OBSERVATIONS ON THE 2’Zi‘."SIOi-OGIC EFFECTS UPON ANIMALS OF FREE AQL’EOUS IODINE iNTRAVENCUSLY INJECTED Thesis. for the Degree of M. S. StilCchAN STATE COLLEGE D avid S. R L11"; 6 1937 4‘. 1h.- , ‘ '(A‘ . .I .‘ . A}. . .- .o . ‘ 4.1;. IQ. 1“- all lo- ‘; ...... Il‘j '2 A \ !)T‘f :' 1 | ’ ‘I ‘3'“? I ‘ T ‘1 .1;1-1'-"r . (.T / ! ~"I :l‘ 1"'1' 1{"If1.’:l ODJHLt ALiaLU‘.~J v.1} 4..-.4 .L‘.LLJIV¢JO\J q ..44....‘-1\.J.L..) "-"x' "—-. -‘r r x "“‘~ '. 1 ,- ‘."‘ }..~- ' -" ‘1 Uqu “MLMALJ J: rfigg nwpdgug IuJihu "m 1- -\ rm". * ILiAILJ'L' .J..f'-.I‘~J~)L[ *‘ 'J .L—I “UL-34 O is for degree or M.3. Th 3 chigan otate College '1 7 ' I I ‘-A David Sieger Ruhe ,, l 9 5 7 THESIS 3. }3. 2314.1) 1. I: Table of Contents RGVieWOfLiteratureooooo00.000000.coco.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . TeChniunSEmployedo0.0000000000000000. \OkON General . . . . . . . . . . . . . . . . . . . . . . Specific . . . . . . . . . . . . . . . . . . . . . ll Protocols. with Commentary Experimental Series I: Blood Pictures . . . . . . . 15 Red Cell Changes . . . . . . . . . . . . . . . .16 Leucocyte Changes . . . . . . . . . . . . . . . 19 Differential Variations . . . . . . . . . . .21 Gum Acacia Injection Response . . . . . . . .23 Experimental Series II: Pathology . . . . . . . . . 26 Gross and.Microsc0pic Findings . . . . . . . . .26 Hemoglobinuria . . . . . . . . . . . . . . . . 129 Temperature Response . . . . . . . . . . . . . .31 Experimental Series III: Blood Pressure and Respiration . . . . . . . . . . . . 33 Toflcn' Experimental Series IV: Minimum Lethal Dose . . . . U2 Discussion of Observations . . . . . . . . . . . . . . . . . ”5 Summary of Observations . . . . . . . . . . . . . . . . . . .MS Bibliography................ooooo...50 .Review of Literature Iodine is the heaviest, the least toxic, comparatively the least active, yet scarcely the lowliest of the halogen brethren. Even before it was isolated as an element it had been shown by kelp-burn— Me ing Irishman-to own almost magical powers in the treatment of goiter. Its function in endocrinology as key atom in the hormone thyroxine has given it great importance in the modern sphere of medicine. Beyond its combination in an indispensable animal hormone, the use of disin— fectant iodine solutions in former days had achieved great pepularity with physicians, though based more upon clinical and empirical re- sults than upon experimental data. Iewer comparative studies of dis— infectants (Eye, 1937 (1)), included within an accumulating mass of literature dealing with various preparations of iodine, have empha- sized the great general superiority of elemental iodine. Among the many and motley iodine compounds prOposed for use, solutions of aque- ous iodine are today awarded leadership. Unbiased information points to these solutions of aqueous iodine, with or without solvent iodides, as being most satisfactory for general utility disinfection, when judged from the multiple angles of low toxicity to animal cells (Salle and Lazarus, 1935 (2), Nyiri and Dubois, 1931 (3)), bactericidal efficiency and penetrability (Simmons, 1933 (M)), low cost (Eye, (1)), and diverse possible usage. Elemental iodine is a general protein precipitant, probably de- naturing by its addition of a heavy ion to the protein molecule in its colloidal state. It combines with lipoidal materials very readily, uniting chemically with the unsaturated lipoids by breaking the double _ 2 - bondings within the molecules of the fatty acids. The familiar bio- chemical iodine index of unsaturated fatty acids is application of the latter fact. Altogether it exhibits an extraordinary ability to combine physically or chemically with a great range of chemical sub- stances. The halogens all possess this chemical versatility, but io- dine is least toxic, and least corrosive of the fqur. It is this manifold activity that is responsible for thefifungicidal, protozoici- dal, and vermicidal powers of iodine. Sufficient quantities of iodine can kill mammalian cells, but the cells alive and intact in the body, or in tissue culture as well (Salle and Lazarus, (2)), apparently have considerably greater iodine resistance than invertebrate pro- tOplasm. Of all the preparations of iodine so far produced by chemical device, within our knowledge only one embodies the need for a true aqueous iodine without strong foreign solvents such as the iodides. Iodine is poorly soluble in water, and foreign solvents had been necessary to bring the solutions to a reasonable potency. The col- loidal iodine (Chandler and Miller, 1927 (5)) is prepared by the very rapid precipitation of iodine from its salts---sodium iodohypoio- dite commercially...in the presence of the protective colloid gum acacia, known familiarly as gum arabic. The precipitation is carried out by acidification with hydrochloric acid at zero degrees Centi- grade. The precipitated iodine is captured by the gum arabic as particles ranging downward from diameter 0.5u, just within the newer arbitrary limits for colloidal disperse phase particles. These finely -3... divided globules of iodine are held rather permanently in the col- loidal state, can be desiccated and resuspended, and at an unvarying temperature in a closed container do not materially lose strength nor crystallize, though there is a strong tendency to a concentration at the vessel bottom. In the suspensoid there is a constant equili- brium maintained between the iodine particles and a water solution of iodine just below the point of saturation (.O3Mfi at 25°C.). Loss of iodine from the water solution results in outflow of iodine from the colloidal fragments to maintain the equilibrium concentration in the water. This movement of iodine occurs up to the limit of avail- able iodine, which may be as high as sixty percent or more in special concentrates. The gum acacia is very inert, and is never found in large concentrations in the true suspensoid. Huffman (1929 (6)), in his report of cases of surgical shock in which large volumes of gwn acacia (600 to 800 cc. of a 6% solution) were introduced into the circulation, reports a definite rise in blood pressure as the desired salient feature of such injection; physiologically otherwise, he shows, the acacia is inert, non-toxic even in the large volumes em- ployed, and is excreted as a gummy mass by the kidneys. The residual Mum acidAmAeemingly unimportant as a factor in the iodine usage, amid“ washed out to a fair degree by removal operations. So large a volume of available elemental iodine readily liberated from a water solution, in a condition apparently ideal for use in the many possible applications, brings up new aspects of iodine physiology and toxicology, with new speculations as to possible action against - h _ disease. The use of the colloidal iodine (Chandler)"l as animal ver- micide has been demonstrated by Stafseth, 1935 (7), and Chandler, 1923, 192M (8,9). Chandler, 1927 (10) has indicated its potentiali- ties in dermatomycoses. Hesseltine anduNoenen, 1935 (11) have shown its usefulness in vulvo-vaginitis caused by pathogenic yeasts. But the effects of iodine when introduced into the gut per as or per rectum, or when applied to the skin or mucous membranes (Chandler, u- ‘c (9.10), Simmons (u), Hesseltine and-mom (11), Nyiri and Janitti, 1932 (12)) are markedly different from those systemic effects brought on by parenteral introduction. In this work only the single phase of intravenous injection has been directly considered, though the facts cursorily inquired into can doubtless readily be linked to the diverging paths of iodine research. 30 fact has anywhere been encountered concerning the effects of aqueous iodine as such upon the blood of animals. The physiologic groundwork is in any event fragmentary. Scarborough (1926 (13)), in his extensive monograph on the blood pictures of normal laboratory animals, a compilation from investigations prior to 1926 only, has supplied a working hematology. Heinz in 1899 (1h) has described a leucocytosis with cases of iodism. Marine, 1915 (15), in his thy- roid studies has described, following iodide feeding, a loss of the histologic hyperplasia of the thyroid regularly seen in normal dog glands. Boehm, 1876 (16), quoted by Sollmann (17). had determined * For simplicity it will be understood that in all cases the"colloi- dal iodine" is that of Chandler. "iodine" refers to free aqueous iodine derived from the colloidal iodine. No other iodine was used anywhere in these experiments. -5... that NO milligrams per kilogram of body weight was the minimal lethal dose for alcoholic solution of iodine, using experimental animals. -4ndtSu1gp9hxt Recently Nyiri and Dubois (3) concluded, after inconclusivebexperi- ments, that the minimal lethal dose of the aqueous iodine (colloidal, Chandler) was the same as that of the tincture: in short MO milli- grams per kilogram. Salant and Livingston, 1916 (18), working with frog heart perfusions, demonstrated that the passage of dilute aqueous iodine through the heart produced irregularities and depression; ‘fhe speed of heart recovery depended solely upon whether free iodine was passing through the heart. They noted greater resistance of the "fresh heart" to perfusion with iodine, and review literature that directly links the heart variations to iodine combination with the unsaturated lipoids of the endocardium. The iodine number of the dog's heart is 70, evidence of the presence of unsaturated, physiologically active fatty acids whose sudden conversion perhaps shocks the heart. They 'report also that heart depression by iodine injection occurs in cats but not in dogs. Jobling and Petersen, lglu (19), suggest that the action of iodine in the body may be due to combination with unsatur- ated fatty acids, the filling of the unsaturated valences causing a corresponding neutralization of the anti-ferment activity of these lipoids.“ McLean, 191h (23), found that in experimental animals the lipoid fraction of the tissue contained 32% of introduced iodine, the water-soluble fraction 67%, and extracted protein no iodine. Musser and Hermann, 1925 (20), quoted by Sollmann (17), noted fibril- lation and flutter, ventricular tachycardia and death from auriculo- ventricular block by the application of alcoholic iodine to the ex- _ 6 _ ternal heart surfaces. Much of the information compiled upon the phenomenon of iodism is perhaps pertinent to the study of the toxicologic action of free aqueous iodine in high concentrations. But here the volume of literature is very extensive and impossible to suitably correlate. It has been the hOpe thatsmme of the pre- vious observations may b%codrdinated in these preliminary studies of the physiologic qualities of a new iodine preparation. _ 7 - Introduction It was the purpose of the experimentation to show what differ- ences, if any, were to be observed in the effects of intravenous injection from those already recorded by Nyiri and Dubois on the toxicity of colloidal iodine, in addition to a comparison with re- ports where the alcoholic tincture has been the form of iodine em- ployed. Exploratory work was done in hematology, pathology and physi- ology in effort to unearth some clues as to the potential powers and limiting factors of this relatively new preparation of iodine)" Specu- lation pointed to possible chemotherapeutic action upon blood parasites and bacteria based upon the large quantity of readily available iodine, now divorced from the alcohol and iodide that heretofore had been complications. Sollmann, (17) in his Manual of Pharmacology, says that "iodine is partly adsorbed, partly loosely bound, and partly converted to iodide ions; this precipitation causes irritation and perhaps corrosion." It was conceivable, then, that iodine in this stated condition of adsorption-combination might be a source of useful free iodine for bactericidal or other function. Its effects upon the host's body had necessarily to be known, the degree of irritation, the relative toxicity as compared with iodine quantities injected, the chemical and physiologic reactions. Confirmation with or divergences from the results of other workers in the field, with differences per- haps attributable to the different materials and methods brought into play, has at least been the outcome of the experiments. The first aspects of the problem were to study the blood changes, to yield light upon the hemopoietic reaction to the iodine. The gross WWWMWI-nn "MWW knead-Mm, wax—www.mm ”WWMMW‘WMW Mu». .. 5 —-——-—fl -g- and micros00pic pathology of injection into animals was closely followed, together with a collection of all the symptomSLdscernible by an inept eye. Attempt was made to link these observations to iodism, acidosis, and hypothetical uses. Systemic physiologic re- actions to injection were surfacely studied. Beginnings were made upon the in vitro effects of the free iodine as a clue to iodine usage as a protein precipitant. The work in all has been explora- tory and preliminary, indicative rather than conclusive. Technique: General The colloidal iodine used in a major prOportion of the cases was made in this laboratory by the author, was purified by concen- tration and resuspension in distilled water to a pH 1.6”, approxi— mating a .OSN solution of the residual acid; later washings of a portion of the iodine yielded samples of considerably decreased acidity, but results were identical in all observable respects. Different samples of commercially-manufactured Merck suspensoid were introduced into the experimental picture from time to time to check the possibility of iodine variation as a significant factor. Gum acacia concentration was close to minimum from the continued wash- ings, existing possibly only as the thin 'shell' of protective colloid over the iodine particles. Calculations of dosage were first made upon a blood volume basis, on the assumption that the known speed of activity of the iodine would cause it to unite with blood elements alone without regard to other body tissues. Blood volumes being only inaccurately known, varying from animal species to species, and from method to method of measurement, all figures have been converted into milligrams per kilogram of body weight. The curious fractional dosages have been the result of this conversion from blood concentration doses based on an estimate of blood volume as 6.2% of body weight (Dukes). Com- plete delivery of all measured iodine from the syringe into the ani- mal was technically impossible because of the particle form of the iodine; save for this the doses are in all cases as notated. Four dogs, a cow, and twenty-eight rabbits were subjects of heaviest - 10 _ observation. Many of these were used over and over after rests of varying interval, some being used for controls in new sets of obser- vations, for different types of observation, or for variations in technique. There ffixgjLsibility of error in the reworking of animals, a possibility that was not in any degree borne out by the evidence from fresh rabbits. It has been the experience of numerous authors that iodine is almost completely eliminated from the body after a week; Nyiri and Janitti (12) found that 70—75% of the iodine was el- iminated.3 days after intravenous injection, by which mode of intro- duction excretion was most rapid. The animals varied greatly in age and weight. Some had been used in dissimilar experiments by other men of the laboratory, had been rested, and to all intents were en» tirely normal. For blood series, only very normal animals were con- sidered in judgments; blood abnormal animals, that is, animals with high leucocyte totals above Scarborough's (13) limit for physiologi- cal variation, were followed as well, and correlation has been listeds A series of preliminary normal observations filled the re- quirements for precaution. \ , inn. le-uLdE Mal WW ~ The sequence of edema and thrombosisAfollowing injection into the marginal ear vein, the signs of pain and uneasiness during in- jection, demanded heart injection for satisfactory introduction of the iodine. Speed of injection could be well controlled, there was no factor of anesthesia, the ear inflammation was absent, and the ear remained available for bleeding; there was assurance as well that the iodine would be brought into as quick and thorough blood contact as possible, without excessive loss to the tissues. For noting heart -11- effects the method could not be surpassed. Vein introduction was used.upon several occasions to check the customary technique. Techniques in specific fields of observation. filggd methods were those in standard usage. Total red and white cell counts were performed with blood pipettes and .lmm. depth Neubauer— ruled.hemocytometers; the differentials were done by Ehrlich‘s two cover-slip technique, stained with Wright's or Hastings' stains, one hundred leucocytes enumerated; the hemoglobin was measured, for speed and convenience, upon a Dare hemoglobinometer; blood sedimentation rate was observed using standard Westergren pipettes, readings taken every half hour because of the very slow sedimentation of rabbit blood, Thrombocyte and reticulocyte counts were discarded as being unreliable in our hands. Fragility tests were after Pepper and Farley, (21) using intervals of .02 and .OBfi in the hypotonic salt solutions; that is, .h§-.M2%-.M5fl etc. For obtaining blood from the rabbits a spring lancet was used to puncture one of the ear veins; the vein was con- stricted below the stab to produce an abundant blood flow. Ox blood was drawn from the jugular into vials. All considerable blood samples were citrated with .2co. sodium citrate (2053) to 5 cc. whole blood. Eping tests for hemoglobin were two, benzidine in glacial acetic acid with hydrogen peroxide, qualitatively.and quantitatively by the Sheard— Sanford photelometer, centrifuging for 10 minutes at moderate speed to drive down the abundant rabbit urinary sediments, using then the blood hemoglobin key for enumeration of hemoglobin in grams per 100 cc. - 12 - of"blood". For albuminuria the simple heat and nitric acid test was used. Temperature recordings were made per rectum, holding the thermometer 1% to 2 minutes deep in the rectum before removing. Physiologic records were recorded as fully as possible upon a smoked kymograph drum. Blood pressure was got by cannulation of the common carotid artery into a mercury manometer system, with scriber. Cannu— lation of the trachea into a Marey tambour recorder supplied respira- tory readings. The ureters were cannulated to observe effect upon the excretion of urine, its volume, the presence of albumin and hemo- globin. In three animals the common bile duct was successfully tapped to observe the secretion of bile as a measure of liver function. To prevent the drying out effect of the numerous incisions, a constant- flow needle with burette attached was inserted into the femoral vein, and physiological salt solution was continuously poured into the body. Most of the injections were made into one or another of the femorals, as being specially accessible, or into the marginal ear veins of the rabbits. Withdrawals of blood samples for total white cell counts, and for erythrocyte fragility changes were made from the same veins. The animals were anesthetized in various ways to eliminate as well as possible the factor of anesthesia. In four cases sodium barbital alone was used, in two nembutal(sodium pentobarbital Abbott)alone, and in two a combination of light nembutal anesthesia plus ether to bring complete unconsciousness. To eliminate the chance that the _ 13 - other two ingredients of the colloidal iodine were in any way respon— sible for the observed reactions, there was injected.prior to the iodine injections a series of volumes of freshly prepared 1% gum acacia solution (far more'than would ever be found in any iodine solution of the strengths used, but infinitely less than the gum acacia solutions used therapeutically), followed.by a single dosage of .l B. hydrochloric acid. The iodine was then given at various speeds of introduction here as throughout the work.at three concen- trations per body weight of the animal; both the laboratory-made iodine and a Merck commercial iodine were used. host observations were made after injecting the approximate minimum lethal dose. All animals that survived the first lot of iodine were allowed to re- turn to a seeming normal as shown upon the kymograph drum; then, after conclusion of blood, bile and urine observations, generally a lapse of three hours, a second quantity was administered and the response again recorded. The animals were killed by pneumothorax, the splanchnic blood vessels of the viscera examined for vasodilata- tion, the organs grossly viewed, and tissues taken for frozen section. Iistologic preparations were secured by fixation in Zenker's solution or 2% formalin as soon after death as possible. Tissues were para- ffin-embedded after dehydration and clearing. Material§for frozen sections were all formalin-fixed. Sections Su. thick for paraffin blocks, 20u. for frozen sections were mounted upon slides, and stained with eosin and hematoxylin without recourse to the special stains, regardless of the information such procedure might have obtained. A _ 1h _ tincture of iodine made from the colloidal iodine was used through- out for the removal of mercury albuminates resulting from the mer- curic chloride fixation. Normal tissues were prepared for comparison. Minimal Lethal Dose totals have assembled partly from specific measurements, partly as end observations in experiments pointed main- ly in other directions. Animals injected with known quantities of iodine were watched for periods up to two weeks for critical symptoms, and were pronounced normal and recovered if at that time they fed normally, had normal urine and feces and were outwardly sound., In all cases the dose and theeventual culmination were the only signi- ficant factors. 1: 9.; WM w w—hé my: m3»: opt: _ 15 _ Protocols Experimental Series I: Blood Changes. In any experiment to observe blood response, working normals are necessary. Scarborough (1}) has compiled much available data on rabbits as experimental animals, and has averaged his findings. For comparison with data later tabulated the normals are included. Erythrocytes physiologically vary between M.5 and 7 million cells per cu. mm., the average of 7M investigators being 5,620,000. Leucocytes physi010gically vary even more widely under a great range of factors extraneous and inherent, from H.000 to 13,000 per cu. mm., the average of 82 investigators being 7,900 per cu. mm. Differenti- ally the average normal and departures from that average are as follows: Cell type Percent Range of percent average . Polymorphonuclears M3.'% 30-50; (Pseudo-eosingophiles) . Lymphocytes ul.8fi 30-50fi Mononuclears and transitionals 9.0% 2-16% (large) ‘ Eosinophiles 2.0% .5-5; Basophiles h.3§ 2—85 Our own normals for leucocytes as obtained in pre-injectional readings are shown in the tabulations later compiled. The hemo- globin index did not enter as a significant change. Blood fra- gility normal has varied with authorities; the estimate shown by previous work is that complete hemolysis begins at .h%, and that complete non-hemolysis, or beginning hemolysis, occurs between .5% and .55% hypotonic NaCl solutions. Sedimentation rates have been _ 16 _ very irregular, and have only been used to test changes from normal on the same animal without recourse to an average. Because of the insignificance of much of the material, representa- tive cases only are given in full. These cases show the points in ques- tion, and are honestly considered to be as representative as any cases in the slim body of my data. In all blood data BBC; total red blood cell count, WBC: total white blood cell count, Diff.: differential smear for leucocytes, b.i.: before injection, a.i.: after injection, iodine: colloidal iodine (Chandler), and dates are year 1937. The irregularities in the collection of data have made tabulation diffi- cult. Red Cell Change, 5 animals. a Rabbip_l, Effect of a single dose upon BBC. Kale, 2.6 Kg., immature, in good condition externally. 5/1 injected into ear vein, slowly, 6 mgms./Kg. of iodine. Ear congested and edematous, 7 hours after injection. Ear could not be used for bleeding, nor vein for injection. 5/1 6 mgms./Kg.12 BBC. 6,300,000 6,510,000 b.i. RBC. 5,850,000 5,160,000 h hrs. a.i. RBC. n.9eo,ooo h,65o,ooo 7 hrs. a.i. RBC. n.990,ooo 10 hrs. a.i. 5/8 EBC. 5,220,000 5,600,000 7 da. a.i. The count does not go below physiologic variation, and the apparent drOp here is not confirmed upon average observations. The error of method might account as well for the changes. Rabbit Lg, Effects of continued iodine dosage upon RBC. hale, u Kg.. adult, in good condition superficially. High initial leucocyte count, but animal otherwise normal. 5/23 injected approximately 6.2 mgms./Kg. - 17 - iodine suspensoid slowly into heart (60 sec. for 6 cc.); iodine sus- pensoid 1 cc. 2.h§ iodine diluted for injection with 5 cc. warm saline. WBC. rose, but EBC. held normal. 5/27 injected .7cc. 3.2§ iodine into peritoneum at slow rate; 6.2mgms./Kg. There was a slight granulocyto— sis only. 5/31 injected .52 cc. h.hfi iodine rapidly into ear vein, WBC. did not markedly increase, RBC. normal. Animal 6/23, 6/29, 6/30. 6.2 mgms./Kg. has lost weight, but is surfacely quite healthy. 7/1, 7/2. 7/3, 7/H consecutively introduced into the rabbit .5u cc. of h.2% iodine, 6.2 mgms./Kg., all injections into heart. Animal died immediately following injection upon last date, of ventricular hemorrhage. Kidneys were grossly and micros00pically normal, very unusual. The two other animals subjected to similar treatment pos- sessed grossly blotched, micros00pically degenerating kidneys. Weight had dropped to 3 Kg. before death, but otherwise animal seem- ed in good health. Continual handling etc. may have been respon- WBC. did not subside from a point of sible for loss of weight. definite leucocytosis. RBC. had lowered to sub-normal. 5/23 RBC. 5,h7o,000 5,220,000 b.i. Inj.6.2 mgm./Kg. RBC. 5,610,000 5,u30,000 6 hrs. a.i. REC. 5,370,000 5,670,000 12 hrs. a.i. 5/27 RBC. 5,6ho,ooo 5,980,000 b.i. Inj.6.2 mgn./Kg. RBC. 6,330,000 5,220,000 12 hrs. a.i. 5/31 RBC. u,510,000 5,020,000 b.i. Inj.6.2 mgm./Kg. HBC. u,suo,000 h,810,000 5 hrs. a.i. RBC. 5,1u0,000 5,170,000 10 hrs. a.i. 6/281nj.6.2 mgm./Kg. REC. 6,020,000 5,h7o,ooo b.i. 6/29Inj.6.2 mgm./Kg. RBC. 5.6uo,ooo 5,320,000 b.i. 6/3OInj.6.2 mgm./Kg. RBC. 5,320,000 5,590,000 b.i. 7/1 Inj.6.2 mgm./K0. RBC. 5,u90,ooo 5,500,000 b.i. 7/2 Inj.6.2 mgm./Kg. RBC. h,2so,ooo u.hso,ooo b.i. 7/3 Inj.6.2 mgm./Kg. n30. 3,550,000 3,550,000 b.i. 7/u Inj.6.2 mgm./Kg. RBC. 3,150,000 3,110,000 b.i. -18- Not at all a conspicuous dr0p in view of the large quantities of iodine administered; BBC. normal up to two days before death. Rabbit §4_ Effect of minimal lethal dose of iodine: iodine toxemia. Male, 3.68 Kg., adult, condition superficially good“ Animal normal in blood picture, but with tendency to leucocyte variations. 7/ll injected 1.2 cc. U.l% iodine, l2.u mgms./Kg, into heart, in one minute. Animal normal externally. Leucocytosis noted. 7/18 in- jected 2.16 cc. h.lfi iodine, 25 mgm./ Kg. into heart. Animal had lost weight, down to 3.57 Kg., and survived second injection though definitely sick. Temperature dropped warningly. Strong hemoglo- binuria. Eyes inflamed, bowels loose, mucoid casts from anui coat rough, some eye discharge. 7/21 temperature climbed back to normal. Blood thin. Hemoglobinuria has lessened in degree. Animal somewhat brighter. 7/22 greenish thin urine has replaced the redwater flow. Weight has dropped to 2.9 Kg., seems bony and emaciated. Pellets are small and moist. WBC. continues as a leucocytosis. 7/25 animal off feed completely, thin, almost moribund. Urine very thin, with- out sediments, pellets small and distOrted in shape. 7/26 killed animal by breaking neck, so weak it could not support itself. Kid? neys almost black, streaked from cortex to pelvis upon cutting; viscera apparently normal. 7/6 BBC. 5,200,000 5,850,000 Normal 7/11 Inj. l2 mgm./Kg. 7/18 Inj. 25 mgm./Kg. RBC. 1,820,000 1,800,000 2M hrs. a.i. 7/21 RBC. 1,720,000 1.660.000 RBC. 1.990.000 2,130,000 _ 19 _ There are unfortunate gaps in the data included but the figures are perhaps significant. Three cases of this type. Leucocyte Changes: eleven rabbits. These eleven animals, together with five representative animals within the group, have been listed to illustrate total and time changes, ages, weights, and representative case histories included. Summary of Leucocytosis Observed Animal No. Dosage. - mgms./Kg. 3.1, 9300 normal to 13,700 in 10 hrs. 6.2 13,000 normal to 3h.000 in 10 hrs. 12.h (after rest of four weeks) 3.111, 7800 N. to 10,100 in 8 hours. 6.2 6600 N. to 9,200 in 11 hours. 6.2 (after rest of one week) R.VI, 8900 to no.000 in 12 hours. back to 6.2 normal in 2H hours. R.VIII, 8500 to 20,000 in 12 hours, back to 6.2 normal in at least 72 hours. l2,h00 to 19,100 in 20 hours .5 R.IX, 13,000 to 27,500 in 12 hours. 6.2 11,h00 to 13,750 in 10 hours. 6.2 3.x, 19,300 to 3u.200 in 8 hours, back to l2.u 'normal' in 20 hours. 19,000 to 25.900 in an hours. 2h.8 (after rest of one week) R.Xl, 8200 to 10,600 in 8 hours. 18.6 R.XIII, 8200 to 12,700 in four hours. .31 R.X1v, 8600 to 33,000 in twelve hours, back 2h.7 to normal in 20 hours. R.XVIII, 13,000 to 18,600 in 21 hours, slow to 18.6 return to normal. R.XVII, M600 to 23,000 in 10 hours. 15.5 _ 20 _ White Cell Total Count“ Change. - Five Animals. Total W.B.C. with times of readings. Babbitt III Age M m0. - Wt. 1.76 Kg. Dose 6.2 m./Kg. Rabbit VI Age 5 mo. Wt. 2.27 Kg. Dose 6.2 m./Kg. Rabbit VIII Age 1 yr. Wt. u.6 Kg. Dose 6.21n./Kg. Rabbit XIV Age 1 yr. Wt. 3.23 Kg. Dose 2M.7m./Kg. Rabbit XVII Age 8 mo. Wt. 3.57 Kg. Dose l5.5m./Kg. Norm. 7350 8200 Norm. 8500 8900 Norm. 8650 8300 Norm. 8200 9100 Norm. 6000 5900 h hrs. 7750 7950 5 hrs. 10,700 13,800 6 hrs. 13,700 19,800 12 hrs.‘ 32.000 35.800 5 hrs. 3900 5300 9 hrs. 10,200 10,000 9 hrs. 17.900 19.300 12 hrs. 18.750 21,650 20 hrs. 11,800 11,700 10 hrs. 23.200 1“ hrs. 8600 8650 11 hrs. M1,700 39.200 72 hrs. 12,600 11.250 21 hrs. 23.200 17.000 Several of the rabbits illustrated above possessed 'normals'. those of acceptable standard normal counts. sis.is, however, in most cases substantiated. 26 hrs. 9200 10,200 2M hrs. 10.700 11,200 very high Compare, however, the 1euco¢ytosis above that level with The fact of a leucocyto- Continued injections of small quantities of iodine (four cases) produced a continuing leucocytosis, that swung back to normal upon discontinuance of the injections. Rabbit IX, previous case history, is representative, though its normal is at the upper limit of the allowed range. 5/23 6.2 mgm./Kg. WBC. 13,000 13,000 b.i. WBC. 5,250 6,200 6 hrs. a.i. NBC. 26,330 29,150 12 hrs. a.i. 5/27 6.2 mgm./Kg. WBC. 8.500 10.350 b.i. wee. 11.050 12,250 12 hrs. a.i. 1.. 12,100 5/31 WBC. 10.800 b.i. 6.2 mgms./Kg. WBC. 13,200 19,200 5 hrs. a.i. WBC. 13,950 12,u00 10 hrs. a.i. 6/28 6.2 mgms./Kg. WBC. 10,000 b 1. (2M hrs. a.i.) 6/29 6.2 mgms./Kg. WHO. 10,200 9,600 b i. (2h hrs. a.i.) 6/30 6.2 mgms./Kg. WBC. 15,000 12,200 b.i. (2h hrs. a.i.) 7/1 6.2 mgms./Kg. WBC. 8800 9,600 b i. (29 hrs. a.i.) 7/2 6.2 mgms./Kg. WBC. 13,000 19,300 b 1. (2h hrs. a.i.) 7/3 6.2 mgms./Kg. WBC. 15,h00 15,500 b.i. (29 hrs. a.i.) 7/u 6.2 mgms./Kg. WBC 16,900 16,500 b.i. (29 hrs. a.i.) Study of the return to apparent normal from an iodine leucocyto- sis has been only incidental to other observations. At that status, yet judging from the scant material available from five animals, thirty to forty—eight hours is the probable span of time before re- turn of total leucocytes to-a normal number. Differential Leucocyte Change. Rabbit XI, Effect of a Single Iodine Dosage. Female, approximately five months old, weight 2.27 Kg., oxyurids in cecum and beginning ear mite infestation, otherwise normal. No previous injections prior to blood picture. Injected 1 cc. 2.55% iodine suspensoid, 6.2 mgms. /Kg., a sub-lethal dose. Killed animal upon conclusion of blood records, found only right kidney streaked and blotched, viscera otherwise completely normal. Rabbit VIII. Effect of a Single Iodine Dosage. Male, mature, approx- imately h.6 Kg., fat and in very good condition. No previous iodine- administrations. Injected 1 cc. 2.55% iodine suspensoid into heart, time sixty seconds, 6.2 mgms./Kg., a sub-lethal dose. Animal much affected by dosage, became ill, with temperature depression and hemo- globinuria, but returned to normal in two days. - 22 - Table of Differential Leucocyte Counts: Representative Animal Cell type Horn. 5 hrs. 9 hrs. 11 hrs. 29 hrs. Rabbit VI P.E.s. 33) 66) 32) no) 21) st. 8)1+1 16182 28)60 29)69 2h)“5 my. 0 O 2 O 0 E. 0 2 0 1 1 B. 3 3 2 3 3 L 55 13 36 27 50 11. 1 0 0 0 1 Total WBC av. 8500 12,300 18,600 h0,200 10,900 Rabbit VIII P.E.s. 20 no 26 36 st. 15 31 M9 , 6 my. 2 0 1 1 E. 0 0 1 1 B. 1 0 0 1 L. 61 29 2M 52 M. 1 0 0 3 Total WBC.Av. 8500 19,300 20,000 11,900 A pseudo-eosinOphilic (neutrOphilic) leucocytosis is shown. More specific interpretation is impossible because of the obvious in- accuracies of the readings. To check the factor of the gum acacia present, a series of ex- periments using various amounts of sterile fresh 1% acacia solution were run. The acacia was introduced intravenously, the leucocyte response followed for approximately twelve hours thereafter. Rabbits VIII, IX and XI were new rabbits; I and III had previously been given iodine doses. All five were superficially sound and normal. (2 Table key, above: P.E.= pspdo-eosinophile s.= segmented nucleus st.= stab form my.= myelocyte E.= eosin0phile B.= basophile L.= lymphocyte M.= monocyte. _ 23 _ Leucocyte Changes Following Injections of Gum Acacia Solutions Rabbit No. Amt. gum acacia Leucocyte counts mgms./Kg. Normal 6 hrs. 10 hrs. l.a. M60 mgms./Kg. 7900 10,900 10,200 7300 9,000 8,900 III.a. uh mgms./Kg. 9150 17,600 lu,800* 11,150 _ 18.150 N hrs. 12 hrs. XI. 8 mgms./Kg. 11,300 7700 11,100 8700 10,500 6900 11,800 7u00 h hrs. 10 hrs. 1x. .2 mgms./Kg. 6900 9000 10.500 8950 7100 7550 9350 I.b. .31 mgms./Kg. 11,700 11,150 10,500 15.500 10.350 10.850 I.c. .31 mgms./Kg. 15,600 18,000 20.300* 15,h00 18,900 III.b. .25 mgms./Kg. 7800 9800 9600 11,900 7950 9700 9900 1h,150* VIII. .25 mgms./Kg. 13.500 13,800 lu,u00 13,150 - 13,900 13,900 13,100 12,700 * In only three cases are there real signs of leucocyte response. Several modifying comments are also required. Rabbit III, in a. ex- periment had a remarkable lymphocyte count of 802, with only 10% poly- morphs: this was verified by two check counts; rabbit III also devel- Oped an infected ear for Experiment IIIb. Rabbit I in check experiment c. to explain the previous figures, showed a decided rise. Most of the changes are possibly physiologic. foreign body res- ponses perhaps. It is significant that in most of the cases there is indication of a rise from the determined 'normal'. In only tw:,*, are there genuine signs of count increase. - 2h _ Fragility tests have been only preliminary. In six cases blood was drawn from the heart for normals the day previous to injection. 20-30 minutes after injection of the iodine a sample was taken from the heart to compare with the normal. N0 marked changes, if any, were noted, but the gradation of hypotonic solutions was coarse, and the accuracy of dilution was not great. Addition of quantities of iodine to washed red cells results ultimately in their fixation, so that they cannot be hemolyzed in distilled water. The relative amounts of io- dine necessary to prevent hemolysis, with the known general activity of the free iodine, make measurable fragility change unlikely. Sedimentation tests fall intozfientical category, as being mere- ly suggestive. In six cases the sedimentation rates did not measur- ably alter. Rabbit blood normals in these six were at best very ir- regular, and very slow. .Addition of increasing quantities of iodine to the whole blood produced the strange blotting out of all sedimenta- tion as such. No explanation can be offered. In the course of differential examinations the curious granula- tion of a small proportion of the red cells was commented. No attempt was made to ascertain in how long these changed red cells remained in the peripheral blood before being destroyed. In vitro experiments were outlined to study the effects of free iodine upon the blood. Addition then of iodine in large amounts to whole citrated blood brought about a general precipitation of the blood elements, but the agglomerates were so finely grained that filtration through a filter paper was excessively slow. Filtrates, when neutralized with sodium thiosulfate of the iodine present, were cloudy. MicroscoPically the _ 25 - iodine was seen to coagulate the plasma proteins into finely granu- lar masses; the erythrocytes stained golden brown, altering to the granular condition of the stained smears in roughly a third of the cells, while others became crisp and hard of outline. A crude separation of blood elements was performed. Washed red cells took up large relative amounts of iodine and became fixed, refusing to lake in distilled water. Combination there is possible in both stroma and hemoglobin; the lipoidal portion of the stroma is the logical unit of combination in the former. Hemoglobin sols from washed and laked red cells, centrifuged to drive down the 'ghost' stromata, microscopically coagulated upon addition of iodine. The residual ghosts picked up perceptible iodine, though the bulk of these was small. Washed fibrin took up only small quantities of iodine, but as a derived protein it may fundamentally differ from the native blood fibrinogen. Defibrinated plasma did not grossly flocculate, but microsc0pica11y converted to a visible mass of granules. In all but the fibrin there was quick removal of the free iodine as testable with starch paper, free iodine as such apparently vanish- ing a moment after mixing. Attempting then to approximate the blood concentrations computed to be obtained in the regular method of in- jection, but adding the necessary iodine in a weak water solution rather than in colloidal form, no effects were microscopically ob- servable, even up to a maximum 30 mgm./Kg. concentration. The granue 1ar red cells and protein flocculation were conspicuously absent. A local concentration of free iodine around the colloidal particles of iodine to supply enough iodine for cell change, without full blood- volume dilution, may be the answer to damaged red cells in the circulation. - 26 _ Experimental Series II: Pathology, Gross and MicrOSCOpic. The various techniques and quantities of iodine administered have provoked pathologic sequelae which may be of interest in round- ing out the picture of iodine injectional effects. A cachexia from toxic conditions brought on by the injected iodine, those symptoms suggestive of iodism, and histologic kidney sections are mentionable. "U .abbi§_XIV. Iodine cachexia. hale, 3.23 Kg., mature, condition outwardly good. 7/1L injected 2 cc. h.1fi iodine slowly into heart; total iodine 2M.7 mgms./Kg. Animal survived, but hemoglobinuria was striking, and there was slight temperature rise. 7/15 animal fed well, but hemoglobinuria again was very marked (1.7M gm./100 cc. phl.), and eyes were inflamed. Feces small, glistening, pointed at ends. 7/16 again a marked hemoglobinuria in the morning (3.3 and 3.8 gm. /100 cc.Ph1.). 7/17 animal almost completely off feed, weak and listless. 7/20 animal found dead in morning. Kidneys with dark patches of grey-black. Rectum filled with mucoid matter, and drOppings were of this character as well. Suggest possibility of uremic poisoning as result of kidney damage. There are encountered generally throughout the case histories phrases suggestive of the symptomatology of iodism. Rabbit 1.7/27, following l2.h mgm./Kg. injection: "animal showed running nose and a sort of dyspnea, like snuffles (catarrhal irritation), two to ten hrs. following injection." Rabbit Iv,5/2, after 31 mgm./Kg. iodine, had lost appetite, showed "pussy nostrils, labored breathing, an apparent heavy 'cold'" before it relapsed and died as result of further iodine injection. Kidneys here were very dark, "very easily _ 27 _ cut". Rabbit XV,7/15, following 18.6 mgm./Kg. iodine, had "inflamed eyes.“ As did Rabbit XIV, who, close to death, had "inflamed, bloods shot eyes". Rabbit x1,7/11, following injection of 18.6 mgm./Kg. io- dine, was "off feed, sick, has inflamed eyes, slightly running nose, and drools saliva." The mucous membrane symptoms were far from uni- versal, occurring irregularly, but generally following fairly heavy iodine doses; the cow is exception to this last phrase, illustrating the symptoms each of the four times she was injected with 6.2 mgm./Kg. Cow I. A Case History. Mastitis-infected Guernsey, herd of C.S. Bryan, weight about H31 Kg.(950 pounds), infection in all four quar- ters,with knotty lumps of fibrotic tissue in two quarters; otherwise plump and seemingly sound. 5/16 injected lu5 cc. 2.3% iodine sus- pensoid into the jugular vein by means of a hypering flask and hose; speed of injection approximately 50 cc. per min.; total dose 7.7 mgms;/Kg. Animal had a definite uneasiness during injection, be- came diarrheic, the feces from which increased in fluidity through- out the day, had a marked increase in respiration with a temporary fading out of pulse. Temperature did not materially change. After injection her respiratory rate came back to complete normal in three hours, her pulse returned to normal rate and strength in 1% hrs. She had no appetite, twice passed volumes of coffee-brown hemoglobi- nuric urine,showed eye secretions to a slight degree, and develOped a very moist nose which she rubbed continuously against the manger. Apparent return to normal by nightfall, 8 hrs. 5/22 injected 135 cc. 2.52 iodine into the jugular at 20-30 cc. per minute; total 7.6 mgm. /Kg. Again the temporary distress at time of injection, followed by ‘ OQBWD-IIH' . . . . ‘4 . ' ‘I "M | .$ ‘ .0. :I" luu":dl’f u‘I-mmm‘ g . “fin-El #1 ”We ”W ‘ l I ~ I c u. :m " MW...“ Witt": ' Ls mal. % “.fifi M . . ”m‘w. t” has 110». M “MM W“ 3T,” {3 m t “:24 t;- Gls i“ 2... W‘M I h Tan» ‘ ‘ 948' pig/1‘1 -23- apparently complete return to normalcy. Again the running eyes, the nose which she licked and rubbed, the excess saliva, the mild diarr- hea, the hemoglobinuria. Respiration back to normal from high rate in 3 hours, pulse faded temporarily, rose slightly and was normal at least in 3 hours, temperature did not again materially change. 5/29 injected again, same amount, same route, wdth practically identi- cal observations. Periodically she had spells of gasping that seemed attempts to clear her throat. No appetite, but animal back to seem- ing complete normal within 20 hours. 6/28 again injected cow with repeated dose of same strength. Again the gasping, but otherwise a repetition of the first injection. Sollmann (17) describes the paramount symptoms of iodism as consisting of rashes, coryza or 'colds', headache, conjunctivitis, laryngitis, bronchitis etc. A considerable number of these have been noted, particularly in the cow; several are not observable in experimental animals even if present. It is worth pointing out that the four rabbits subjected to continuous dosages of iodine, Rabbits I, III, VIII, and IX,did not show symptoms of iodism throughout the entire series of injections. Elodism as such has been considered in. the outcome of repetitive iodide dosage But iodism as such cannot be concluded from the scant observations, without comparison with the commonly known condition. Its occurrence in this connection is, however, very probable. The kidneys in a good percent of the animals that died of iodine injection were of the grey-black blotched type. In terms of gross patholoér, that organ was the only fairly consistently pathologic - 29 - one. Rabbit IX, which perhaps should have showed the effects of continued iodine injection, had apparently normal kidneys, free from the dark streaking that in the average animal extended from cortex to pelvis in the cut organ. Upon section the kidneys presented two conspicuous conditions. The proximal convoluted tubules in general exhibited a cloudy swelling degeneration, with some vacuol- ation of the cells; and the ascending limb of the loop of Henle showed a stasis of the excreted hemoglobin, and of what seemed to be disin- tegrating red cells. The thelium of the ascending limb is often flattened by the distention. The glomeruli do not show evidence of visible damage. In animals of the "black kidney" type this picture is magnified to an appalling degree, involving a large prOportion of the lOOps of Henle, and virtually all of the proximal convoluted tubules. It is a pathology perhaps associated with Stieglitz'(2h) discovery of localization of iodine in Henle's loop, or more likely the simple fact of graVity, which permits collection of the hemo- globin in the ascending limb. Study of hyperplasia or non—hyperplasia of the thyroid (Marine (15)) waits upon suitable knowledge of normals. It may be that Marine'S'conclusion: "maximum thyroid effects are induced with mini- mum amounts of iodine" is a point of application of intravenous in- jection of iodine. Yet oral administration, if as good, requires testing as the more desirable method, with less hazard. Hemoglobinuria: an invariable symptom following injection of iodine. No red cells_were observed to hemolyze in vitro as direct con- sequence of added iodine; the opposite was true. And yet a surprising- WMWP “WWW sin W‘- WmutqummI-w WMMMWMW WWW WWW“ Wmfimwwqp - 3Q - "m1 male“ ly severe hemoglobinuriaAwas the outstanding single external sign of the iodine injection at the quantities employed. This oeuld not but mean excessive destruction of red cells as seen in hemolytic jaundice, Texas fever, and malignant tertian malaria. The redwater urine was watched, and proved an invariable post-injectional finding. It was associated with albuminuria, which zould predicate a damaged renal filter. Benzidine tests were sharply positive for hemoglobin, and heat with nitric acid confirmed the presence of moderate albumin quantities. Urinary sediments were apparently devoid of red cell ghosts. The first micturition after injection was consistently hemoglobinuric; later measurement of the time of hemoglobin-albumin excretion by direct ureter cannulation confirmed the speedy presence of pigment in the urine. First traces appeared from MO minutes to 3% hours after injection, averaging slightly over an hour in four anesthetized animals. Centrifugation prior to quantitative estimation of hemoglobin with the SheardeSanford photelometer threw down sedi— ments that were very dark in color; it is likely that much hemo— globin was lost, precipitated perchance with the albumin. Photelo- meter readings then record only such hemoglobin as was found in the supernatant urine. Such figures do not, probably, truly show the amount of pigment present, but suffice as a quick and standard method. Normal centrifuged.urines range from .05 to .3 gms./lOO cc., using the blood hemoglobin chart. In severe cases the hemoglobin was con- tinuously excreted.up to ME hours following injection, this time fig- ure obtained by micturition only, rather than by the direct tapping of ureters. - 31 _ Hemoglobinuria: Salient Observations Animal No. Dosage Hb. first observed Phl.readings Duration of Hburia mgm/Kg. gms./lOO cc. RXV 18.6 First micturition 3.“ (not cent.) 32 hrs. RXIV 2U.7 First micturition 1.7% (first pass.) 50 hrs. RXII 23.7 Bladder full of hemoglobinuric urine at autopsy. RXI 18.6 First micturition 1 MS hrs. RI 12.2 First micturition 1.8M RS hrs. RXVII 15.5 First micturition .92 M8 hrs. RXVIII 18.6 First micturition 1.11 (first pass.) 2h hrs. Cow 6.2 First micturitions following four injections, back to normal in about 2H hours. DI 18.6 Left ureter 2% hrs. Right ureter none at death, 2% hrs. DIV 18.6 Left ureter 1% hrs. 1.2 Right ureter M hrs. .83 RXXII 18.6 Both ureters MO min. 1.2 (first pass.) 1.} (sec. pass.) R=rabbit, D=dog, cent.=centrifuged, pass.=passage,Ph1.=photelometer, Hb.=hemoglobin. The last three animals tabulated were killed N hrs.a.i. The evidence points to breakup of the damaged red cells by body phagocytes, perhaps of liver and spleen, to liberate this cell pigment. It is strange that Boehm, 1876, (16) and others following him have not seen the hemoglobin excretion if it is also seen with alcoholic iodine introduction. It may be unique for free aqueous iodine. Temperature Reaction, following intravenous injection. It was thought that the effect of the iodine upon the tempera- ture might be interesting, as showing temperature control center res— _ 12 _ ponse to the iodine. For a considerable portion of the earliest observations, temperature charts were routinely compiled. Twenty- three charts are assembled in the following table: Animal No. Times Dose 12 Total Rise, or Fall Route of entry used. mgm/Kg. OF Cow 3 6.2 1..80F. 2..70F. 3..50r very slight each jugular vein R.III h 6.2 1. 30F. 2. 1.60F. ear vein 3 3. ,gOF, ear vein 3.1 h. slight change ear vein R.II 1 6.2 1°r. ear vein R.I 2 6.2 1. .MOF. 2. No change. ear vein R.V 1 12.u .6OF. ear vein R.VI 1 6.2 2.5OF. ear vein R.VIII 3 6.2 —h.5°F. heart 5 No change Peritoneally administered. 55.8 No change Peritoneally administered. R.IX 3 6.2 1. -2.7°F. 3. 1.3OF. heart 6.2 2. No change peritoneum R.X 1 2h.8 1..8°F, then dropped -1.6°F. heart R.XII 1 2u.s 1.10s. heart R.XIV 1 2h.8 slight rise. heart R.XVII 1 15.5 1.80r. heart R.XVIII 1 18.6 .50F. heart R.=rabbit, all temperatures in degrees Fahrenheit. -33... Experimental Series III. Observations On Blood Pressure and Respiration. In surveying preliminarily the response of animals to aqueous iodine injections, four dogs and four rabbits were used. Respira- tory and blood pressure reactions were mainly desired. Hemoglo- binuria and bile flow findings were incidentally sought. Animals were anesthetized, adjusted to register responses upon the kymograph, in- jections were given, and readings taken. A representative drum recording is included. Dog II. 8/5. Male, mongrel bull pup, 9.75 Kg., well fed and in good condition, though scary and nervous. Anesthetized with sodium bar- bital, 285 mgms./KV. injection intraperitoneally; deep anesthesia. Cannulated.ureters, bile duct, trachea and carotid artery. Injected 2 cc. 1% fresh gum acacia solution into femoral vein, in 15 seconds. There was a very surprising dr0p in blood pressure, and increase in respiratory rate. Return to normal was prompt. Second and third doses of acacia, varying the rate of injection from slow (#0 sec.) to fast (3-5sec.) produced no changes from normal. Injected 1 cc. .1N.HCl slowly (at pace of iodine injection) into femoral vein, with- out graphic response. Two small dips in B.P. due to femoral artery reflexes from handling of the artery, (Brewer). Injected n.53 cc. n.7fi (18.6 mgms./Kg.) iodine intra-femorally, in 80 sec., and wit- nessed a considerable drOp in B.P. Respiration was characterized by a sudden stOppage for lO-l5 sec., then a rapid increase in rate and amplitude. B.P. never quite returned to normal. The second Injection 3 hrs. later was of the same size dose, at the same speed, into the femoral vein. There was a long-drawn drOp in B.P. a tem- _ 3h _ porary recovery to a normal, then a steady fall. Respiration increased again in rate and amplitude. The viscera were exposed during the latter low B.P. period, and some dilation of blood vessels was seen; the blood pressure fall may be due to splanchnic pool onen- ing, perhaps a capillary or arteriolar effect of the iodine not seen before. Albuminuria at 2 hrs., but only faint hemoglobinuria at 3 hrs. Bile flow showed a temporary increase directly following both injections. WBC. unchanged over three hour period, expected. DQg;Ifl;_ 8/10. Male, shepherd mongrel, 13.6 Kg., in fairly good con- dition. Anesthetized with nembutal 30 mgms./Kg.=h08 mgms.; deep anesthesia. Ureters, carotid, trachea cannulated. Animal showed tendency to hemorrhage and so omitted bile flow determinations. Animal showed irregular respiration, a modified Cheyne—Stokes effect, without an apneic phase. Injected h cc. 1% gum acacia without effect. Again injected the same quantity of acacia; there may have been some slight respiratory increase, in amplitude only. Again injected the same amount of gum acacia, without effect. Injected M cc. .lK.HCl, and noted irregular breathing with several dropped respirations. In- jected 12.65 cc. 2.08% iodine (55-60 sec. total time of injection) into femoral vein; 18.6 mgms./Kg. dose. This iodine was a Kerck commercial product diluted with saline from 20% suspensoid. There appeared grossly to have been considerable damage to the vein wall, but sections were not taken. No.3.P. change was noted, but res— piration markedly increased in rate and amplitude. M hrs. later in- jected 7.7 cc. M; iodine (65 sec.) into opposite femoral vein; 18.6 mgms./Kg.dose. Noted short drop in blood pressure with concomitant -35... respiratory increase in amplitude and rate. Killed animal while blood pressure was yet at ebb, and found viscera congested. Took thyroid, kidneys, congested liver, and normal spleen for section. Urine volumes uncertain; hemoglobinu~ia with albuminuria one ureter 1% hrs.; other ureter anuric, and hemoglobinuria not observed until M hrs. No change observed in blood fragility. Rabbit XXII. 8/2. Female, 1.87 Kg., superficially sound. Deep anesthesia with sodium barbital, 285 mgm./Kg.=605 mgms. Cannulated ureters, carotid and trachea. Injected 1 cc. 3.h§ iodine (70 sec. total time of injection) into marginal ear vein; approx. 18.6 mgms. /K~. dose. Immediate blood pressure drOp and respiratory increase in amplitude. Blood.pressure returned to normal in 30-35 minutes. Hemoglobinuria observed at no min., and albuminuria simultaneously. Reinjected iodine in same quantity at the approximate same speed, and again observed a drop in B.P. and increase in respirational amplitude and rate. Killed animal, and noted a few lesions of coccidiosis of liver, a streaked kidney upon cutting sagittally; viscera uncongested and normal. Rabbit XX. 58/3. Male, 2.5 Kg., animal to all intents sound. Deep anesthesia with sodium barbital, 275 mgms./Kg.=785 mgms., injected intraperitoneally. Ureters, carotid and trachea cannulated success— fully. No flow from bile duct. Injected into marginal ear vein 1 cc. H.02fi iodine, (MO-M5 sec. total time of injection); 16 mgms. /Kg. dose. There was no change of any sort noted. Injected one hour later the same quantity, by the same route, in roughly the same time. No response, singularly enough. hemoglobinuria with albuminuria _ 36 - occurred 1 hr. 15 min. after first injection. Killed animal, vis- cera apparently normal, some traces of past lesions upon the liver. Took thyroids, kidneys, liver and spleen for frozen section. The usual number of laboratory technique difficulties was responsible for omission of one or another of the observations in each of the experiments. The regularity of results, more apparent upon the graphs than upon a table, has been marred by resistant rabbit XX, from which no response could be elicited, and by dog II and rabbit XXV, both of which showed surprising reactions to the admin- istration of gum acacia; with the rabbit blood pressure dropped but slightly; with the dog it dipped very abruptly a distance of Ml mm., the respiration modified simultaneously, but recovery was almost as rapid as the fall, and indications were of an unimpaired system. It is worthwhile to note the unresponsiveness shown by dog III to a light dose, for it is perhaps within this dose range that suc- cessful and virtually harmless injections might be made if there were any warrant for them. The observed data show that iodine in a free state when direct- ly placed in the blood stream is capable of causing to occur a dyna- mic response of short duration. Blood pressure falls rather sharply, the respiration either attempting to compensate by raising intra- thoracic pressure, or reacting to a stimulus upon the central nervous control area (Hacht and Hooker (22)) in either possibility the out- ward recording is an increase in rate and amplitude. The recovery, depending again, in all likelihood, upon the animal susceptibility, is fairly rapid. Electrocardiographs would be very helpful. Albumi— -37.. nuria and hemoglobinuria occurred almost simultaneously. Bile flow could not be estimated. Splanohnic pool dilatation is ir- regular, and questionable. In a few observations to check musser and Hermann (20), using colloidal iodine instead of the tincture, the beating frog heart in situ was treated by a drop or two of concentrated colloidal iodine. There was an immediate staining of the tissue, fibrillation and resumption of beat in one, temporary halt of a few moments with re- sumption in another, no effect upon a third, but in any case no fatal action, such as might have been presupposed by the experi— ments of the previous workers. Introduction of a drop or two of iodine into the venous sinus produced irregularity of function such as the stOppage of the ventricle only, then complete cessation of beat. Two rabbits that had accidentally received iodine into the pericardium at autOpsy showed fibrinous inflammation, but no other heart impairment. 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The Graphic Effects of Aqueous Iodine Injection Graph I. Response to First Injection of 18.6 milligrams of iodine per kilogram of body weight. Vertical lines with timer recordings indicate speed of injection; timer interval 10 seconds. Corresponding points have been indicated. 8.: start of injection. F.= finisn of injection. Graph II. Response to Second Injection after lapse of three hours; 18.6 milligrams of iodine per kilogram of body weight dosage. Note no signs of beginning recovery in the blood pressure recording. Vertical lines indicate speed of injection; timer interval 10 seconds. Note corresponding points. 5.: start of injection. F.= finish of injection. Graphs from kymograph record of Dog II. See corresponding notations of time effects, etc. upon preceding table. The unusual reaction to gum acacia, in addition to hydrochloric acid reaction shown in complete record, on file with Dr. W. L. Chandler. ‘\ A, . #0 m Er -142- Experiment Series IV. Determination of Minimal Lethal Dose In Rabbits. Knowing in advance the statistical difficulties involved in establishing a minimal lethal dose for a compound for one route of entry into the animal body, and recognizing that such figures ob- tained hold only for the one animal experimentally used, yet it was desirable to gain an approximation of that dosage to better study extreme effects...where probably the real fruits of possible usefulness do not lie...and to derive an idea of how far one might go in introducing the free iodine into the blood stream. A minimal lethal dosage of any chemical substance has been ruled to be that quantity of iodine which will kill a few animals, though not all of them by a considerable deal. But dosage below that quantity of the substance will spare all normal animals, even though they chance to show temporary toxic symptomatology. Deaths from iodine around the point of M.L.D. are in general of a lingering, cachectic typef'described fully previously. The data concerning this determination of an approximate M.L.D. has grown partly from experiments designed to show the lower limits of deadliness, partly as end result of experiments upon other phases of the work. In compiling the table from twenty-five rabbits, the facts only of dose and survival or death have been considered. - L3 - Determination of Minimal Lethal Dose Animal No. Dosage Remarks: Case Conclusions. mgms./Kg. R.I,XXI,XXIII, 12.u All animals recovered XXVIII Cow I 6.2 Recovered completely, four injections R.XVI,XVIII,XXII, 15.5 All animals recovered XXVII,XVII R.XV,XVIII 18.6 Animals recovered R.XXIV 18.6 Cachexia,8 days,died. R.XI 18.6 Steady cachexia, 5 days, died. R.XVII 18.6 Died of shock, 15 min.a.i. P.VII 21.7 Died in 3 hrs. R.IV 2H.8 Died in 26 hrs., after showing ca- tarrhal symptoms. R.X 2N.8 Steady cachexia, 8 days, died. Had had 12.“ mgms./Kg. previously. R.XIV 2h.8 Cachexia 6 days, died. R.XXV 2M.8 Died of shock, 10 min.a.i. R.XXVI 2n.g Died of shock, 2 min. a.i. R.XIII 31 Died of shock, s hr. a.i. R.III* 35 Recovered. Had had two previous doses of 6.2 mgms./Kg. R.VIII"”‘l 8x6.2 Died of hemorrhage following last in- ject. Last six injections on successive days R.IX"”'I 10x6.2 Died of shock following last inject. .Last seven inject. on succ. days. R.=rabbit a.i.= after injection. I"Note remarkable resistance of this animal. l""“foote how resistant these animals were to small doses. Recovery was universal following injection of 6.2 mgms./Kg. intro- duced at moderate speed into the circulation, at least where a single dose is concerned. It is likely, from these figures on the rabbit, that minimal lethal dose of free aqueous iodine (colloidal) is about one-half that of the amount set for tincture of iodine by Boehm (16). Confirmatory experiments on his work, and on that of Nyiri and Dubois 13) who roughly confirmed him, are lacking. Failing more promising results in the fields at present defined, it has not been considered worthwhile to further refine the limits of -1414- minimal lethal action in rabbits, and to determine M.L.D. in other animals as well. The Minimal Lethal Dose of free aqueous iodine then lies in the vicinity of 18.6 mgms./Kg.; let us say approximately 20 mgms./Kg. Discussion of Observations In studying changes in blood picture following intravenous introduction of free aqueous iodine, a relatively small number of total red and white cell counts, differential leucocyte counts, hemo- globin determinations, sedimentation rates and blood fragility estima- tions were made. The recording of these few experiments shows that the red cell picture does not significantly change either with light or heavy doses, within at least the first 1+8 hours. Such slight changes as are seen can all be accounted for as purely physiological variations. If, however, the dose has been very toxic to the animal, there is indication that the erythrOpoietic tissues are depressed. In one very cachectic animal the RBC. drOpped to one million cells per cubic millimeter just preceding death, and in continued-dose animals the EEG. was seen to fall perceptibly. On the other hand, the white cell response is notable. There is a sharp increase in pseudo-eosin- ophiles, rising to a numerical peak in l2—2M hours,...a peak which may be three to four times the normal count...ebbing to normal in two or three days depending probably upon the susceptibility of the animal to iodine, and upon the size of the dose. .A slight proportion of the leucocytosis may also be due to the gum acacia found in very small amounts in the colloidal iodine. This leucocytosis may be comparable to the iodide leucocytosis of Heinz (1“). Injection into the perito- neum in three cases suggests a low toxicity by that route. Hemoglobinuria coupled with albuminuria has in all animals been the invariable consequence of all doses of iodine down to a minimum of six milligrams per kilogram of body weight; this symptom has not before | _ M6 - been reported. Temperature response of the seemingly normal rabbit has been seen to be erratic, and no construction can be put upon the figures other than the susceptibility differences of individual animals, seen universally in pharmacologic studies. The kidneys seem to bear the brunt of the toxic effects; microscopically there is cloudy swelling degeneration of the proximal convoluted.tubules. with stasis of excreted hemoglobin and albumin in the ascending arms of the loops of Henle, while the glomeruli remained visibly whole and normal. The known fact of kidney excretion of iodine not stored in other body tissues (Nyiri and Janitti (12)), in the lipoids and the thyroid mainly (McLean(23)),(LIarine (15)), accounts perhaps for the localization of iodine toxemia in that organ. In sum the patholOgic effects seem to indicate an ioidne combination with blood substances in a form irritating, toxic, and poorly excretable. Kidney irritation may partially be explained by Stieglitz'(2h) histochemical determination of iodine localization in the loop of Henle of the kidney. There may be symptoms of iodism following injection of free aqueous iodine intravenously. A marked drop in.blood pressure and an increase in respiratory rate and amplitude were observed. following injection. Reason for- such responses may be that of respiratory center stimulation by iodides (Macht and Hooker (22)),be heart weakness thru iodine combination with unsaturated lipoids (Salant and Livingston (18)). Electrocardiographs might fill a needed gap in the information. Study of minimal lethal dose has progressed only to disagreement with the conclusions of Nyiri and Dubois (3), to comparison with the report of Boehm (16), to a definition of the probable highest limits of aqueous iodine usage intravenously. Minimum lethal dose is shown to _ M7 _ be close to twenty milligrams per kilogram of bodynweight, for the rabbit, or approximately one-half of Boehm's determination for tincture of iodine. Additional work mapping the reactions of blood to addition of iodine has only been included as groundwork, as preface to further approach. It is to be emphasized that the dosages here worked with are relatively non-toxic in a pharmacologic sense. The character of a large portion of the work in defining limiting factors of intravenously; injected free iodine (colloidal iodine, Chandler (5)) has made the results to a degree forbidding. Uses have not been anticipated from these preliminary observations. Many major aspects of approach lie untouched; the effects upon metabolism, and the purely chemical studies are two. A large number of factors entering into experimental consideration were omitted; the results are then necessarily Open to severe criticisms. None of the conclusions is substantiated from the point of statistical numbers. It has been the hope of the worker that he might plot a field, indicate promising lines of further labor, disclose investigative blind alleys, and givé to himself a training in biologic thought. Summary of Observations The effects of free aqueous iodine( the colloidal iodine (Chandler)) upon rabbits by direct circulatory introduction are indicated tentatively to be several. 1. There is a pseudo-eosinOphilic (neutrophilic) leucocy tosis, probably as response to a foreign irritant. The granulocytosis rises g' ,ikzwr‘ to a 12-2N hour peak, returns to normal in 2-3 days. There is n. A erythrocytic change, except where general toxemia has resulted, and is there due possibly only indirectly to the iodine. A.small pro- portion of the leucocytosis may be due to the gum acacia present. 2. Virtually all blood elements to some degree combine with free aqueous iodine. Apparent denaturation of proteins and fixation of red cells are pointed out. 3. Free aqueous iodine causes heart depression, fall in blood pressure, and rise in respiratory rate and amplitude, invariable hemoglobinuria due to other than direct iodine hemolysis, and degenera- tion of the proximal convoluted tubules of the kidney. Effects ob- served in dogs. rabbits, and a cow. h. The minimum lethal dose of free aqueous iodine for rabbits is approximately twenty milligrams per kilogram of body weight. I 5. Iodine toxemia is characterized by cachexia, and irritative phenomena. Toxicity depends upon dose size and individual suscepti- bility to iodine intravenously introduced. Free iodine intravenously injected is irritative, and shows little promise of application. TM m m‘tpmm W W Mt‘fl an. MM”. Small numbers of animals used and omission f numerous variables remain sources of criticism and weakness. As an amateur in the fields of biological investigation, I have unashamedly attempted to tap the personal knowledge of the college specialists, recognizing the difficulties of wide reading without suitable background of critical judgment based.upon working experi- ence. and.permitting also the opportunity of learning of the valuhble personal experiences of these men. I cannot sufficiently, nor volubly enough, thank the workers in my own Department of Bacteriology, Hygiene and.Parasitology for assistance of many kinds. Dr. Wallace L. Chandler has been my continued teacher and stimulant throughout the year's work, supplying abundantly yet unbiasedly from his wide familiar- ity with the colloidal iodine, his own physico—chemical development. Dean Ward.Giltner has given encouragement toward the probing of some of the ideas outside the specific field of bacteriology. Dr. B.B. Roseboom has generously given the use of his Physiology Department laboratory. Dru Nathan.Rs Brewer, veterinary physiologist, has been very largely responsible for the physiologic data; his knowledge and experience have solely made possible the exploratory work in this field. Dr. C.A.Hoppert, Mr. E.C.Tabor, and.Mr.T.L.Canniff have assisted in the merely tentative chemical attack. Dr. E.T.Hallman has been unfailingly helpful in the histo—pathologic observations. Dr. I.Forest Huddleson has overseen the blood work, and his staff of the Central Brucella Station have given freely of their skill. References Cited (1) Nye, 11.11. The Relative In Vitro Activity of Certain Disinfect- ants in Aqueous Solution. Journ. Am. Med. Assoc. 108, Jan. 23, 1937. (2) Salle, A.J. , and Lazarus, A.S. A Comparison of the Resistance of Bacteria and Embryonic Tissue to Germicidal Sub- stances: V. Proc. Soc. Exp. Med. B iol.32:9, 1935 (3) Nyiri, w., and Dubois, L. The Toxicity of Colloidal Iodine. Journ. Am. Pharm. Assoc. XX:6, June 1931. ()4) Simmons, J.S. The Comparative Bactericidal Action of Mercuro- chrome and Iodine Solutions as Local Tissue Disin- fectants. Surg. ,Gyn.and Obst. . 56, 1933. (5) Chandler, W.L., and Miller. E.J. Colloidal Iodine. Journ. Phys. Chem. m1, July 1927. (6) Huffman, L.D. Gum Acacia as Hemorrhage Therapeutic. Journ. Am. Med. Assoc. 93. Nov. 30, 1929. (7) Stafseth, H.J. On the Control of Tapeworm Infestation In Chickens. Tech. Bull. 111:8, 1935, 11.5.0. (8) Chandler, W.L. Iodine as a Vermicide and Disinfectant. Black _ Fox Magazine, Dec. 1923. (9) Chandler, TM. The Vermicidal Action of Iodine. Quart. Bull. Mich. Ag. 0011., Feb. 192+. (10) Chandler, ILL. Experimental Treatment of Dermatomycoses With Iodine. Journ. Mich. State Med. Soc.,April 1927. new I'- u. (11) Hesseltine, H. C., andw. A Study of Yeast—Formed Conditions of Vulvo-Vaginitis. Journ. Lab. and Clin. Med. 21. 3, 1935. ‘ (12) Nyiri,W. , and Janitti, M. About the Fate of Free Iodine Upon Application To the Unbroken Animal Skin. J ourn. Pharm. and Exp.'Ther. XLV:1, May 1932. p (13) Scarborough, R.A. Blood Pictures of Normal Laboratory Animals. Monograph, Yale University Press 1926. (IN) Heinz,R. (from Sollmann) ABch. Path. Anat. 155, Mt, 1899. (15) Marine, D. Quantitative Studies On the In Vivo Absorption of Iodine In Dogs' Thyroids. Journ. Biol. Chem.22, 1915. (16) Boehm, R~(from Sollmann) Arch. Exp. Path. Pharm. 5, 529, 1876. (17) Sollmann, T. A Manual of Pharmacology. Saunders, 1926. (18) Salant, w., and Livingston, A.E. The Effect of Iodine and Sodium Iodide Upon Circulation. Journ. Pharm. 8, 119, 1916. Am. Journ. Physiol. #1, 23h, 1916. (19) Jobling, J.W., and Petersen, W. The Therapeutic Effect of Iodine. Journ. Am. Med. Assoc. 63, 1930, l9lh. (20) Musser, J.H., and Hermann.(from Sollmann) Iodine. Proc. Soc. Exp. Med. and.Biol. 23, 212, 1925. (21) Pepper, O.H., and Farley, D.L. Practical Hematological Diagnosis. , Saunders, 193%. (22) Macht, D.I., and Rocker. The Respiratory Center. Journ. Pharm. 11, 63, 1918. (23) McLean, F.C. The Present Status of Organic Iodine Preparations. . Journ..Am. Mods Assoc. 63, 1382, 191M, (2H) Stieglitz, E.J.(from Sollmann)(17) Iodides. .Journ. Pharm. and.Exp. Ther. 22, 89, 1923. .| 1 ‘1‘? A r... \ . 63% met jumpb9 ”/10!“ 1d 16 1940 DE 1053 An 24 '5‘