MPO deficiency

Abstract

Enzyme Therapy (ET) has been practiced successfully for a wide range of disease states. Often, the enzymes used are fungal based because they are effective over a wide range (mostly acidic due to the digestive environment they are formulated to work in) of pHs. This has been fruitful. In fact, many of the enzymes used are touted for their ability to function well in the gastric environment and are considered useful for that purpose. However, what has been ignored is that there are diseases that can be associated with having too low a pH in the body. This paper discusses the formulation of an enzyme supplement that can be used to alter the pH of the body. The implications of this are staggering and will certainly garner much attention over the next few years. Additionally, data is presented demonstrating that the special, food grade enzymes can work in an alkaline environment. The present work demonstrates the feasibility of creating an enzyme system capable of being functional at higher pHs and the possibilit y that the pH change can have a genomeceutical effect. (1)

Introduction

Acidosis is a state where the pH of the blood is abnormally low (blood is usually at a pH of 7.3). Webster's states it is "an abnormal condition characterized by reduced alkalinity of the blood and of the body tissues." (2) There are a large number of clinical conditions which have been associated with acidosis. (3)

For instance, inhibition of mesangial iNOS by reduced extracellular pH is associated with uncoupling of NADPH oxidation. Chronic renal failure is associated with metabolic acidosis and down-regulation of intrarenal nitric oxide (NO) synthesis. It has been hypothesized that acidosis may impair the intrarenal NO synthesis. The effects of alterations in extracellular pH were examined on inducible NO synthesis in murine mesangial cells (MMC) in culture. It was shown that acidosis impairs iNOS activity in MMC by a post-translational mechanism that involves uncoupling of NADPH oxidation.

Acidosis has also been implicated in childhood diseases. (4) Mitochondrial cytopathies are caused by genetic alterations of nuclear- or mitochondrial-encoded genes involved in the synthesis of subunits of the electron transport chain. Mutations of mitochondrial DNA are associated with a wide range of clinical presentations. Typically, the pathology is so complex it has been hard to ferret out exactly what is happening. However, it is well known that nucleic acids require a defined pH range to function correctly. Indeed, if the pH around DNA is altered too significantly, the actual shape and therefore function of the DNA can be affected. During metabolic acidosis, increased renal ammoniagenesis and bicarbonate synthesis are sustained by the increased expression of various transport proteins and key enzymes of glutamine metabolism. Altering the pH may be a way of using a genomceutical (1) to change the expression of desirable enzymes. (5)

Also in children, initial screenings include tests for acidosis. Neuro-degenerative diseases in children (6) and other diseases have been linked to acid-base problems. (7) Genetic disorders of acid-base transporters involve plasmalemmal and organellar transporters of H(+), HCO3(-), and Cl(-).

Interestingly, acidosis is not always one of the first things checked. There have been reports of dyspnea, which actually appeared more as renal tubular acidosis (RTA). It may be that some diagnoses of congestive heart failure, asthma exacerbations or both, may be due to acidosis. (8)

In addition to children, it should be emphasized that renal tubular acidosis (RTA), renal insufficiency, aldosterone deficiency, in old age with reduced renal mass and function, and angiotensin-converting enzyme (ACE)-inhibitor therapy, may be at high risk of developing these severe and potentially life threatening complications from acidosis. (9)

Historically, renal tubular acidosis (RTA) has been classified on a clinical basis, without any reference to the underlying disorder. Here we review the normal mechanisms of renal acidification and we identify disorders of specific transporters (genetic, disease-related or drug-induced) that lead to the main categories of distal RTA. We also describe the approach to diagnosis and the current treatment of distal RTA.

Acidosis has also been linked to intestinal injury (10) by investigating the pathogenic mechanism(s) of small intestinal injury during acidosis in relation to circulating nitric oxide (NO) in an experimental rat model. They showed that the pathogenic mechanisms of acidosis-induced small intestinal lesions involve up-regulation of NO production by increased expression of iNOS and augmentation of superoxide radicals and MPO activity.

Rarer still are reports of other forms of acidosis such as those caused by Multiple acyl-CoA dehydrogenase deficiency. (11) Treatment options that could raise the systemic pH and also supply the afflicted enzymes would be highly advantagious.

Certain antiseizure or antiepileptic drugs, such as Topiramate, also appear to be able to induce acidosis as a possible side effect. Such side effects could be eliminated if the drugs were administered white the system was being buffered.

Attempts to Block Acidosis

There have been a variety of attempts to block acidosis. Some such attempts have been rather unique and exotic such as the use of antisense mRNA to reduce lactate dehydrogenase. (12)

Another attempt has been to simply take antacids or bicarbonates. (13-14) These can be effective but carry their own side effects such as constipation, diarrhea, etc. In infants, typically a bicarbonate level of oral sodium bicarbonate (1-2 mmol/kg) can be used. (15)

At present, the science of Enzyme Therapy (ET) is very limited. Usually, ET consists of using enzymes that function optimally over an acidic range. This is done because most of the therapy is thought to be derived from digestion of substrates along the lumen of the GI tract. (16) This has proven to be very successful. However, at present, there is also a need for enzymes to be able to function, in diseased states, and ideally raise the blood pH at the same time. For this reason, an enzyme formula was created to address this issue. The idea of using a base for the treatment of acidemia is not novel, (17) however, the combination with ET is.

AIDS is a disease which often leads to states of acidosis and eventual complications including liver failure. Treatment options have been limited and there is a dire need for products which can rebalance the body's pH. (18) High doses of riboflavin and thiamine may help in secondary prevention of hyperlactatemia. These naturally occurring compounds have found a place in the treatment of complications due to AIDS. (19,20)

During exercise-induced metabolic acidosis, intravenous administration of bicarbonate increased the buffering capacity of blood and attenuated the decrease in intracellular muscle pH, although there has been observed a small increase in the arterial carbon dioxide pressure.(21,22) Again, the side effects are still rather unpleasant.

The present study looks at a novel enzyme composition consisting of an amylase, lipase, two proteases (one alkaline and one acidic), Phytase, and a buffer system. The purpose of the first four enzymes is to mimic the body's own pancreatin. The Phytase is added to assist in the liberation of divalent cations, often needed by enzymes for proper functionality. The buffer system contents are similarly designed to include many of the nutritional factors required by enzymes (e.g., zinc and other metals) while at the same time maintain a slightly basic pH. The theory being that if a body is in a state of acidosis, the endogenous enzymes may not be functioning properly. These enzymes need to be exogenously supplied. Since we are also trying to support an increase in pH, the enzymes chosen, function over a broad pH range. We also designed a buffer system that complements the functionality of the enzymes.

Data is presented on the functionality of the entire system on various substrates. Additionally, data is presented on the ability of the system to raise the pH of urine from a volunteer.

Materials and Methods

The measurement of pH: All measurements of pH were done using a pHydrion INSTA-CHEK 0-13, Micro Essential Laboratory, Brooklyn, New York 11210 USA.

Enzymes: A Vegetarian enzyme Makzyme-Ezmed, was formulated from a fungal source to have four times the activity of that Listed by USP/NF for pancreatin. This formula, Makzyme-Ezmed, has an effective pH range from 3-8 for the amylase, protease, and lipase in it. However, because the protease does not function very well at higher pH (above 5) an additional protease was employed. An alkaline protease (MAKAP) (MAK Wood, Grafton, Wisconsin) was supplied with an effective pH of 610. Also, a Phytase was added to the formulation at 25PU.

Buffer system: A buffer system had to be created that could support the activities of the enzymes and that would also be able to raise the urine pH after oral ingestion of the enzymes. The buffer, MAKTech [TM] Enzymeoptimizer (MAK Wood, Inc. Grafton, Wisconsin) was chosen because it is known to not just have an alkaline pH but also to be a good buffer containing many of the cofactors (calcium, magnesium, zinc, etc) that many enzymes require.

Substrate digestion: In the first Step, precooked, packaged noodles were obtained from a local store in bulk. Roughly, one quarter of the material was used for each of the two runs. Each run consisted of testing either plain noodles in water or plain noodles in water plus the Enzyme Preparation above. The reaction beakers were allowed to incubate at 37C for varying time from 0-8 hours.

Because of the data from the first experiment, another step was taken. The second step was to add 20X the amount of enzyme to the mixture (test beaker) and allow the noodles to continue to incubate.

Because little or no breakdown of the noodles was observed, it was reasoned that even though in theory all the enzymes required to break down a product such as dried, fried noodles, there was little observable effect, even after eight hours of incubation. This surprising result suggested several possibilities. The first was that the enzymes were no good. The second was that something in the buffer inhibited all the enzymes present. The third was that the substrate was, for whatever reason, not digestible by typical enzymes used in ET.

Because it was known that the second enzymes to be added were extremely functional (data not shown) in a similar assay but different substrate, we ruled out number one. The second and third possibilities were addressed by a simple third experiment. Here, a cracker was added to both a beaker with the Enzymeoptimizer Buffer and a beaker of water. Pictures were taken at various intervals. Photographs were scanned on a Microtek Scanner using the highest resolution setting.

Urine analysis: The pH of a volunteer was checked pre- and post-supplementation with the system. The pH paper was held in the urine stream until saturated and then recorded photographically. Photographs were scanned as above.

Results

The initial step in creating a digestive enzyme formulation that can function at a basic pH is to decide on the enzymes to use. Fungal enzymes known to function over a broad enough pH range to include that of the buffer system to be used, were chosen. Those chosen were a Vegetarian Pancreatin and an Alkaline protease.

Having chosen the enzymes, simple chemistry dictates that a basic buffer would be required to raise the pH of a solution from an acidic level to one which is slightly basic. As stated in the introduction, common methods of doing this are to use a buffer such as sodium bicarbonate.

Figure 1 shows pH paper that has been dipped in either [H.sub.2]0 (left) or [H.sub.2]0 plus Buffer. As can be seen, the buffer is able to raise the pH of the solution above that of ordinary water. More importantly, the buffer raises the pH above that of 7.0 to a basic level.

However, because the objective was not only to raise the pH of a system but also to have functional enzymes, many of which require metal cofactors, a buffering system was chosen that contains many of those nutritional requirements. Because enzymes themselves can be acidic, it was necessary to make sure the buffering system plus enzymes were still able to provide a basic solution when orally consumed. Figure 2 shows the results of pH paper dipped into two solutions. The paper on the left is that of only [H.sub.O]. The paper on the right has been dipped into [H.sub.O] with buffer and the enzymes. As can be seen, the test solution (one with buffer and enzymes) is able to maintain a basic pH. Figures 2C and D are replicates of A and B, respectively. This was done to ensure reproducibility.

To assay for functionality of the enzymes in the buffer, Asian noodles were chosen because it was thought they would provide an easily viewed qualitative form of digestion. Figures 1A and B show the appearance of the noodles at time zero, either in plain water or in 1120 plus buffer and enzymes. As can be seen, there was no initial visual difference in appearance between the two groups.

Figure 2 shows the results of pH paper dipped into two solutions. The paper on the left is that of only H2O. The paper on the right has been dipped into H2O with buffer and the enzymes. As can be seen, the test solution (one with buffer and enzymes) is able to maintain a basic pH. Figures 2C and D are replicates of A and B, respectively. This was done to ensure reproducibility.

Figures 3 A and B show the Asian-style noodles at the beginning of the experiment. As can be seen, there is no observable difference in the integrity of the control (A) or the experimental (B) beakers.

Figures 4A,B,C,&D show the same reaction beakers at: A) H2O and noodles after 15 mins; B) H2O, buffer, Enzymes, after 15 minutes; C) same as (A) but after one hour; D) Same as (B) but after one hour. As can be seen, there was very little visible digestion of the noodles even after one hour.

Because it was conceivable that the enzymes were not functional, another group of enzymes known to work in a similar system were added to the noodles and the reaction was allowed to proceed for eight hours with 20 times the previous levels of enzymes added to the mixture. Again, there was little noticeable digestion of the noodles. However, the noodles in the enzyme-treated group did appear slightly more separated and the solution became slightly more cloudy with some sedimentation.

At this point, it was suspected that the buffer was inhibiting the enzymes. To test this, a second round of incubations, exactly the same as the first were performed, with the exception that the test material was a Ritz cracker and not the Asian-style noodles. Figure 6. A,B,C,D,&E show the following results: A) The pH is buffered. The strip on the left is plain H2O. The strip on the right is H2O plus Makzyme-Ezmed and Maktech Enzymeoptimizer. B) Shows the cracker is not digested at all after 15 minutes in H2O. C) Shows that the cracker is visibly digested in H2O, Buffer, and Enzymes and demonstrates that the enzymes are indeed functional in the buffering system. Figures 6 D&E are side views of B&C, respectively. Clearly, the pH buffer and enzymes function in concert to digest some foods. Discussion is given below to the inability to digest the Asian noodles compared to the cracker.

Discussion

Supplemental enzymes have been shown effective in prior systems for conditions ranging from autism, to vitamin and mineral deficiencies.23 The present work explores the possibility of using a novel buffer system to allow enzymes to function at higher pH and at the same time, work to alter the body's pH level.

The treatment of clinical acidosis has been shrouded in controversy for decades. This is principally due to the primary treatment being bicarbonate infusion in response to indirect measurements that are calculated rather than obtained directly. While both sides of the debate have valid points, it makes sense that given a standard blood pH being slightly alkaline, then ingestion of a neutral or slightly basic solution would not aggravate acidosis and could only assist. Also, if the solution was able to assist with digestion, that may be altered by an exacerbated acid-base imbalance in the body, that too would be beneficial.

In the present study we attempted to look at the feasibility of creating an enzyme supplement consisting of a variety of enzymes that reflect a typical pancreatic output in response to food intake. A fungal-based enzyme supplement that was functional at slightly basic pH was established along with a novel buffer system to maintain the slightly basic pH.

Initial tests of the functionality of the enzymes suggested that perhaps the enzymes would not work in a basic environment, even though theoretically, they should. The substrate for the enzymes consisted of Asian-style noodles. Typically, these noodles are boiled before eating to ensure they are cooked. This was not done and is suspected to be the problem. It had been assumed that the noodles would be at least partially digestible in the relatively massive amount of enzymes that were applied, however, this was incorrect. Little if any, digestion of the substrate occurred. As it turned out, this made for a good negative control of the process.

What is really striking is the need to boil these types of noodles first. At least, it is presumed that boiling would correct the inability to digest them. The inability of the enzymes to digest the uncooked noodles was truly not expected. The authors will let the readers extrapolate as to the implications of this result, however, it should be said that much of the modern food supply is processed. Such processing obviously can interfere with the endogenous enzymes and their functionality. That should be considered when designing a proper diet.

The second half of the study looked at the same principle but used a different substrate. This time, one that was thought to be much easier to digest was chosen, a Ritz cracker. Under the same experimental conditions as with the first substrate, the cracker was easily digested within 15 minutes. This demonstrated that both the pH was maintained to be slightly basic and that the enzymes were functional. As to what degree of functionality, it could be argued that it is limited based on the first substrate. However, we believe this is incorrect as the initial substrate was fortuitously not properly prepared and as such, was a good control. The slightly cloudy solution of the test beaker and the sedimentation in the same are suspected to be the result of partial digestion and just the enzymes falling out of solution.

In summary, a novel enzyme preparation has been created and established to be functional in a slightly basic, buffered system. In theory, the enzymes could be altered to include or exclude a large variety, though those with a basic pH optimum would function better. In addition, the present system of buffer and enzymes could be used not just for digestive support, but also in cases where acid-base imbalances are thought to be affecting metabolic activities, especially those involving enzymes. The afflicted enzymes could be exogenously supplied along with the buffering system. Future experiments should be designed to look at the feasibility of this. For instance, the conversion of creatine to creatinine in the body occurs rapidly in an acidic environment. Such a situation would deplete the phospho-creatine pool that acts as a reservoir for high-energy phosphates. Adding creatine kinase to the buffer system would be one such possibility.

Given these results, we have begun designing enzyme supplements to support the digestive functions of autistics, AIDS, cancer, diarrhea, Down Syndrome, lactose maldigestion/intolerance, and a wide variety of other conditions. It should even be possible, based on simple tests, to tailor one, two, or three supplements to an entire subpopulation. The next 20 years will be exciting times for those of us who have been involved with enzymes from the beginning.

References

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(2.) ref http://www.m-w.com/cgi-bin/dictionary?acidosis.

(3.) Prabhakar SS 2002 Inhibition of mesangial iNOS by reduced extracellular pH is associated with uncoupling of NADPH oxidation. Kidney Int Jun;61(6):2015-24

(4.) Gropman AL 2001 94 Diagnosis and treatment of childhood mitochondrial diseases. Curr Neurol Neurosci Rep Mar;1(2):185-

(5.) Huang TT, Carison EJ, Kozy HM, Mantha S, Goodman SI, Ursell PC, Epstein CJ. 2001Genetic modification of prenatal lethality and dilated cardiomyopathy in Mn superoxide dismutase mutant mice. Free Radic Biol Med Nov 1;31(9):1101-10.

(6.) Wong V. 1997 Neurodegenerative diseases in children. Hong Kong Med J. Mar;3(1):89-95.

(7.) Alper SL. 2002 Genetic diseases of acid-base transporters. Annu Rev Physiol; 64:899-923.

(8.) Bomann JS, Peckler BF. 2002 Type IV renal tubular acidosis presenting as dyspnea in two older patients taking angiotensia-converting enzyme inhibitors. Ann Emerg Med Jan;39(1):73-6

(9.) Margassery S, Bastani B. 2001 Life threatening hyperkalemia and acidosis secondary to trimethoprim-sulfamethoxazol treatment. J Nephrol Sep-Oct;14(5):410-4

(10.) Pedoto A, Nandi J, Oler A, Camporesi EM, Hakim TS, Levine RA. 6 2001 Role of nitric oxide in acidosis-induced intestinal injury in anesthetized rats. J Lab Clin Med Oct;138(4):270-

(11.) Grice AS, Peck TE 2001 Multiple acyl-CoA dehydrogenase deficiency: a rare cause of acidosis with an increased anion gap. Br J Anaesth Mar;86(3):437-41.

(12.) Jeong D, Kim TS, Lee JW, Kim KT, Kim HJ, Kim IH, Kim IY. 2001 Blocking of acidosismediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression. Biochem Biophys Res Commun Dec 21;289(5):1141-9

(13.) Nielsen HB, Hein L, Svendsen LB, Secher NH, Quistorff B. 2002 Bicarbonate attenuates intracellular acidosis. Acta Anaesthesiol Scand May;46(5):579-84

(14.) Ammari AN, Schulze KF. 2002 Uses and abuses of sodium bicarbonate in the neonatal intensive care unit. Curr Opin Pediatr Apr;14(2):151-6.

(15.) Philippi H, Boor R, Reitter B. 7 Topiramate and metabolic acidosis in infants and toddlers. Pilepsia 2002 Jul;43(7):744-

(16.) Brudnak MA 2000. Enzyme Therapy - Part I Townsend Letter for Doctors & Patients. December 209:88-92.

(17.) Rosival V. 2002 Use of base in the treatment of severe acidemia. Am J Kidney Dis May;39(5):1125-6

(18.) Read AIDS 2002 May;12(5):222-4

(19.) Briggs JM, Drabek CA McComsey GA, Lederman MM. 2001 Metabolic complications of HIV and AIDS. Orthop Nurs Jul-Aug;20(4):41-50.

(20.) Grace A. McComsey, MD; Michael M. Lederman, MD 2002 High Doses of Riboflavin and Thiamine May Help in Secondary Prevention of Hyperlactatemia AIDS Read 12(5):222-224.

(21.) Nielsen HB, Hein L, Svendsen LB, Secher NH, Quistorff B. 2002 84 Bicarbonate attenuates intracellular acidosis. Acta Anaesthesiol Scand May;46(5):579-

(22.) Unwin RJ, Shirley DG, Capasso G. Urinary acidification and distal renal tubular acidosis. 2002 J Nephrol Mar-Apr;15 Suppl 5:S142-50.

(23.) Brudnak MA, et al. 2002 Enzyme-based therapy for autism spectrum disorders - Is it worth another look? Med Hypotheses. May;58(5):422-8.

Correspondence:

Mark A. Brudnak, PhD, ND

Healthy Living Naturally, LLC

Wisconsin, USA

Email: Mark.Brudnak@alumni.usc.edu

COPYRIGHT 2002 The Townsend Letter Group
COPYRIGHT 2003 Gale Group