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TRACE ELEMENTS IN ATHEROSCLEROSIS, EFFICACY OF ZINC MEDICATION AS A THERAPEUTIC MODALITY

by J. H. Henzel, M.D., B. Holtmann, M.D., F. W. Keitzer, M.D., M. S. DeWeese, M.D., and E. Lichti, Ph.D.Department of Surgery, University of Missouri - ColumbiaMedical Center University of Missouri - Columbia. In: Trace Substances in Environmental health, Conference 2nd proceedings 1967-68.

ABSTRACT

Certain trace elements are now being investigated as possibly having a role in atherosclerosis, and recently administration of zinc has been described as beneficial treatment in a significant percentage of patients with severely symptomatic atherosclerotic disease. A study was therefore initiated in which certain patients with severe atherosclerosis were evaluated, medicated with zinc sulfate, and then re-evaluated at bimonthly intervals in order to assess the efficacy of therapy. Objective patient evaluation included treadmill walking, electrocardiography, plethysmography, and positional photography of ischemic limbs. Objective improvement was observed in 12 of 16 patients;however, six improved patients were discounted since factors such as weight loss and cessation of smoking may have contributed to improvement. Assessment of objective findings', subsequent to subjective improvement, and comparison of pre- and post-treatment serum and urine zinc concentrations, suggests that zinc acts as a therapeutic pharmaceutical (rather, than as replenishment for subnormal biologic stores) in those patients with occlusive vascular disease who experience improvement when medicated with this micronutrient.

INTRODUCTION

The incidence, pathogenesis, and development of atherosclerotic occlusive vascular disease are known to be related to numerous factors and variables.(8) However, in spite of millions of research dollars, untold man-years of laboratory investigation, and exploitation of hundreds of initially promising leads, the number of answers which have been "interlocked" as far as the prevention of atherosclerosis is concerned are indeed few in number and tenuous in stability.

It is particularly fitting to present and discuss investigative results pertinent to this disease at an environmental health conference. The reasoning behind this statement is realistic and sound. Albeit the clinical specialties are able to recognize and "palliate" or "delay" the progressively incapacitating symptoms of atherosclerosis; however, it has been the public health or environmental medicine approaches which have defined certain geographic, hereditary, and dietary "constants" that hold the key to prevention rather than prophylaxis.

The history of science in general, and medical research in particular, is dotted with periodic spectacular developments in knowledge which have enhanced man's healthful existence on this planet. However, there have been few areas for new investigation which can compare with the trace substances as far as a real need for an interdisciplinary approach to research in concerned. Being cognizant of this fact, and recognizing that every field of scientific endeavor either directly or indirectly benefits man's health and longevity, I feel comfortable discussing a clinical disease before an audience which contains representatives from such varied fields of specialization as analytical chemistry, agriculture, horticulture, physics, and nutrition. I feel at ease not because of some macabre satisfaction that all of us within this auditorium have some degree of atherosclerosis, and certainly not because most of us suppress the fact that "if something else doesn't get us, " hardening of the arteries will. Nor is it because of the prevalence of this disease that I feel justified in discussing a clinical study; rather, it is primarily because of a conviction that with respect to the trace substances, meaningful investigation, as well as interpretation and application of study results, requires a cooperative interdisciplinary approach, based on an awareness of and respect for what each of us has to contribute. As clinicians we can never hope to treat or eliminate biologic accumulation of a "toxic" trace substance if we do not have some understanding of its biosphere distribution, route of entry into the organism, and sensitivity of analytical methodology applied to biologic specimens. Along this same line of thought, discovery and/or development of fertilizer-enrichment trace substance combinations developed to enhance crop yield demands some knowledge on the part of agriculture and horticulture specialists of-whether cumulative biologic uptake may eventually produce toxic results.Finally, it will serve little purpose for the analytical chemist to be able to detect tissue concentrations of a particular micronutrient at 15 ppm if he is unaware that his laboratory technicians are accumulating hazardous biologic levels through cutaneous absorption which occurs at 40 ppm.

In preparing a presentation for this conference I learned two valuable lessons. First, it behooves one to check the program for the title of one's own talk before attempting to compose his presentation; and second, it 4 is wise to be punctual when submitting a title to the Program Chairman so that he isn't forced to politely select a comprehensive heading which has one scurrying through a general literature review at the same time that he is compiling his own data. When we started to prepare a presentation for this conference, it was with the assumption that we would limit the material to our own particular area of interest and endeavor. However, we are indebted to our Program Chairman, Dr. Hemp hill, for designating a title which necessitated a literature review, for if this audience presently has any doubt that certain trace substances occupy a role in occlusive vascular disease, it should resolve while listening to the investigative threads which are slowly beginning to interweave. It would not be practical, nor is it necessary, for us to discuss all of the trace substances which have been investigated as potentially having a role in the pathogenesis or development of atherosclerosis. Therefore, we have only attempted to condense those investigative results which have enkindled our research protocols, and which have probably encouraged your own research endeavors. Of the five trace elements which have been strongly implicated in vascular disease, only two (zinc and copper) have been included in our own studies. In considering a possible interrelationship between trace substances and atherosclerosis, it is important to recognize that certain diseases which do not in themselves constitute occlusive vascular conditions nonetheless may provide a "fertile environment" in which development, progression, or sequelae of atherosclerosis is potentiated. Thus we believe it pertinent to allude to copper and arterial aneurysm formation, cadmium and hypertension, and chromium in relation to diabetes.

Copper:The provocative experimental studies which Dr. Boyd O'Dell (9 ,10) has carried out on this campus are well known and stimulated our group (in a very preliminary fashion) to test this investigation on one small group of clinical patients. After observing large artery aneurysm formation with rupture and high mortality in copper-deficient chicks and pigs, Dr. O'Dell found that the deficient levels of elastin (a substance whose presence and structural integrity characterize healthy aortic tissue) present in copper-deficient arteries were related to subnormal activity of a copper-dependent elastin-producing enzyme. The specific biochemical site at which copper deficiency results in retardation of elastin synthesis by inhibiting the formation of lysine-derived crosslinkage units of elastin has since been identified by Hill. (5) Since Dr. O'Dell had called attention to the microscopic structural similarity between the aneurysmal arterial wall lesions which occurred in copper-deficient laboratory animals, and the cystic medial necrosis which is occasionally associated with aneurysm formation in humans, we performed copper assays on specimens obtained from nine patients who required resection of diseased abdominal aorta for either occlusive or aneurysmal changes. Of these nine patients, four had atherosclerosis without aneurysm, three had atherosclerosis and abdominal aortic aneurysm, and two had aneurysm without visible evidence of atherosclerotic occlusive disease. Table 1 presents the copper concentrations found in the resected arterial wall specimens from the nine patients. The average copper concentration in the four patients who had atherosclerosis but no aneurysm was 26.3 ppm, while the average concentration for the three patients with atherosclerosis and aneurysm was 21.2 ppm. However, for the two patients who had aortic aneurysm without visible evidence of atherosclerosis, the average concentration of copper dropped to 14.2 ppm. Although only a small number of patients have been studied, this suggestive evidence that copper deficiency may have a role in development of some of the aneurysms which develop in humans will hopefully stimulate additional investigations along these lines.

Manganese-Cobalt:(14) Studies have revealed that manganese possesses choline-like lipotropic properties, contributes to prevention of experimental atherosclerosis in rabbits, and may have a favorable effect on lipid metabolism in certain patients. Georgel and Curran observed a depression in cholesterol synthesis when cobalt was added to rat liver homogenate. Tennet et al. observed reduction in serum cholesterol when cobalt was administered with the diet, but increased when the cobalt was injected parenterally.

Vanadium:(1,2) This trace metal has been shown to inhibit cholesterol biosynthesis both in vitro and in vivo. Curran and Costello demonstrated that in rabbits "rendered" atherosclerotic with high cholesterol diets, supplementation of vanadium was associated with mobilization of cholesterol from aortic walls. Although the precise role of vanadium in cholesterol synthesis has met with experimental roadblocks and contradictions, it appears that this element can assume a "regulatory" role in experimental cholesterol synthesis, and that for some reason this regulatory role is apparently age-dependent as demonstrated by the observation that vanadium decreases cholesterol synthesis in young rats but actually has the opposite effect in older, heavier animals.

Two diseases which are well recognized "accelerators" of the development and/or progression of atherosclerosis are essential hypertension and diabetes mellitus. For both of these clinical diseases, specific trace substance investigation has transcended from the laboratory animal to the clinical patient. One cannot help but delight in the enthusiasm with which Dr. Walter Mertz describes his work with chromium. (3,4,5,6,7) In similar fashion, a sense of excitement is aroused when one listens to Dr. Henry Schroeder unfold the newly discovered relationship between hypertension and cadmium. (11, 12, 13) Realizing that environmental uptake of cadmium "promotes human hypertension de novo and enhances that arising from organic renal ischemia, (13) and that the impaired glucose tolerance of certain patients with diabetes "showed statistically significant improvement during the oral supplementation with chromium," (6) one cannot help but believe that at least an indirect relationship exists between the development and progression of atherosclerosis in certain patients and these two trace substances.

It would be foolhardy for any individual whose presentation follows one on diabetes and precedes another's on hypertension to say much more then very little about the relationship between certain trace substances and these two diseases. By this same token, it is with some humility that we introduce a description of our own efforts, which thus far have been primarily concerned with studies of biologic and pharmacologic zinc in patients with severely symptomatic inoperable vascular disease.

EXPERIMENTAL MATERIALS AND METHODS

Approximately two years ago we initiated a triple-phase study designed to answer three questions. First, is biologic zinc deficiency a factor in the pathogenesis of atherosclerosis? Second, can the clinical improvement which certain atherosclerotic patients experience when medicated with zinc be attributed solely to supplementation with this micronutrient, rather than to other factors such as a subjectively-positive, objectively-negative placebo effect? Third, if documentable, statistically significant, objective improvement accrues exclusively because of zinc administration, is the improvement in clinical status related to replenishment of subnormal biologic stores of this element or, instead, to a heretofore undescribed therapeutic action of zinc?

As a preliminary step to instituting a rigidly controlled Clinical Research Unit program of zinc supplementation to a group of patients with severely symptomatic, inoperable atherosclerosis, we assayed whole blood, serum, erythrocytes, hair, and arterial wall from more than 100 patients who had clinically demonstrable atherosclerosis, and from an equivalent number of patients in whom no evidence of this disease could be demonstrated. The results of this survey-type effort are summarized in Table 2. We failed to be impressed by any significant difference in hair zinc concentration between the two groups and stopped assaying hair after data were available on about 20 patients in each group since results were variable, as they had also been in two other studies which we were conducting on burns and wound healing. While it is conceivable that study of additional patients may have yielded a "positive" difference, we have eliminated hair assays serum in preference of serum determinations which are not "exposed" to the same potential sources of contamination, and which in actuality constitute a more uniform reflection of dynamic in-vivo availability. It is evident in Table 2 that there was a difference in zinc concentration between healthy aortas and diseased aortas. However, there are two reasons why we are presently uncertain of the significance of this observation. In the first place, our specimens of normal aortic tissue were obtained following traumatic death, and it turned out that the average age of these specimens was 23 years younger than the average age for the atherosclerotic group of patients. Secondly, most of the atherosclerotic arterial specimens which were assayed contained varying degrees of calcific plaque formation, and it became evident that zinc levels in the less diseased portions of some arteries approached the low-normal zinc concentrations which we encountered in aortic tissue from non-atherosclerotic patients. Nonetheless the impetus to proceed with a definitive, controlled Clinical Research Unit study arose out of observations made on arterial wall and serum zinc concentrations in this early phase of our work. There was little doubt that as a group, atherosclerotic patients exhibited lower serum zinc concentrations than did their non-atherosclerotic counterparts.

The remainder of the material which we will discuss today comprises one portion of a broad-scale, long-term investigation which is currently being conducted at the University of Missouri Medical Center. The eventual overall aim of the investigation is to correlate alterations in biologic micronutrient stores with aberrations of blood viscosity, serum lipid fractions, and coagulation processes in patients who have early onset atherosclerotic occlusive disease. A certain number of these patients who have rapidly progressive, symptomatic, inoperable occlusive arterial disease are being medicated with zinc sulfate, and we are attempting to explain why a significant percentage of these patients experience lessening of their symptoms when they are medicated. Table 3 outlines the various parameters which are being ascertained and followed on all patients who are studied. All data are being accumulated and stored for eventual correlation-type computer analysis as disease develops, progresses, and initiates morbidity and mortality during coming years.

Physicians who treat patients with atherosclerosis, and patients who have this disease, realize that in many instances symptoms resulting from atherosclerotic blockage of blood flow to organs and limbs can frequently be controlled and even lessened by such factors as weight loss, selectivity of diet, graded exercise programs, and cessation of smoking. It therefore becomes of particular importance when evaluating the supposed role of any biologic factor in atherosclerosis, or the efficacy of any therapeutic agent administered in the treatment of this disease, to control rigidly (insofar as this is possible) any factors which may simultaneously be influential in altering symptoms and clinical findings. Of equal importance is that the investigator supplement subjective evaluation with sensitive, objective "recorders" which can quantitatively confirm or refute a patient's subjective impression of clinical improvement.

Table 4 outlines the objective studies that are performed on all patients during follow-up visits. In order to minimize bias, all evaluations, including the physical examination, are performed by personnel who are not directly involved with the study.

RESULTS

We are interested in the results obtained when zinc sulfate was administered to 14 patients with extensive, inoperable, symptomatic atherosclerosis, and two patients who had disabling vasospastic disease. All patients gave written consent. to receive an investigational drug and volunteered to return to the Clinical Research Unit at bimonthly intervals in order to assess the efficacy of their treatment. [The investigations described have been approved by the committee of associates of the investigators in accordance with this institution's (University of Missouri - Columbia Medical Center) assurance on clinical research. Patients are studied initially and re-evaluated during follow-up in the General Clinical Research Unit.] Length of treatment has ranged from three to eleven months. In order to be as rigidly objective as possible in evaluating the zinc therapy, we have eliminated from our evaluation all patients who altered their smoking habits, lost more than five per cent of pre-study body weight during the period of medication, and who for one reason or another started progressive exercise tolerance programs on their own. While a particular effort was made to avoid informing patients about the relationship between these factors and alteration of the symptoms associated with their disease, it became evident that some patients who volunteered to participate in this study were individuals who were sufficiently motivated that they undertook dietary restriction and cessation of smoking on their own. Six out of the 16 patients who we have studied are therefore discounted for this reason. Four of the remaining ten patients experienced no improvement, and two of these had rapid progression of their disease to the point of amputation.

We are therefore left with six patients who experienced improvement with zinc sulfate, and who did not undergo any significant change in body weight, alteration of smoking habits, or increased exercise program while receiving medication. Table 5 summarizes serum and erythrocyte zinc concentrations and 24-hour urine zinc excretion for these six patients. It is evident that serum concentrations were subnormal (less than 95 micro-gm/ 100 ml in our laboratory) in five of these six patients. Erythrocyte concentrations were not subnormal in any of the patients. Subnormal 24-hour urinary excretion (less than 350 mg/24 hr) was encountered in only one patient. While being medicated with zinc sulfate, serum zinc levels increased in all patients to an average of 177 micro-gm/100 ml. Urinary zinc excretion increased to levels which usually ranged between 1.5 and 2.75 mg/day; however, levels as high as 4 mg/day were encountered in two 24-hour urine collections. We did not observe any significant alteration in erythrocyte zinc concentration subsequent to medication of these six patients with zinc sulfate.

Table 6 reveals the objective findings, as documented by EKG, plethysmography, and treadmill, in the six patients who experienced demonstrable improvement which we were unable to ascribe to anything besides the zinc sulfate medication. The first three patients were males with severe claudication of their lower extremities. All three of these gentlemen demonstrated marked improvement on treadmill exercise. The first gentleman actually improved to the point where he was able to walk three-quarters of a mile without onset of pain in the calves of his legs. Prior to starting zinc sulfate medication he had experienced severe pain from walking distances as short as 100 yards. The fourth patient is a lady who had almost complete blockage of the artery to her left arm. She presented to us complaining that she was unable to knit or crochet. After five months of medication, she was able to do needlework to the point that "eyestrain" or "headache" are now the limiting factors.

The last two patients represent what we believe are the most exciting observations made to date. Both patients were males who had severely symptomatic vasospastic conditions of the extremities which had resisted all modes of therapy. They had been suggested as candidates for surgical treatment, but were first referred to us for a trial with zinc sulfate. Rather than attempt to describe their response to medication with zinc sulfate, we obtained photos of their extremities which are included below.

Figure 1 illustrates the endstages of the disease which we have been attempting to treat with zinc sulfate. The fact that these photos were obtained immediately prior to amputation represents reason enough to explore completely any new lead which may prevent the ravages of atherosclerosis. Figure 2 includes pre- and post-treatment photos of one of the vasospastic conditions which we mentioned above. The name Raynaud's had been attached to this man's disease at the Mayo Clinic some eight years ago. Every medication known to be even partially effective in this condition had been tried prior to initiation of medication with zinc sulfate. The follow-up photo in Figure 2 speaks for itself. Figure 3 is our second case of Raynaud's disease, and in this instance the condition involved the patient's fingertips. It seemed improbable that any reversal of the changes evident in the first photo could occur. Nonetheless, the dramatic improvement which occurred during short-term treatment with zinc sulfate is evident in Figure 4. This gentleman has now returned to active farming, something that he had been unable to do for two years prior to being referred to this institution for surgical treatment of his disease.

CONCLUSION

The material which we have discussed this afternoon, and the results of the studies which we have presented, were not described to startle or excite any audience. The findings have been presented, rather, as an example of what a cooperative effort by clinicians, analytical chemists, environmental health specialists, and others can accomplish when an interdisciplinary approach to trace substances research is utilized. Our results to date utilizing zinc sulfate in atherosclerotic patients seem to indicate that in vascular disease this micronutrient may act as a pharmacologic agent allowing; increased blood volume (in some as yet undescribed manner) to reach areas of the body in which diseased blood vessels have restricted arterial perfusion.

The entire field of the trace elements or micronutrients is new, exciting, and enticing. If medication of human patients with a metal that is used to galvanize washbuckets can also produce results in wound healing like the ones evident in Figure 5, one can anticipate with excitement the number and magnitude of beneficial discoveries which continued investigation of the trace substances will yield. We are optimistic and confident that cooperative, coordinated interdisciplinary research in this relatively new field of "investigative fertility" will yield findings far more exciting than what we have presented today.

FIGURES

The figures have been omitted due to the inability to reproduce their color and clarity by the journal.

LITERATURE CITED

  1. Curran, G.L., and Burch, R.G. 1967. Biological and Health Effects of Vanadium. Proc. First Annual Conference on Trace Substances in Environmental Health, p. 96.
  2. Curran, G.L., and Costello, R. L. 1956. Reduction of Excess Cholesterol in the Rabbit Aorta By inhibition Of Endogenous Cholester Synthesis. J. Exptl. Med. 103:49.
  3. Glinsmann, W. H. , Feldman, F.J., and Mertz, W. 1966. Plasma Chromium During Glucose Loading. Science 152:1243.
  4. Glinsmann, W.H. , and Mertz, W, 1966. Effect of Trivalent Chromium on Glucose Tolerance. Metabolism 15:510.
  5. Hill, C.H., Kim, C.S. 1966. Studies on the Role of Copper in Elastin Formation. Internatl. Congress Nutrition, Hamburg.
  6. Mertz, W. 1967. The Role of Chromium in Glucose Metabolism. Proc First Annual Conference on Trace Substances in Environmental Health, p. 86.
  7. Mertz. W. 1967. Biological Role of Chromium. Federation Proc. 26: 186.
  8. Moses, C. 1963. Atherosclerosis, Mechanisms As A Guide To Prevention. Lea and Febiger, Philadelphia.
  9. O'Dell, B.L., Bird, D. L. , Ruggles, D.L., and Savage, J.G. 1966. Composition of Aortic Tissue From Copper-Deficient Chicks, J. Nutr. 88:9.
  10. O'Dell, B.L., Elsden, D.F., Thomas, J. , Partridge, S.M., Smith. R.H. , and Palmer, R. 1966. Inhibition of the Biosynthesis of the Crosslinks in Elastin by a Lathyrogen. Nature 209:401.
  11. Schroeder, H.A. 1964. Cadmium Hypertension in Rats. Am. J. Physiol. 207:62.
  12. Schroeder, H.A. 1965. Cadmium as a Factor in Hypertension. J. Chronic Dis. 18:647.
  13. Schroeder, H.A. 1967. Some Prospects for Research on Biologically Active Trace Elements. Proc. First Annual Conference on TraceSubstances in Environmental Health, p. 20.
  14. Volkov, N.F. 1963. Cobalt, Manganese and Zinc Content in the Blood of Atherosclerotic Patients. Federation Proc. Transac.Supplement 22:897.