The Kinetics of Acetylcholinesterase Inhibition and the Influence of Fluoride and Fluoride Complexes on the Permeability of Erythrocyte Membranes
Author: By Johannes Westendorf. Hamburg, Germany – 1975
Dissertation to receive Ph.D. in Chemistry from the University of Hamburg
Reviewer: Prof. Dr. A. Knappwost
Co-Reviewers: Prof. Dr. Malorny, Prof. Dr. Strehlow, Prof. Dr. Hilz, Prof. Dr. Gercken.
The oral defense took place on 2/18/1975
A Foreword intended to place the Westendorf Research in current context indicating why it is relevant to a wide range of contemporary health and behavioral problems has been prepared by Myron J. Coplan and Roger D. Masters whose credentials are also attached.
A similar PDF version of this work is available: Westendorf PDF (link opens in a new window).
II. Presentation of the Problem
III. Procedure and Results
A. Inhibition of Acetylcholinesterases
A1. General Information
A2. Theoretical Treatment of the Enzymatic Kinetics
A3a. Description of the TracerMethod
A4. Carrying out the measurements
A4a. Acetylcholinesterase Inhibition by NaF
A4b. pH Dependence of the Inhibition
A4c. Dissociation Behavior of Several Fluoride Complexes / Properties of the Fluoride electrode
A4d. Inhibition of Acetylcholinesterase by Complexed Fluorides
B. Effect of Fluoride on the Permeability of Erythrocyte Membranes for Na+, K+,Ca2+, F-, HPO42-, and Glucose
B1. Description of the Isotopes Used
B2. Presentation of the Permeability Experiments
B2a. Potassium Permeability
B2b. Calcium Permeability
B2c. Sodium Permeability
B2d. Fluoride Permeability
B2e. Phosphate Uptake
Foreword (by MJ Coplan and RD Masters, April 2001)
Westendorf’s 30-year old PhD research work is important for reasons beyond its specific scientific findings. First, his work was motivated by the assumption that ingested fluoride was beneficial. Knappwost, his thesis supervisor, believed that fluoride in saliva afforded protection against tooth decay and was seeking a means of enhancing the output of fluoride-bearing saliva for that purpose. Therefore, it can hardly be said that Westendorf’s work was biased against water fluoridation.
Second, Westendorf’s research was based on knowledge that fluoride ion is an enzyme inhibitor. Indeed, that feature of ingested fluoride seemed to offer multiple benefits. Knappwost believed that ingested fluoride, by inhibiting cholinesterase, could achieve both greater expression of total saliva and an increase in its fluoride content. The research of his student quite logically examined different forms of ingestible fluoride for their effect on several variants of cholinesterase. Westendorf’s results showed that fluoride in the form of the silicofluoride complex (SiF), as well as several other complexes, was a substantially more powerful inhibitor of cholinesterases than the simple fluoride ion released by sodium fluoride (NaF). This was simply an objective finding.
Third, to account for the more powerful inhibition effect of SiF, Westendorf studied the course of its fluoride release in fine detail. He found that under physiological conditions, dissociation was no more than 66% in the concentration range considered “optimum” for fluoridated water by United States health authorities. If the released fluoride came uniformly from all of the initially injected SiF, the molar concentration of the residual non-dissociated species would be the same as that of the injected SiF. It would follow that dilution of fluosilicic acid to a nominal 1 part per million of free fluoride in water at pH 7.4 induces each [SiF6]2- to release 4 fluorides to be replaced by hydroxyls. The partially dissociated residue would be the ion [SiF2(OH)4]2- which would then be present in the water at the same concentration as the originally introduced SiF. The biological consequences of ingesting such a species are probably not innocuous, with enzyme inhibition being only one of several possibilities.
Westendorf’s visualized course of SiF dissociation, based on actual experimental evidence, is materially at odds with the dissociation route assumed by US EPA and CDC, based on theory. In judging the reliability of the theoretical approach and claims of health safety presented by these government agencies, one should be aware that both the nature of the complicated mixture called “fluosilicic acid” and the course of its dissociation upon dilution remain unresolved despite nearly a century of research. Two recent documents demonstrate this. In the first, an expert in the recovery of fluoride in phosphate rock processing, addressing a group of his peers at a 1999 International Fertilizer Association[a] meeting held in the former USSR, said:
“The chemical formula of fluosilicic acid is H2SiF6. However, things are not as simple as that due to the fact that rarely is fluosilicic acid present as pure H2SiF6…There are well reported references to the existence of H2SiF6 . SiF4 …Hereon in this presentation, FSA [fluosilicic acid] means a mixture of HF, H2SiF6 and H2SiF6 . SiF4.”
[a] Smith PA; “History of Fluorine Recovery Processes”: Paper delivered at the IFA Technical Sub-Committee and Committee Meeting[a] in Novgorod, Russia; Sept 15-17, 1999. (http://www.fertilizer.org/PUBLISH/tech0999.htm)
This is a highly significant statement coming from someone who ought to know the subject under discussion. It means that a key intermediate dissociation product postulated by CDC and EPA theories to be transient species only fleetingly after SiF is introduced into the water at the water plant, may be present in concentrated fluosilicic acid before dissociation begins. Such a starting condition would cast serious doubt on the postulated theoretical equations predicting “virtually 100%” dissociation that supposedly “guarantee” no adverse health effects from undissociated SiF residues in drinking water treated with these compounds.
Equally important is a letter[b] dated March 15, 2001, written by the Director of the EPA Water Supply and Water Resources Division, which concludes with the statement:
"In January, representatives from the [EPA] Office of Research and Development (ORD) and the Office of Science and Technology and Ground Water and Drinking Water met to discuss a number of water related issues including Fluoridation. Several fluoride chemistry related research needs were identified including; (1) accurate and precise values for the stability constants of mixed fluorohydroxo complexes with aluminum (III), iron (III) and other metal cations likely to be found under drinking water conditions and (2) a kinetic model for the dissociation and hydrolysis of fluosilicates and stepwise equilibrium constants for the partial hydrolysis products.”
In plain English, senior EPA research staff now believe their staff needs to go back into the lab for at least another year or two to find out if the EPA’s longstanding confidence in the “virtually total” dissociation of SiFs may have been misplaced. Whatever the outcome may be of their new study of SiF dissociation, it is clear the EPA does not intend to perform animal tests to ascertain health effects of chronic ingestion of SiF treated water under controlled conditions.
Animal experiments according to accepted toxicology testing protocols would be the logical way to examine health effects of enzyme inhibition by SiF that Westendorf observed at the cellular level. Three published reports bearing directly on this matter should be noted. In the early 1930s, the Ohio agriculture department wanted to develop a replacement for bone meal as a source of calcium and phosphorus in the feed ration of farm animals. Natural “rock phosphate,” comprising largely calcium phosphate, was a candidate, but it was known to carry about 2 to 5 % of fluoride bound in some chemical form. Thus it was necessary to study possible adverse health effects due to ingestion of fluoride from several sources.
A report[c] issued in 1935 comparing health effects primarily from calcium fluoride, sodium fluoride, and rock phosphate. Highly significant for present purposes was one small experiment that included sodium fluosilicate. With equal dosage and equal amounts of fluoride retained, rats fed sodium fluosilicate excreted three times as much non-retained fluoride in urine as rats fed sodium fluoride, who eliminated more fluoride in feces. Apparently about three times as much fluoride had crossed the gut/blood membrane into the bloodstream from SiF than from NaF.
[b] Gutierrez SB (signed by Thurnau RC); Letter from the Director of the US EPA National Risk Management Laboratory to Roger D. Masters, dated March 15, 2001.
[c] Kick CH et al; “Fluorine in Animal Nutrition”; Bulletin 558, Ohio Agricultural Experiment Station; Wooster, Ohio; November 1935; pp1-77.
A second report, this one by the US PHS[d], was published about ten years after water fluoridation had begun. The study compared the time, starting from the date of fluoridation either with sodium fluosilicate or sodium fluoride, for urinary fluoride level to reach equilibrium with ingested fluoride from fluoridated water. The study populations were boys and men. There were two noteworthy results. First, for either fluoridating agent, urine fluoride levels in older males reached equilibrium with ingested fluoride levels sooner than in younger males. The longer time for young males can be accounted for by the fact that the weight of the older males was essentially constant, while the younger males were adding bone mass over the several years of the experiment. The bodies of younger males were therefore providing a time-related increase in storage compartment capacity for ingested fluoride.
A more important finding was that for the younger males it took longer for their urine level of fluoride to reach equilibrium with ingested water fluoride from SiF than from NaF. Apparently in growing boys SiF fluoride must have been metabolizing differently from NaF fluoride.
A third relevant study[e], conducted around the same time as Westendorf’s research, involved feeding water treated with the same fluosilicic acid used to fluoridate the local water supply to squirrel monkeys for up to 14 months. Morphological and cytochemical effects were reported for the liver, kidney, and nervous system due to ingestion of 1-5 ppm of fluoride in water. Although the study did not compare results from exposure to NaF, the report emphasizes the fact that the kidneys of monkeys ingesting SiF treated drinking water “…showed significant cytochemical changes, especially in the animals on 5 ppm fluoride intake in their drinking water.”
The report later observes that work by others in the 1940s and 1950s “..showed that fluoride has an inhibitive effect on the activity of succinate dehydrogenase. These studies indicate that under the effect of fluoride intake, a serious metabolic distress may develop in the kidneys.” In concluding, the report notes that “Earlier, some workers had also indicated that inorganic fluorides have a strongly adverse effect on the activity of some enzymes and of these, mitichondrial enzymes, acid and alkaline phosphatases and ATP-utilizing enzymes and aldolase may be the most affected (Batenburg & Van den Bergh, 1972; Katz & Tenenhouse, 1973).”
This study of squirrel monkeys is a rare (possibly singular) American experiment with SiF. If the research team had known that Westendorf was finding greater effects of silicofluoride than sodium fluoride on enzyme activity at virtually the same moment, the U.S. study might have taken a different turn. In any case, two of these three American experiments compared effects from NaF and SiF, and both found that SiF and NaF do not produce the same effect. Moreover, all three studies found the strongest adverse clinical effect of silicofluoride in the kidney. But damage to the kidney is hardly the only possible health effect of ingested SiF.
[d] Zipkin I et al; “Urinary Fluoride Levels Associated with Use of Fluoridated Water”; Pub Hlth Rpts 71 pp767-772; 1956.
[e] Manocha SL, et al; “Cytochemical response of kidney, liver and nervous system to fluoride ions in drinking water”; Histochemical Journal, 7 (1975); 343-355.
“Life” involves an incalculable number of chemically active molecules initiating, continuing, and terminating a bewildering variety of chemical events. Throughout this panoply of events and in every organ where they occur, various enzymes play crucial roles. A particularly important example is the quenching by enzymes of muscle stimulation induced by the neurotransmitter acetylcholine (ACh), an ester comprising the acetyl moiety bound by an oxygen bridge to the choline molecule. The principal “quenching” enzyme, acetylycholinsterase (AChE), comes in several variations and the ACh/AChE dyads operate in numerous ways in many organs. Related enzymes called pseudocholinesterases are found in serum and include the butyrylcholinesterases.
At latest count over 7,000 enzymes have been detected and catalogued[f], and there is no reason to suppose that the effect of SiF is limited only to a small sub-class. In any event, one would be hard put to identify a more important enzyme sub-class than “esterases,” which cleave molecules called “esters” at the right time and place in the healthy organism. While a great deal is known about many of the ways these enzymes function, there are still large knowledge gaps to be filled.
To do just that, an extensive survey of contemporary knowledge about cholinesterases has recently been published[g] by an employee of the Office of Prevention, Pesticides and Toxic Substances in EPA’s Health Effects Division. The published article carries this disclaimer:
“Although this article was written as part of the author’s official duties as an EPA scientist, the opinions and conclusions expressed in it are his alone, and do not reflect the position of the Environmental Protection Agency.”
Because this comprehensive review deserves a great deal of attention, one wonder’s why it was not published as official work of the EPA. Moreover, the fact that over 91% of fluoridated water in the US today is treated with SiFs heightens the importance of Westendorf’s findings very significantly. The EPA has officially acknowledged[h] that it has no data on health effects of the SiFs, even though it has been allowing dissemination of 200,000 tons per year of SiFs into the public water systems. The relevance of the issue is illustrated by a recent published[i] hypothesis that might explain Fibromyalgia, Multiple Chemical Sensitivity, Chronic Fatigue Syndrome, ADD/ADHD, poor impulse control, and even Gulf War Syndrome.
One would like to believe that the following English language translation of the Westendorf thesis will shed some light on the possible health effects of what has become a virtually ubiquitous enzyme inhibitor in the daily diet of over half of US residents. With millions of people suffering from one or the other of several poorly understood conditions with likely roots in environmental toxins, it is time to reexamine entrenched governmental doctrines in the light of Westendorf’s research which, while 30 years old, has received little or no attention.
[f] On February 7, 2001, the Brookhaven Registry of Enzymes listed 7,164 enzymes on their web-site, http://www.biochem.ucl.ac.uk/bsm/enzymes/.
[g] Dementi B; “Cholinesterase Literature Review and Comment”; Pesticides, People and Nature; 1 (2); 59-126; 1999.
[h] Letter to the Honorable Ken Calvert, Chairman of the Subcommittee on Energy and the Environment, US House Committee on Science, from EPA Assistant Administrator J. Charles Fox, June 23, 1999.
[i] Laylander J. “A Nutrient/Toxin InteractionTheory of the Etiology and Pathogenesis of Chronic Pain-Fatigue Syndromes: Parts I & II,” Journal of Chronic Fatigue Syndrome; 5 (1), 67-126, 1999.
Synopsis of Foreword Authors’ Relevant Professional History
Roger D. Masters, Ph.D., is President of the Foundation for Neuroscience and Society and Nelson A. Rockefeller Professor of Government Emeritus at Dartmouth College. For the last 30 years, he has studied the implications of modern biological science in understanding human behavior. He serves as editor of the "Biology and Social Life" section of Social Science Information (an international journal published at the Maison des Sciences de l'Homme in Paris) and member of the Council of the Association for Politics and the Life Sciences. He is a published expert in the history of Renaissance politics, especially the contribution of Niccolo Machiavelli.
After undergraduate studies at Harvard (where his instructors included Henry Kissinger), he served in the U.S. Army before graduate studies at the University of Chicago. Despite his work in other areas, he retained a strong professional interest in military and international affairs. In addition to writing The Nation is Burdened: American Foreign Policy in a Changing World (Knopf, 1967), he served as U. S. Cultural Attaché to France. Among his many other books are The Political Philosophy of Rousseau (Princeton, 1968), The Nature of Politics (Yale, 1989), Machiavelli, Leonardo, and the Science of Power (Notre Dame Press. 1996) and Fortune is a River: Leonardo da Vinci and Niccolò Machiavelli's Magnificent Dream to Change the Course of Florentine History (Free Press, 1998). Before turning to issues of environmental pollution, health and behavior, he also published widely on the effectiveness of leaders' nonverbal behavior on television (working with colleagues on experiments in France and Germany as well as in the U.S.).
Among many other publications on biological factors in human behavior, he was co-editor (with Michael T McGuire) of The Neurotransmitter Revolution, Serotonin, Social Behavior and the Law (Southern Illinois University Press, 1994); senior author (with Brian Hone and Anil Doshi) of "Environmental Pollution, Neurotoxicity, and Criminal Violence," in J. Rose, ed., Aspects of Environmental Toxicity (London: Gordon & Breach, 1998), pp. 13-45; and co-author (with.MJ Coplan) of "Water Treatment with Silicofluorides and Lead Toxicity" International Journal of Environmental Studies, 56: 435-449 (July-August 1999).as well as of other publications.
In addition to an earlier teaching position in political science at Yale, he served as U.S. Cultural Attaché to France, Fellow of the Hastings Center, Chair of the Executive Committee of the Gruter Institute for Law and Behavioral Research (a foundation specialized in linking biology to the study and practice of law), a visiting professor at Yale Law School and Vermont Law School, and a consultant to Upjohn Corp, to the Commissioner of Corrections of Vermont, and to several agencies of the Federal Government. As a result of these varied professional activities, Dr. Masters has had extensive experience applying new scientific research in the biology of human behavior to the establishment of successful government policies.
Myron J. Coplan, PE is a consultant in chemical engineering and chemical sciences, doing business as “Intellequity” after retirement in 1987 as Vice President and General Manager of the Albany International Co. Membrane Development Venture. The fruits of this latter activity include a product line of membranes now used by a major multi-national company to supply a market for industrial gases measured in the $ billions.
Coplan’s working career started during WW II first as a civilian employee of the US War Department and then as a production chemist for a firm supplying the military with two crucial commodities: DDT, without which the S. Pacific campaign might not have been successful, and a wire insulating chemical, without which the US Navy’s capacity to deal with disastrous convoy damage by Nazi mines might not have been achieved. He was one of the few civilians deferred throughout WW II for his critical occupation status.
Post WW II, while pursuing his own advanced degree studies, Coplan headed an academic chemical engineering department, supervising doctoral research of others. This was followed by a 37-year relationship with an independent consulting and r/d firm specializing in material sciences (chemistry, polymer systems, statistical analysis, physics, fluid dynamics, statistical mechanics, etc.) which eventually became the central research laboratory of a large multi-national corporation.
Coplan is recognized in American Men of Science, holds 32 patents, is a member of several professional organizations and has published many technical papers. He authored a series of bench-mark articles on mathematical probability statistics and wrote a manual on statistical quality control for internal corporate use. He also personally carried out a wide range of laboratory research and engineering tasks and supervised the work of as many as 35 other professionals of many disciplines. He has been consulted by research staffs and corporate executives from some of the world’s largest corporations. To mention only one example, over about ten years he had 28 assignments from GE.
His services were also engaged by NASA, USDA, EPA, Interior Dept, Post Office Dept and several other government agencies, including virtually every branch of the DOD. In these assignments, Coplan was cleared on a “need-to-know” high level security basis several times for consulting and research work in such diverse fields as “decoy” chaff used to frustrate radar-tracked anti-aircraft fire to protective measures for ground-troops at risk of exposure to chemical, biological and nuclear attack.
In due course, Coplan’s activities became more focused on the interests of the large company which in 1972 had acquired the firm he had joined in 1951. After 1972, he took on the corporate mission of identifying and exploiting science-based new business opportunities, including direct management of scientific entrepreneurial r/d for new products and technologies. He became Senior Corporate Scientist and then Vice President and General Manager of a membrane development venture that eventually licensed his patented inventions to other large corporations. Membrane treatment of phosphate waste pond waters was among the applications studied. Coplan, therefore, has first-hand knowledge of the processes from which the principal water fluoridating agents (the silicofluorides) are derived.
Notes and Credits
NOTE I. The following English language text, translated from the German in which it was written by Dr. Johannes Westendorf, (Toxicology Department, Eppendorf-Hamburg University Hospital) was submitted to him in March 2001 for his comments with a series of questions. This was his response:
“With respect to my thesis I finished this kind of work in 1976, when I changed to the Medical faculty, where I still am. After my thesis I continued the work on fluoride for another year and we especially worked on the stability of hexafluoro complexes of silicon and iron. We used radioactive isotopes, such as F-18 and Si-31… when we analyzed the electrophoretic mobility. In the presence of silicon and iron, fluoride ions showed a different mobility compared to fluoride [ion] itself. Unfortunately I have no access to these old experiments and we did not publish it.
... During hydrolysis we got a continuous shifting of the mobility, indicating that the different forms of hydrolysis with 2-6 fluorine at the Si are present at the same time, ending up at the more stable form of Si(OH)4F2. If we increased the pH to 9 and higher, a total hydrolysis occurs.
... In answering your final paragraph I can say:
The English translation of my thesis is excellent
I have no evidence from others that contradict to my old findings
Your idea of the enzyme inhibition by the complex could be right, however slight changes in the pH, caused by the hydrolysis of hexafluorosilicate, would also result in an increased inhibition of acetylcholinesterase. Nevertheless, I agree with you that the toxicology of hexafluorosilicate should be investigated because it may be different from simple fluoride.
Please let me know if I can be of further assistance to you.
Johannes Westendorf” [email@example.com]
NOTE II. Although the main body of the Westendorf thesis was not published in a circulating journal as such, three short articles based on this work were. Copies of the two most relevant ones appear at the end of the English text of the full thesis.
CREDITS. The thesis was called to our attention and photocopied from the document on file in the archives at the University of Hamburg by Peter Meiers (Weissenburgerstr. 28, D-66113 Saarbrucken; the translation was prepared by Jakob von Moltke (Dartmouth College); final proof editing was done by Myron Coplan with the aid of Norman Mancuso.