,18]. To explore the question of whether DCA and bicarbonate is a viable therapeutic intervention in metastatic disease we conducted an interventional survival study in mice bearing MDA-MB-231 metastatic breast tumor xenografts divided into four treatment groups 1) untreated; 2) bicarbonate; 3) DCA and 4) DCA-bicarbonate (DB).

Methods

Animal experiments

All animals were maintained under Institutional Animal Care and Use Committee (IACUC) - approved protocols at the University of Arizona. Six to eight-week-old female SCID mice received orthotopic injections of 5 × 10⁶ MDA-MB-231/eGFP tumor cells in a mammary fatpad. Tumor bearing animals were randomized into 1) untreated; 2) sodium bicarbonate (Sigma, St. Louis, MO), 200 mM (equivalent to 3.2 g/kg/day in mice); 3) sodium dichloroacetate (DCA) (Sigma, St. Louis, MO), 5 mM (equivalent to 112 mg/kg/day); and 4) sodium bicarbonate (200 mM) plus sodium dichloroacetate (5 mM) combination (DB) water six days following tumor inoculation. Starting cohort sizes were 16 per group. Animals were monitored and maintained by the Experimental Mouse Shared Services (EMSS) core facility of the Arizona Cancer Center, Tucson Arizona. Volumes of primary tumors in mammary fat pads were measured twice weekly and calculated from orthogonal measurements of external dimensions as (width)² × (length)/2. Surgical resections of primary tumors occurred between days 28-50 when tumors were approximately 500 mm³. Mice were euthanized when tumor burden was excessive (primary, intraperitoneal, or lymph node > 2000 mm³) or when mice progressed to a moribund state. The EMSS technician, who was blind to the study, monitored the mice twice weekly. Notification to euthanize was received by the investigator on the day of a monitoring visit and study termination endpoints did not exceed 48 hours. Animals were euthanized by cervical dislocation.

Urine was obtained by applying gentle abdominal pressure against the mouse for 10 sec over plastic film. Urine was collected by micropipette and transferred to a 0.6 mL tube for pH measurement. Serum was collected by heart puncture after mice were euthanized. Blood was centrifuged and serum was collect for pH measurement. Urine and serum pH was measured using a Mettler Toledo pH meter with an InLab^® Micro probe.

Metastases measurements

Upon termination of the survival experiment, gross necropsy was used to identify tumor metastases. Green fluorescent tumors were detected by the Illumatool Bright Light System (LT-9500) using a 470 nm/40 nm excitation filter (Lightools Research) and imaged using a mounted PowerShot SD750 digital camera (Canon, Lake Success, NY). The images were captured at the same focal plane in the presence of 480-nm excitation and > 490-nm filtered emission. Whole lung image data were analyzed with Adobe Photoshop 5.0 using the "magic wand" tool to select lung area and green fluorescent tumor lesions. Pixel area of the selected images was measured using ImageJ (http://rsb.info.nih.gov/ij webcite/).

Cell culture experiments

MDA-MB-231 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's Modified Eagle's Medium/F-12 supplemented with 10% FBS. Crystal violet assay: Cells, growing in log phase, were plated in 96-well culture plates at a density of 5 × 10⁴cells/mL. The following day cells were exposed to titrating doses of DCA suspended in fresh growth media from 80 mM to 2.5 mM at 20% (normoxia) or 1% O₂ (hypoxia). Untreated cells in growth media were included as negative controls. After the 24 hour dose period, cells were fixed with 0.025% gluteraldehyde then stained with 0.1% crystal violet. Stained cells were washed with water then resuspended in 10% acetic acid. Absorbance was measured at 590 nm using a Victor3™ plate reader (PerkinElmer, Waltham, MA). Lactate production measurements: Cells growing in 24-well plates were washed once with phosphate buffered saline then cultured with 100 μL in 10 mM D-(+)-glucose-supplemented, serum-free RPMI for 1 hour incubation at 37°C in a humidified 5% CO₂atmosphere, 20% or 1% O₂. Lactate levels were quantified using an enzymatically coupled lactic acid detection reagent (Sigma). Ten microliters of lactate supernatant were assayed with 90 μL lactate reagent in 96-well plates. Mixtures were incubated for 5 minutes in the dark at ambient temperature. Absorbance of colorimetric assay was measured at 450 nm. Lactate production rates were calculated from an internal standard curve and expressed as nmol/min/mg protein. Cellular protein was obtained by lysing cells with 100 μL 0.1 N NaOH for 1 minute then neutralizing lysate with an equal volume of 0.1 N HCl. The protein concentration was determined using and internal bovine serum albumin standard curve in a Bradford Assay (Pierce, Rockford, IL). Absorbance of colorimetric assay was measured at 630 nm.

Statistics

Statistical calculations were determined using the analysis feature in GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego CA, http://www.graphpad.com webcite). Unpaired, two-tailed t-tests were used to determine if means were significantly different between untreated and treated groups. Categorical analyses were carried out using a two-tailed Fischer's Exact test. Ap-value of less than 0.05 was considered to be statistically significant. Numerical data values are represented as mean ± SEM.

Results

Chronic oral administration of DB has no adverse systemic effects

Our previous studies have found that chronic oral bicarbonate significantly increases urine pH in tumor bearing mice by approximately 1.4 fold over a 21 day period. This is a consequence of excess systemic bicarbonate and raises concerns about the safety of long term use in humans (Martin N, Robey I, Gaffney E, Gillies R, Gatenby R, Maini P: Predicting the Safety and Efficacy of Buffer Therapy to Raise Tumor pHe: An Integrative Modeling Study, submitted). Urine pH in DCA treated mice was the same as measured in untreated mice. Urine pH was similar between bicarbonate and DB treated mice (Figure 1A). In the post-survival study there were no statistically significant changes in serum pH between any of the groups. A marginal, but significant serum pH difference (p < 0.03) was observed between DB and DCA treated mouse serum (Figure 1B).

Figure 1. Comparison of urine and serum pH in treatment groups. A) Urine samples were collected from tumor bearing mice at eight different time points over a 3 week period from 3-10 mice in each treatment group. At time zero before starting treatment, urine pH in all groups averaged 5.59 ± 0.2 with no statistical difference between groups. The first collection time after starting treatment was at 24 hours. Urine pH measurements from all collection times were averaged. Asterisks (_*) designate average urine pH values that were statistically significant from untreated mouse group by student's two-tailed t-test (p < 0.001). The cross (†) designates average urine pH values between the DCA and DB treated groups were statistically significant (p < 0.03). B) Serum pH was measured in euthanized mice. Differences in serum pH between groups were not statistically significant. Error bars are expressed as SEM.

Primary tumor growth was unaffected by treatments

Previous studies have shown that oral administration of DCA can slow the rate of tumor growth in vivo [17]. In our study, treating MDA-MB-231 tumor bearing mice with DCA and DB did not impact primary tumor growth. Tumors in all cohorts grew at an equal rate before they were resected (Figure 2A). The primary tumor growth rates in the bicarbonate and DB treated groups were consistent with earlier findings in bicarbonate treated mice. The results confirm that bicarbonate buffering of tumor acidity does not function to inhibit primary tumor growth [16]. In animals where primary tumors re-occurred, growth rates were not statistically different between groups (Figure2B). After resection, most primary tumors grew back in the untreated and DCA treated mice. In bicarbonate and DB treated mice, less than half of the tumors grew back after tumor resection (Table 1). Primary tumor reoccurrence was not statistically significant when comparing individual treated cohorts to the untreated group (Figure 3A), but non-bicarbonate treated cohorts (untreated and DCA) had a significantly higher rate of tumor reoccurrence after survival surgery than bicarbonate treated cohorts (bicarbonate and DB) (p < 0.05) (Figure 3B)

Figure 2. Effect of bicarbonate, DCA and DB treatments on primary tumor growth. Growth rates of primary tumors are expressed in mm³/time (days). Plots indicate none of the treatments exhibited a measurable effect on primary tumor growth at the beginning of the study (A) or from the time of the tumor resections to study termination (B). Error bars are expressed as SEM. Two-tailed, unpaired t-test, between these groups yielded a p > 0.9.

Table 1. Fraction and percentage of mice in each treatment group with re-occurring primary tumors, and with intestinal, mesentery, lymph node, and pulmonary metastatic involvement

Figure 3. Primary tumor re-occurrence after survival surgeries. Survival surgeries occurred between day 28 and 50. Average resection day occurred on day 37 ± 8. Primary tumor sizes in all treatment groups averaged 490 ± 12 mm³. Primary tumors started to re-grow around day 50. Primary tumor re-occurrence is plotted over the survival time course as Kaplan-Meier curve. A plot point represents the day when a primary tumor was first measured after surgery. In mice where no primary tumor was observed in post-study necropsy experiments, a value of zero was recorded for day 120. A) Plots for all experimental cohorts (p = 0.29). B) Curves comparing groups that were not administered bicarbonate (untreated and DCA) and groups treated with bicarbonate (bicarbonate and DB) (p = 0.046).

Oral administration of bicarbonate and DCA-bicarbonate improves survival over DCA and untreated mice

Because our study design chronically administered DCA over a 17 week period, we limited the drinking water concentration to 5 mM (0.75 g/L) to avoid potential development of hepatic carcinogenesis [19]. Treatments were started 6 days after tumor injections and were continued for the remainder of the study. Mice received survival surgeries when tumors reached approximately 490 mm³ (± 95). On average, these surgeries occurred on day 37 (± 8). Mice that died from surgical procedures were omitted from the study. The mice were maintained on cohort-specific treatment regimens until conditions related to tumor burden required animals to be euthanized. Mice were then analyzed to score and measure tumor metastases. Spontaneous deaths occurred in all groups. Complete postmortem analysis could not be obtained in some of these deaths. These include one in the untreated group, two in the bicarbonate group, two in the DCA group, and one in the DB group. The survival study was terminated at day 120, at which point those mice surviving beyond that time were euthanized for post study analysis. Mice treated with bicarbonate (p = 0.03) and DB (p = 0.01) had significantly improved survival over untreated and DCA-treated (p = 0.2) mice (Figure 4). Three of 15 untreated mice survived after day 120. In the bicarbonate treated group, 8 of 13 mice remained after day 120. Six of 15 mice remained after day 120 in the DCA treated group. In the DB treated group, 10 of 15 mice remained after day 120. The mean survival day was 100, 110, 104, and 112 for untreated, bicarbonate, DCA, and DB treated mice, respectively.

Figure 4. Effect of bicarbonate, DCA, and DB on and survival. MDA-MB-231 cells were stably transfected to express neomycin-resistant pcDNA3/EGFP (16). MDA-MB-231/eGFP cells (5 × 10⁶) were injected into inguinal mammary fat pads of animals that were randomized into bicarbonate, DCA, DB, and control groups (n = 16 per group) 6 days post inoculation. Tumors were allowed to grow for 5 to 6 wk (to a volume of approximately 500 mm³), at which time they were surgically removed. After survival surgeries, the disease model was allowed to progress until tumor burdens or morbidity criteria warranted the mice to be euthanized. The survival experiment proceeded to day 120. At time of sacrifice, mice were necropsied by examination with a fluorescence dissecting scope. Data from this experiment are plotted as Kaplan-Meier survival curves (A) bicarbonate (p = 0.03), (B) DCA (p = 0.2), and (C) DB (p = 0.01). Treated mice are represented as solid lines compared to the untreated group represented as a dashed line. The difference in the survival curve for the treated versus untreated animals was evaluated by log-rank test.

Analysis of metastases

Metastatic involvement was observed in the intestines, mesentery, lymph nodes, and lungs in all groups (Table 1). All treated mice sustained a lower or equal amount of intestinal, mesentery, and lymph node metastases compared to the untreated group, but no differences were statistically significant. Metastases were present in mice where primary tumors re-occurred after tumor resection and in mice where primary tumors did not re-occur. Overall, there was a significant correlation between tumor re-occurrence and development of metastases (p = 0.028). There was no statistical significance between metastases and mice that did not have re-growth of primary tumors. There was no positive correlation between >120 day survival and the presence of metastases (Table 2).

Table 2. Metastases observed in mice that survived greater than 120 days

Most (11 of 14) untreated mice developed pulmonary metastases. Lung metastases were found in 1 of the 11 bicarbonate treated animals. Pulmonary metastases were counted in 6 of 13 DCA treated animals, and 9 out of 15 DB treated mice (Table 1). Average pulmonary lesion sizes in DB treated mice were significantly smaller than lesions from the other groups (p < 0.001). The sum area of the pulmonary lesions in DB treated animals was less than half the sum of the lesions in the other groups (Table 3). Representative lung images from each treatment group are presented in Figure 5. The average lesion area in the single bicarbonate treated mouse lung was significantly larger than lesions measured in the untreated mouse lungs (p < 0.023) (Table 3). Lung lesion sizes did not correspond with time of death.

Table 3. Summary of pulmonary lesions in treatment groups

Figure 5. Representative pulmonary lesions from the four study cohorts. Green fluorescent lung tumor metastases from necropsies were detected by the Illumatool Bright Light System (LT-9500) using a 470 nm/40 nm excitation filter (Lightools Research). Whole lung images were captured in the frame of view at the same focal plane in the presence of 480-nm excitation and > 490-nm filtered emission.

Cytotoxic and lactate production assays suggest loss of anti-cancer efficacy of DCA in hypoxic tumors

DCA monotherapy in MDA-MB-231 tumor bearing mice produced no measurable effects against the tumors or metastases. Although previous studies testing lower doses of DCA on tumor growth in vivo were effective using other cell lines [17], the tumors in this study were comparatively unresponsive. The effect of titrating doses of DCA under normoxic conditions (O₂ = 20%) in vitroshowed that low doses in culture from 2.5 to 20 mM appeared to stimulate cellular viability as demonstrated by a significant 30% increase in viable numbers of cells at these doses after 24 hours. MDA-MB-231 cells exhibited a 25% decrease in viability at 40 mM and an 80% decrease at 80 mM. Viability was 50% at 58 ±5 mM under normoxic conditions (Figure 6A). Similar results were seen after 48 hour incubations with DCA (data not shown).

Figure 6. MDA-MB-231 breast tumor cells were cultured for 24 hours (n ≥ 3) with titrating doses of DCA starting from 80 mM and diluted 2-fold to the lowest concentration of 2.5 mM. Percent viability for all groups of replicates was normalized by dividing absorbance (590 nm) values by the absorbance values obtained from sham treated (growth media) control cells. Cytotoxicity by DCA in a crystal violet assay was carried out under A) normoxia (O₂ = 20%) and B) hypoxia (O₂ = 1%). Asterisks (_*) designate viable DCA treated cells percentage that were significantly different from 100% viable sham treated cells. Error bars are expressed as SEM.

Microenvironmental conditions such as hypoxia (O₂ = 1%) can influence drug efficacy against tumor cells. This condition was tested evaluating the cytotoxic effect of DCA on cultured MDA-MB-231 tumor cells. Under hypoxic conditions, DCA doses between 2.5-20 mM had no effect on MDA-MB-231 cell viability. Similar to MDA-MB-231 cells grown under normoxia, a 40 mM DCA concentration significantly reduced viability by 25-30%, but cells were more resistant to increasing concentrations under hypoxic conditions. A DCA dosage of 84 ± 8 mM was required to reduce cell viability to 50% under hypoxia (Figure 6B).

We tested DCA in its ability to inhibit glycolysis under normoxia and hypoxia in MDA-MB-231 cells by measuring lactate production. All DCA concentrations from 2.5 to 80 mM inhibited glycolysis between 19% and 29% in MDA-MB-231 cells cultured under normoxia. Reduction in lactate production was significant in all doses (p < 0.05) with the exception of the 40 mM concentration (p= 0.059) (Figure 7A). Alternatively, under hypoxic conditions, DCA at the same dose range had no measurable effect on lactate production (Figure 7B).

Figure 7. MDA-MB-231 breast tumor cells were cultured for 24 hours with titrating doses of DCA starting from 80 mM and diluted 2-fold to the lowest concentration of 2.5 mM (n ≥ 3). Supernatant lactate was measured as a ratio of the total cellular protein. Lactate production was measured under A) normoxia (O₂ = 20%) and B) hypoxia (O₂ = 1%). Asterisks (_*) designate lactate production in DCA treated cells percentage that were significantly different from untreated control cells. Error bars are expressed as SEM.

Discussion

The premise for these experiments was based on mathematical models that suggest the buffering capacity of bicarbonate against tumor acidity could be safely enhanced by addition of DCA (Martin N, Robey I, Gaffney E, Gillies R, Gatenby R, Maini P: Predicting the Safety and Efficacy of Buffer Therapy to Raise Tumor pHe: An Integrative Modeling Study, submitted). The model indicated that inhibition of tumor proton production by a relatively small amount could cause substantial reductions in tumor acidity, without any adverse effects on blood pH. A reagent like DCA fits this criterion because it can reduce extracellular tumor acidity by inhibiting lactic acid production. Moreover, DCA has been shown to selectively target tumor cells [17,18]. The design of this study was to treat our mouse model for breast cancer as similar to a patient study as possible by carrying out a survival experiment with surgical tumor resections.

We confirmed from both urine and serum pH measurements that addition of chronic DCA at the doses administered did not cause significant changes in either urine or serum pH. The urine and serum pH results from DB treated mice were statistically comparable to the measurements in the bicarbonate treated group. We conclude from these findings that DCA does not induce any systemic perturbations that could lead to systemic alkalosis.

Our studies show that none of the treatments reduced primary tumor growth. Our bicarbonate treatment group results were consistent with earlier studies [16]. Other studies have shown that DCA significantly inhibits tumor growth rates in vivo [17]. We expected to see this result as well, but found that DCA and DB treated mice had similar primary tumor growth rates to untreated and bicarbonate treated mice. This difference may be explained by the variable effect DCA can have in different tumor cell lines [20,21]. With respect to the metastases results, our findings were similar to a study that administered 5 mM DCA orally to rats injected with a metastatic breast tumor line through the tail vein. There was no measurable effect of the 5 mM dose [21].

It is unknown why DCA treatment did not provide therapeutic benefit against tumors in mice, but the in vitro studies may help to explain our findings. The viability studies under normoxic conditions demonstrate that tumor cells have a biphasic response to titrating DCA concentrations. This occurrence is known as hormesis, where low doses can be agonistic and higher doses are toxic to cancer cells [22]. The normoxic in vitro viability results suggest DCA concentrations at 20 mM or lower could have a stimulatory effect in vivo, though this was not measured. Only at concentrations exceeding 20 mM did DCA reduce viability under normoxia. Under hypoxia, DCA concentrations at 20 mM or lower had no effect on viability. There was a marginal, but significant reduction in viability at DCA concentrations greater than 20 mM under hypoxia. An 80 mM concentration was required to reduce viability 50%.

In the lactate production studies, DCA doses at 20 mM or lower significantly reduced MDA-MB-231 cell glycolytic metabolism under normoxia by approximately 19%. Doses higher than 20 mM inhibited lactate production by 29% under normoxia. No concentration of DCA tested reduced cellular lactate production under hypoxia. The in vitro studies suggest; 1) that DCA concentrations greater than 20 mM may have a non-specific effect on MDA-MB231 cells and 2) hypoxia may be a factor contributing to the in vivo findings.

MDA-MB-231 mammary xenografts are known to develop regions of hypoxia due to the development of necrotic lesions. Also, unpublished experiments (Robert Gillies laboratory at the University of Arizona) imaging MDA-MB-231 tumors in mice with bioluminescent reporters linked to promoter regions for hypoxia response elements such as VEGF or CAIX demonstrate that tumors develop regions of hypoxia (exhibit bioluminescence) as soon as tumors become palpable (between 100-200 mm³). Other studies have shown the link between DCA function and hypoxia. DCA inhibits pyruvate dehydrogenase kinase-1 (PDK1) which inhibits cellular metabolism and causes oxygen consumption resulting in increased hypoxia [23,24].

Although the metastatic load observed in bicarbonate treated mice appeared markedly lower than reported in DB treated mice (especially mesentery and intestinal), the data is insufficient to conclude that bicarbonate monotherapy was more effective at reducing the spread of metastases. The major impact on survival in our cancer model is lung metastases. As seen in the untreated group, most (79%) of the animals showed evidence of metastatic lung lesions. Only one of the bicarbonate treated animals in the analysis was found to have lung metastases, but there were two (of the four) spontaneous deaths that occurred in this group and one in the untreated group where we were unable to analyze the lungs. Nonetheless, a two-tailed Fischer's exact analysis shows that including the bicarbonate treated group spontaneous deaths as lung metastases positive (23%) and the untreated as negative for lung metastases, frequency of lung metastases would still be significantly lower in the bicarbonate treated mouse group (p = 0.02). It was surprising that the percentage of DB treated mice with lung metastases was 60%, and not significantly different from the occurrence of lung metastases in the untreated mouse group, indicating the bicarbonate monotherapy as a more favorable treatment modality at least in this tumor model. The findings suggest a potential risk in the DB treatment that would need to be investigated further in other models for cancer.

Analysis of the pulmonary lesions revealed that DB treatment resulted in significantly smaller mean tumor metastases compared to all other groups. The mean lesion diameter in DB treated mouse lungs was no larger than 30% of the lesion diameters in the other treatment groups. In a previous study it was reported that smaller tumor metastases in lungs was correlated significantly with bicarbonate treatment alone, but this was a 30 day experiment using β-galactosidase expressing MDA-MB-231 cells and the mean tumor diameters were about 87% of the lesion diameters measured in the untreated group [16]. The comparison of tumor lesion sizes between the metastasized lungs in all the study groups appears to suggest that DB treatment inhibits the growth of these metastases (Table 3). However, given the prevalence of non-metastatic lungs in the bicarbonate treated group, it could be argued that DCA has more of a stimulatory effect, especially if O₂ concentrations are more normoxic, and may even compete with bicarbonate therapy. This conclusion is supported by the in vitro findings which show that lower concentrations of DCA increase cell viability under normoxia.

Improved survival was a significant outcome in all groups treated with bicarbonate. There seemed to be little measurable therapeutic effect in DCA treated mice, suggesting bicarbonate was a driving component in the survival study. The results are consistent with earlier findings [16], but the mechanisms are not well understood. More than half the primary tumors in bicarbonate and DB treated mice did not return after surgical resections. We attribute the lower frequency of primary tumor re-occurrence to the improved survival rates in these groups. The difference between bicarbonate treated groups (bicarbonate and DB) and non-bicarbonate treated groups (untreated and DCA) in tumor re-occurrence after surgeries is statistically significant (p < 0.05). It may be possible that the effect of systemic bicarbonate could potentiate the wound healing process after surgical disruption of the tumor region. The role of bicarbonate ions (HCO₃^-) in wound repair was first reported in a study investigating gastric mucosal repair in cats systemically administered sodium bicarbonate. It was concluded that systemic excess of HCO₃^- facilitated superficial mucosal repair [25]. The molecular mechanisms driving this effect are unknown, but other reports have shown that blocking the Na⁺-HCO₃^- co-transport (NBC) with isothiocyanate (ITC) inhibits epithelial restitution, the process of epithelial migration involved in wound healing. Additionally, cell migration during restitution is dependent on glycolysis for energy [26]. This is notable because NBC functionally cooperates with monocarboxylate lactate transporter (MCT-1), a glycolysis-associated enzyme. Extracellular HCO₃^- increases NBC activity, which in turn enhances MCT activity leading to an increase in intracellular pH [27], a physiological condition conducive to glucose metabolism [28]. It is thought that the upregulated glycolytic activity of tumor cells offers a competitive advantage over surrounding host cells [14]. The epithelial wound healing processes after tumor resection in conjunction with an increase in extracellular bicarbonate may improve the competitive state of some host cells in the tumor microenvironment, and result in prevention of primary tumor re-growth. Although further evidence for these processes is required, it suggests that an optimal time to administer systemic bicarbonate would be after a tumor surgery.

Conclusions

This study confirms earlier reports about the role of systemic bicarbonate in the inhibition of metastatic spread from primary tumors, but highlights the limitations of this approach. While DCA has been shown both safe and effective against tumor cells in other studies, the findings reported here concur with the most recent investigations warning of potential pitfalls with DCA use [23,24]. First, it is not universally effective against all cancer cells. Secondly, tumor hypoxia serves as a confounding micro-environmental factor against DCA efficacy. The unpredictable effect of DCA against tumors therefore signals caution against using this agent as a therapeutic approach until new studies can determine the molecular role of DCA in different tumor microenvironments.

Abbreviations

DCA: dichloroacetate; DB: DCA-bicarbonate; PDH: pyruvate dehydrogenase; PDK: pyruvate dehydrogenase kinase; IACUC: Institutional Animal Care and Use Committee; SCID: severe combined immunodeficient; EMSS: Experimental Mouse Shared Services; GFP: green fluorescent protein; NBC: sodium bicarbonate co-transport; ITC: isothiocyanate; MCT-1: monocarboxylate lactate transporter.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

IR performed all experiments. IR and NM performed the statistical analysis. IR drafted the manuscript and NM revised the manuscript. IR and NM were involved in conception and design of the study and participated in the discussion and interpretation of the results. All authors read and approved the final manuscript.

Acknowledgements

We thank Libia Luevano, Katie Morgan and Gillian Paine-Murrieta at University of Arizona for their contributions to this work. IR: National Centers for Complementary and Alternative Medicine, NIH grant K01AT004678. Arizona Cancer Center: CCSG grant (CA 023074).

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