STUDIES ON CHROMIUM EXCRETICN IN THE DOG Thesis for the Dogm of Ph. D. MICHIGAN STATE UNIVERSITY Robert James CoIIIns 1958 R 19515 This is to certify that the thesis entitled STUDIES ON CHRCMIIM EXCRETION IN THE Dm presented by Robert James Collins has been accepted towards fulfillment of the requirements for _£h.L_degree in_P_h.1§1_°]-_‘?8Y and Pharmacology /%J Wu), Major professor? Date Se tember 6 58 0-169 LIBRARY Michigan State University STUDIES ON CHROMIUM EXCRETION IN THE DOG By ROBERT JAMES C OLLINS A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Physiology and Pharmacology 1958 ABSTRAC T No demonstrable biologic function has been reported for chro- mium although it is omnipresent in trace quantities in man, other animals, food products, soils, and water supplies. In addition, ex- tensive use of chromium in industry frequently results in exposure of animal tissues. Toxicity and tissue distribution of chromium compounds is well documented but little information exists on its excretion. Objectives of this study were to investigate the routes and mechanisms involved in chromium excretion as well as to com- pare some of the physical and chemical properties of chromium in biological fluids to its characteristics before injection. Routes of excretion were studied in acute anesthetized dogs over a four-hour period and in bile fistula dogs for four days after intravenous injection of Cr51 as chromic chloride or sodium chro- mate. These studies showed that urine was the major route of ex- cretion of chromium of either valence after intravenous injection; excretion in bile and feces was negligible. Mechanisms employed in renal excretion of chromium were studied by the renal clearance technique. Renal clearance of chro- mium decreases exponentially with time after a single intravenous ii a.~——-vIw————fl_ ~1m—h'ufi—tflnfl - ,J , injection of Cr51Cl3 from 2.5 or 3 ml. per min. at one hour after injection to less than 1 ml. per min. eight hours postinjection. Measurement of plasma-dialyzable chromium by equilibrium dialysis showed that the percentage not bound to plasma proteins in vivo also decreased exponentially with time after injection. These observations ). led to determination of a dialyzable chromium clearance (C d-chr The mean and standard deviation of C d-chr. on eight dogs measured over periods 11p to ten hours after injection was 36.3 a: 16.6 ml. per min. This clearance indicates that glomerular filtration and tubular reabsorption are two mechanisms of renal function involved in the handling of unbound chromium. Tubular excretion of dialyzable chro- mium may occur; however, no reduction in C at high plasma d-chr. levels favors the conclusion that tubular excretion is of minor im- portance. Simultaneous creatinine and PAH clearances were normal, demonstrating that renal function was not impaired by large doses of chromium given intravenously. Measurement of trivalent and hexavalent chromium in urine after the intravenous injection of sodium chromate showed that re- duction readily occurs in vivo. Chromium excreted in urine and bile \or present in a plasma dialyzate is anionic as shown by ion ex- change absorption after intravenous administration of cationic chro- mic chloride. iii Paper chromatography and precipitation procedures on urine . . . 51 , and plasma dialyzate indicate, but do not prove that Cr 18 excreted in part in organic combination. iv AC KNOWLE DG ME NTS The author wishes to express his gratitude to Dr. W. D. Col- lings and Dr. P. O. Fromm for their council and interest during the course of these experiments and in preparation of this manuscript. Special thanks are also due to J. F. Wagner for many fruitful dis- cussions and assistance in preparing some of the experimental ani- mals. In addition the writer is indebted to the United States Public Health Service for funds in support of this work. ' .I. «wruuvww “"'._'s——"_'_"v.~ .u. w- . . ”an... ---.. - TABLE OF CONTENTS Inorganic Chemistry of Chromium ............... Nature of Chromium-Protein Bond ............... The Binding of Chromium by Tissue Proteins ....... Absorption, Distribution, and Elimination of Chromium ................................ METHODS ................................... I. Routes of Chromium Excretion ............... General procedure ..................... Acute experiments on anesthetized dogs ...... Prolonged experiments on bile fistula dogs II. Mechanisms Involved in the Renal Excretion of Chromium ........................... Renal clearance procedure ...... . ........ Dialysis procedure ..................... Creatinine and PAH procedures ............ III. Some Physical and Chemical Properties of Chromium in Biological Fluids ............... vi 14 22 22 22 24 25 27 27 29 30 33 Dialysis procedures .................... Reduction of hexavalent chromium .......... Ion exchange procedure .................. Chromatographic procedure ............... Chromium precipitation proc edure .......... II. Mechanisms of Renal Excretion of Chromium ..... III. Some Physical and Chemical Properties of Chromium in Biological Fluids ............... DISCUSSION .................................. I. Routes of Chromium Excretion ............... II. Mechanisms of Renal Excretion of Chromium ..... III. Some Physical and Chemical Properties of Chromium in Biological Fluids ............... SUMMARY AND CONCLUSIONS .................... REFERENCES CITED ........................... APPENDIXES ................................. A. Four-Hour Excretion Values of Chromium in Acute Anesthetized Dogs, Four-Day Excretion Values of Chromium in Bile Fistula Dogs, and Data Used to Calculate Chromium Clearances vii 35 35 37 39 39 44 50 62 62 64 76 79 82 89 90 APPENDIXES B. Detailed Procedures for PAH, Creatinine, and Chromium Analyses, and for Oxidation of Cr51Cl3 to NazCr5104 ................. C. Renal Clearance and Statistical Formulas ...... viii Page 99 105 TABLE LIST OF TABLES Renal Clearance Values ................. Per Cent Dialyzable Chromium in Urine ...... Reduction of Hexavalent Chromium in vivo and in vitro .......................... Ionic Properties of Chromium in Biological Fluids .................... . ......... The Rf Values of Cr“, Ninhydrin-positive Substance, Creatinine, and PAH in Samples Taken from Dog 12 .................... Precipitation of Chromium from Biological Fluids Using Ammonium Hydroxide ......... Dialyzable Chromium Clearances in Milliliters per Minute per Square Meter Surface Area as Calculated from Per Cent "Free" Chromium in Plasma ......... Relationship between the Dose of Chromium Injected and the Rate Constant (k) for the Decrease in Per Cent Dialyzable Chromium in Plasma ......... . ................. ix Page 45 53 57 58 59 61 67 69 W A 7 __ ‘—.—-— FIGURE 10. LIST OF FIGURES Method Used to Determine Plasma Concen- tration at Three Minutes before Midpoint of Urine Collection Period .................. Accumulated Excretion of Hexavalent Chromium in Urine ............................. Accumulated Excretion of Hexavalent Chromium in Bile .............................. Accumulated Excretion of Trivalent Chromium in Urine ............................. Accumulated Excretion of Trivalent Chromium in Bile .............................. Comparison of Excretion of Hexavalent and Trivalent Chromium in Urine and Bile ........ Comparison of Four-Day Excretion Values of Hexavalent and Trivalent Chromium in Urine ............................... Comparison of Four-Day Excretion Values of Hexavalent and Trivalent Chromium in Bile and Feces ........................ The Decrease of Chromium Clearance with Time after Injection ..................... The Decrease in Chromium Clearance of Dogs 8 through 17 with Time after Cr51 Injection ............................. Page 32 40 4O 41 41 42 42 43 48 49 FIGURE 11. 12. 13. 14. 15. 16. The Decrease in Per Cent Dialyzable Chromium in Plasma with Time after Injection .......................... The Decrease in Per Cent Dialyzable Chromium in Plasma of Six Dogs with Tim e after Injection .................. Dialysis of Cr51 in Saline and Urine of a 10.5 kg. Dog after Intravenous Injection of N32CI‘5104 ooooooooooooo e o ......... Dialysis of Cr51 Added to Urine in vitro and Urine after Intravenous Injection of NaZC r5 1 O4 ........................ Variation in Clearance Ratios with Time after Chromium Injection .............. Variation in Clearance Ratios with Plasma Concentration of Chromium ....... . ..... xi -3‘ I Page 51 52 55 56 73 75 .' I“ " ""13"? v.7 r“ INTR ODUC TIO N No demonstrable biologic function has been reported for chro- mium; however, trace quantities have been found in tissues of man, other mammals, and food products (Grushko. 1948; Van der Walt and Van der Merwe, 1938; Koch et al., 1956; Udy, 1956). Access of chromium compounds to animals and plants is provided by its wide distribution in soils and water supplies (Braidich and Emery, 1935; Davidson and Mitchell, 1940; David and Lieber, 1951; Udy, 1956). Walsh (1953) has reviewed hazards to workers in industry where chromates are used. In chrome plating and metal anodizing by electrolysis of a chromic acid bath, hydrogen bubbles produce a mist of chromic acid which causes ulcers and respiratory inflamma- tion. Chromate solutions used to control corrosion in various water recirculating systems are responsible for dermatitis and chrome ulcers. Chromium ore dust as well as chromate have been impli- cated in liver damage and lung cancer. Chromate workers have a death rate due to lung cancer twenty-nine times greater than non- chromate workers (Brinton et a1., 1952). Chromate hazards may also result from its use in batteries,gcement, and primer paints. The despicable practice of coloring food with yellow lead chromate has fortunately been discontinued (Monier-Williams, 1949). EXposure to sufficient quantities of chromium can also cause dermatitis, ulceration (Sollman, 1957) and possibly cancer among workers in the leather-tanning industry. The finished leather con- tains 3.9 to 6.9 per cent chromium that occasionally causes irritation and sensitization. With the advent of radioisotopes, chromium compounds con- taining Cr51 have been used in research, diagnosis, and therapy. These uses of radio-chromium result in exposure of animal tissues even though large quantities are not used. Colloidal chromic phos- phate has been used in therapy of blood diseases (Fields and Seed, 1957) and as a reticulo-endothelial function test (Gabrieli, 1951). Hexavalent Cr51 has found application in the diagnosis of hemolytic anemias (Korst 9:31., 1955), measurement of red cell volume (Sterling and Gray, 1950) and half life (Read et a1., 1954), location and quantitation of intestinal bleeding (Owen £3.31." 1954) and as a tag for leucocytes (McCall 3131., 1955). Trivalent chromic chloride is used for measuring plasma volume (Frank and Gray, 1953). Although toxicity and tissue distribution of chromium com- pounds is reasonably well documented in mammals, there is little information available on routes and physiological mechanisms involved in the excretion of chromium once it gains entrance to ani- mal tissues. For this reason a study of the following aspects of chromium excretion was made: (1) the quantitation of excretion of trivalent and hexavalent chromium in urine, bile, and feces after intravenous injection; (2) plasma binding of chromium in vivo; (3) physiological mechanisms of chromium excretion by the kidney; (4) reduction of hexavalent chromium in vivo; and (5) investigation of the excretion of ”bound” chromium. Mongrel dogs were used in all experiments. Investigation of the routes and relative amounts of chromium excreted by each route were done in two ways: (1) acute experiments on anesthetized dogs in which the ureters and bile duct were cannulated, and (Z) experi- ments on unanesthetized dogs with bile fistulas. Determination of plasma binding and renal mechanisms involved in chromium excre- tion were done by dialysis and the renal clearance technique. An- alysis for hexavalent chromium, paper chromatography, ion exchange, and dialysis procedures were used to elucidate chromium reduction and the possible excretion of "bound" chromium. LITERATURE RE VIE W Inorganic Chemistry of Chromium For didactic reasons a paragraph on the valence states of chromium is presented here. Chromium is a member of Group VI of the periodic table and is characterized by its ability to serve both as a metal and as a nonmetal. Generally it behaves as a metal at lower valences while the characteristics of an acid grow stronger as the valence is in— creased. Chromium forms compounds in which it is divalent, tri- valent, or hexavalent. In both the trivalent and hexavalent state it forms two groups of compounds which differ in degree of hydration. Consequently the following five classes of chromium compounds exist: (1) chromous (basic), (2) chromic (weakly basic and amphoteric), (3) chromite (feebly acidic), (4) chromate (acidic), and (5) dichromate (acidic) (Hopkins, 1942). In addition. univalent and pentavalent chro- mium are encountered in certain reactions, but only the trivalent and hexavalent states exhibit sufficient stability to exist in biological systems. Of these two, trivalent is more stable than hexavalent at pH's below 7. Hexavalent chromium, when dissolved in water, exists in true solution regardless of the hydrogen ion concentration or the presence of other ions. However, trivalent chromium, when added to water, exists in solution as a complex or as a colloidal or flocculent pre- cipitate depending on the pH and dissolved substances. The chem— istry associated with solvation of trivalent chromium is complex and remains in part theoretical. A brief resumé of the interactions of chromium with aqueous solvents is pertinent (Gustavson, 1956; Udy, 1956). When chromic chloride is dissolved in water, hydration or coordination occurs so that the complex [Cr(HZO)6]+3 + 3Cl- is formed. This simple hydration does not satisfy the binding forces of the chromium atom; consequently. one or more oxygen atoms are drawn within the coordination sphere of the chromium atom. The oxygen atom of the water molecule becomes positively charged and so it releases a proton (H+). The net result is that the chromium cation formed has an ionic charge of +2 instead of +3. H +3 +2 [Cr(HZO)6] C13 # [Cr (H20)5] C12 + HCl Protolysis or hydrolysis, as this process is called, is favored by the removal of hydrogen ions from the system. The tendency of the chromium atom is to attain the electronic structure of krypton. This can be accomplished by further hydrolysis so that chromium complexes with charges of +1, 0, -1, -2, and -3 are produced by the successive hydrolysis if protons produced by the reaction are removed. In addition to water, trivalent chromium readily forms coordinate complexes with a large number of neutral or charged groups, some of which are NH3, (N03)-l, CZHSOH’ N02, (OH)-l, and (504)-2 (Udy, 1956). These complexes can also undergo succes- sive hydrolysis. A further complication in chromium chemistry is that divalent chromium cations produced by a single hydrolysis may interact to form a diol with two chromium atoms per molecule and an ionic charge of +4. Z[Cr OH(H 0) 1+2 c1 g [(H 0) Cr/ Cr (H 0) ]+4 c1 + 2H 0 z 5 z z 4 \OH/ 2 4 4 z The addition of one mole of sodium hydroxide per mole of chromium hydrate results in a quantitative formation of the diol. More sodium hydroxide results in large polynuclear complexes containing great numbers of chromium atoms in each molecule. Finally. if protons are removed as they are formed, the chromium complexes become so large that they can no longer be accommodated by the solvent and precipitation occurs. Thus the solvation of chromium can result in a conglomeration of chromium molecules varying greatly in size and ionic charge. Nature of Chromium-Protein Bond The tanning of leather with chromium salts has led to exten- sive investigation of the interaction of chromium complexes with collagen and gelatin. Bowes and Kenten (1949) demonstrated the importance of carboxyl groups in the fixation of chromium by pro- teins. Esterification of these groups led to decreased fixation of chromium by proteins. Strakhov (1951) reported that collagen binds more chromium than silk fibroin presumably because it has more free carboxyl and amino groups. An e—capralactam polymer, devoid of amino and carboxyl groups, was unable to fix chromium at all. By treating collagen with formaldehyde which blocks free amino groups, the initial reaction of chromium in cationic form was shown to be with carboxyl groups, while anionic chromium was found to react first with amino groups. Early theories of the possible mechanism involved in binding chromium to proteins are reviewed by Shuttleworth (1950) who found 154 articles worthy ‘of mention. While no single theory explains all the data or is accepted by all leather chemists, Gustavson's (1949, 1956) concept of the formation of chelate compounds is attractive. According to this concept, the initial reaction is an ionic attraction +2 of cationic chromium complexes such as [CrZ(OH)ZSO4] with the charged carboxyl groups of collagen represented by COO--P'NH3+ , the sulfate ions being compensated by the NH3 ions. Carboxyl groups, having a great tendency for complex formation and for a direct attachment to chromium, penetrate into the coordinate sphere forming a covalent-coordinate bond. Since several chromium atoms are present in large chromium complexes, there is the possibility of a multipoint interaction of a chromium complex with several carboxyl ions of the collagen lattice resulting in the linking of ad- jacent protein chains by strong bonds by means of the chromium bridge. That this view of the nature of the chromium-protein bond may be incomplete is shown by Green's (1953) observation that hydroxyl and amino groups as well as carboxyl groups of proteins are involved in chromium fixation. He found that increased acetyla- tion of free amino or hydroxyl groups of collagen decreased fixation of either cationic or anionic chromium complexes. The Binding of Chromium by Tissue Proteins Brard (1935) first pointed out the selective partition of chro- mate between erythrOCytes and plasma proteins. However, Gray and Sterling (1950) investigated the fundamental aspects of the bind- ing of chromium to proteins and erythrocytes. They demonstrated that erythrocytes have a marked affinity for anionic chromate. When small quantities of sodium chromate were incubated with a saline suspension of erythrocytes, over 97 per cent of the chromium was taken up by the red cells and a semilogarithmic plot of the uptake indicated a first order reaction. Under their experimental conditions the uptake half-time of chromium was 38 minutes. The constant for this reaction was approximately 0.02, indicating that about 2 per cent of the chromium remaining in the saline was bound each minute. The large uptake and the exponential nature of the reaction suggested that chromium was bound or modified after gaining entry into the cell. This aspect was investigated by lysing red cells tagged with Cr51 labeled chromate and comparing the counts of each fraction with that of the intact cell. Hemoglobin was found to contain 97 per cent of the activity of the red cells, while only 2 per cent of the ac- tivity was in the washed stroma. This indicates that chromium after gaining entry into the red cell is bound to hemoglobin rather than changed to an indiffusable melecule. Fractionation of the hemo- globin into globin-HCI and hemin showed that chromium is bound to the globin and not to the heme. The firmness of the chromium- globin bond was indicated by noting that the addition of nonradioactive chromate to a Cr51 tagged red cell suspension did not result in elution of the tag from the red cells. This observation also shows 10 that chromium binding is essentially irreversible or at least that the equilibrium is largely in favor of the bond. As further evidence of the firmness of the chromium bond it was shown that continuous shaking of a tagged red cell suspension for periods up to 43 hours resulted in little loss of the tag to the saline diluent. Trivalent chromium binding was, also studied by Gray and Sterling. Chromic chloride when injected intravenously in dogs did not enter the erythrocytes but remained in the plasma for a consid- erable time suggesting protein binding. This was verified by study- ing the binding of trivalent chromium by albumin. Radioactive chro- mic chloride was incubated with crystalline bovine albumin and the percentage bound determined by dialysis. At low molecular ratios of chromium to albumin (1:1) 70 per cent of the chromium was bound, while at higher ratios (100:1) only 40 per cent binding oc- curred. The firmness of the chromium-albumin bond was indicated by prolonged dialysis which resulted in little or no further loss of chromium from the albumin, and by the addition of nonradioactive chromium to tagged albumin, with the result that only a small loss occurred on dialysis. As with the tagging of erythrocytes with chromate, the binding of trivalent chromium to albumin is essentially irreversible or the equilibrium is greatly in favor of the bond. 11 Gray and Sterling suggested that hexavalent chromium binds to hemoglobin within the red cell only after becoming reduced to the trivalent state. Several facts can best be explained by this hypothesis. They showed that under identical circumstances tri- valent chromium would bind to albumin to a considerably greater extent than would hexavalent chromium. Also trivalent chromium was much more effective in tagging cell-free hemoglobin. A reason- able explanation of these observations was that hexavalent chromium diffused into the erythrocytes was reduced to the trivalent state and bound to hemoglobin. The reason greater binding occurred within the cell than with cell-free hemoglobin may have been that the chromium, once reduced, could not leave the red cell and so re- mained in close proximity with the hemoglobin. The small amount of hexavalent chromium bound to albumin may be hexavalent chro- mium that had undergone reduction. The work of Grogan and Oppen- heimer (1955) lends support to this concept. They were unable to demonstrate the binding of hexavalent chromium to egg albumin or human plasma proteins by paper electrophoresis at pH 7.35. In dialysis experiments at pH 4.14, where hexavalent chromium is less stable, considerable binding of hexavalent chromium could be dem- onstrated. However, at hydrogen ion concentrations above pH 7 all hexavalent chromium could be dialyzed away from the protein by 12 changing the dialyzate once. Grogan and Oppenheimer suggest that the binding of anionic chromium at higher pH's is electrostatic in nature. Nechels et al. (1953) were interested in using radioactive sodium chromate for the study of the survival of red blood cells in vivo and so investigated the stability of the chromium tag. They found that erythrocytes tagged with radioactive chromate lost ap- proximately 1 per cent of their remaining activity each day. In addition, they reported that hexavalent chromium even in large quantities had little detrimental effect on erythrocytes. They found that whole blood underwent 0.4 per cent hemolysis in four days while the addition of 30 pg. chromium per milliliter of blood in- creased this hemolysis to only 0.6 per cent. Sixty pg. chromium per ml. blood caused only a slight additional increase in hemolysis. The ease with which hexavalent chromium undergoes reduc- tion to the trivalent state is indeed surprising. MacKenzie (1957) introduced radioactive hexavalent chromium by stomach tube into two groups of rats, one starved and the other fed. After four hours blood was collected, centrifuged, and the plasma and erythrocytes counted separately. In the starved animals each m1. of plasma con- tained 4.8 times as much activity as an equal quantity of packed red cells. In the nonstarved animals the plasma contained 8.8 times as .S‘ .. 43 AL’ ,LE _ 13 much activity as an equal volume of erythrocytes. The experiment was repeated using radioactive trivalent chromic chloride; here es- sentially all the activity was in the plasma. In order to interpret these results it is necessary to assume that ingested hexavalent chromium will tag erythrocytes while trivalent chromium will bind to plasma proteins. If this is true then the greater part of the hexavalent chromium taken orally is reduced in the alimentary canal or during the absorptive process. This experiment also shows that food in the digestive tract will cause reduction of hexavalent chro- mium. In another experiment the chromium was injected directly into the duodenum. Much less reduction of hexavalent chromium was found as shown by the increased quantity of chromium tagged to the erythrocytes. This would indicate that considerable reduction of hexavalent chromium occurs in the stomach. Since hexavalent chromium is least stable in an acid medium considerable reduction would perhaps be expected here. Cunningham 3111.. (1957) noted that the addition of sodium chromate to an acid citrate dextrose solution before the addition of red cells resulted in comparatively poor erythrocyte labeling. Fur- ther investigation revealed that labeling was adversely affected by citrate, dextrose, decreased- pH, and by exposing the acid citrate dextrose and sodium chromate mixture to light. Inquiry into the 14 nature of this change by spectrographic analysis for trivalent chro- mium established that chromate was undergoing reduction to the chromic state. Grogan and Oppenheimer (1955), by adding large quantities of chromium to egg albumin. were able to observe by color changes the reduction of hexavalent chromium. Reduction readily occurred below pH 6.4 but not above in these solutions. Absorption, Distribution, and Elimination of Chromium Absorption of chromium compounds from the digestive tract depends greatly on the chromium compound present. Brard (1935) found considerable absorption of chromates that were introduced by stomach tube. Conn 21.11. (1932) found little absorption of chromic phosphate in milk fed to rats as indicated by the trace quantities present in tissues and excreted in the urine. MacKenzie (1957) fed rats small quantities of radiochromate by stomach tube and found a maximum of 5.5 per cent chromium present in the tissues or ex- creted in the urine in seven days in starved animals. In nonstarved animals less than half this quantity was recovered in the urine and tissues. By comparing the tissue levels of two groups of rats fed 25 p.p.m. sodium chromate or chromic chloride for one year he concluded that five times more hexavalent than trivalent chromium 15 was absorbed. In another experiment in which rats were fed vari- ous quantities of chromate in their drinking water for periods of up to one year he noted that levels of chromate below 5 p.p.m. resulted in little accumulation, whereas between 5 and 10 p.p.m., an appre- ciable increase in the rate of accumulation occurred. The absorption of chromium compounds through the respira- tory tract has not been studied, but that some absorption may occur was indicated by the tissue and urinary levels of chromium found in chromate workers (USPHS, 1952). Some chromium compounds are probably absorbed through the skin. White (1934) reports of twelve deaths that followed the use of an antiscabetic ointment in which a chrome preparation was substituted in place of sulfur. The fate of chromium compounds when intravenously injected has been studied by Kraintz and Talmage (1952). They injected rabbits and rats with Cr51 labeled chromic chloride in acetate buf- fer, sacrificed the animals at intervals of up to twenty-four hours, and counted various tissues. At one hour after injection the kidney showed the highest activity per gram of wet tissue followed by the blood, bone, and liver, each with the same activity. The spleen and muscle contained the least chromium. After twenty-four hours the activity in the liver and blood decreased, while that of the spleen I“ A .- “\ ) . . . . . “‘5'” _ . _ : 7, -Ig 7.7 7 Iii-11ft . .. l . ,‘7. ' - i . 1 2| . - R! r I, V‘ I ' _. 7‘ "I , - r' .7: _.. ‘_ .' * 16 and bone increased. Forty per cent of the injected dose was ex- creted in the urine but none in the feces in twenty-four hours. Treatment of the plasma of one animal with trichloro-acetic acid indicated that 80 to 90 per cent of the chromium was associ- ated with the precipitated plasma proteins. Saline extracts of liver and spleen homogenates contained 40 per cent of the radioactivity, 80 to 90 per cent of which was precipitated with trichloro-acetic acid. Visek 33:11. (1953) used a method similar to that of Kraintz and Talmage to study the metabolism of chromium compounds. They employed two hundred rats sacrificed at intervals of up to forty- three days after injection. Further, they correlated the electro- phoretic behavior of each chromium compound present in serum with . . 51 its tissue distribution. Of the total sodium chromite (NaBCr 02) injected, which was colloidal in serum, 90 per cent appeared in the liver and tissues of the reticulo-endothelial system. Chromic chlo- ride also behaved as a colloid in serum and its distribution in liver, spleen, and bone marrow reflected reticulo-endothelial accumulation. The reduced uptake of chromic chloride compared to sodium chro- mate that they observed was probably due to its binding to plasma proteins. Chromic chloride buffered by either acetate or citrate existed in the serum as a complex and was not found in high 17 concentration in any organ. Sodium chromate was ionic in serum and its distribution pattern resembled the buffered chromic com- pounds. The excretion values for each compound in urine and feces respectively were as follows: colloidal sodium chromate 0.6 and 1.6, colloidal chromic chloride 15 and 20, chromic chloride complexed with citrate 75 and 17, and ionic sodium chromate 35 and 17 per cent of the injected dose excreted in four days. The low excretion values for chromate, which was ionic in serum, and therefore should be ex- creted rapidly, was attributed to its binding by erythrocytes. These results on the metabolism of chromium compounds are in general agreement with the concept that the physical and ionic states of chromium in plasma largely govern its tissue distribution and rate of excretion. Chromium present in the serum in particulate form is rapidly taken up by reticulo-endothelial tissues and so is available for excretion. Additional support is lent to this concept by the work of Brauer, Holloway, and Long (1957). They reported that 75 per cent of a colloidal chromic phosphate preparation was re— moved from the blood in a single circulation through the liver. Presumably the colloidal particles of the chromic phosphate were larger than those encountered by Visek _e_t__al. and so were removed more rapidly. 18 Little additional information exists on the excretion of chro- mium compounds other than that already discussed. Edmunds and Gunn (1928) state that chromic acid and its salts seem to be ex- creted through the kidneys and probably to a less extent through the intestinal epithelium. These authors also report that the metal oc- curs in urine in part in organic combination. However, no data or references were presented to substantiate this statement. Mancuso and Hueper (1951) are of the opinion that chromium may be ex- creted through the skin and) intestinal mucosa since chromium was found in the hair and feces of a chromate workman whose last ex- posure to chromium was three years preceding his death. Elimina- tion of chromium through the urine, bile, stomach, and intestinal mucosa of dogs poisoned with sodium chromate or chromic chloride has been reported by Brard (1935). Renal Clearances Ludwig in 1844 suggested, purely on anatomical considerations, that urine formation begins as a passive process of filtration of a protein-free filtrate at the glomerulus. However, eighty years elapsed before a quantitative measure of filtration rate was made. Rehberg (1926) measured urine volume and the concentration of creatinine in plasma and urine after orally ingesting creatinine. 19 By appropriate calculation, not unlike the renal clearance formula used today, he derived the volume of plasma in milliliters per min- ute necessary to provide the quantity of creatinine excreted in the urine each minute. Moller, McIntosh, and VanSlyke (1929) justly deserve credit for using the term "clearance" and for presenting the clearance formula and concept clearly. They defined urea clear— ance as the volume of blood cleared of urea by one minute's excre— tion of urine (UV/ B). No attempt was made to explain urea clear- ance in terms of any particular process of the kidney since they were interested primarily in quantitatively comparing the capacity of normal and diseased kidneys to excrete urea. Jolliffe and Smith (1931) extended the term clearance. to the excretion of creatinine. Since then it has been widely used to describe the excretion of a large variety of substances. The next step in the quantitation of renal function was to find a substance excreted only by glomerular filtration. No one experi- ment has ever been devised to establish that any given substance is filtered but not reabsorbed or excreted by the kidney tubules. However, it has been concluded beyond all reasonable doubt that inulin clearances in vertebrates and creatinine clearances in the dog measure glomerular filtration (Smith, 1951). 20 Concurrently with the search for substances that measured glomerular filtration, compounds which would measure other aspects of renal function were sought. The work of Shannon (1935); Goldring, Clarke and Smith (1936); and Smith, Goldring, and Chasis (1938) sug- gested that phenol red might be a measure of renal plasma flow and tubular function. Subsequently, diodrast and then p-aminohippuric acid (PAH) as the sodium salt have come into use for the measure- ment of renal plasma flow and tubular excretion. Data for glomerular filtration rate and renal plasma flow in an animal or between animals of the-same species show consider- able variation. Houck's (1948) statistical analysis of seventy-five normal unanesthetized female dogs gives a mean and standard devi— ation of 84 :t: 19.1 ml. per min. per sq. meter surface area for creatinine and 266 :I: 66 ml. per min. per sq. meter surface area for PAH clearances. Russo Et__a_l. (1952) also using female dogs gives slightly different figures. For creatinine 95 per cent of all observations in their study could be expected to fall within the limits of 94 :I: 36 ml. per min. per sq. meter surface area and for PAH, 238 :1: 133 ml. per min. per sq. meter surface area. The standard deviation of a single determination on any given dog on any one occasion was 9 ml. per min. for creatinine and 32 ml. per min. for PAH expressed per sq. meter surface area. 21 Anesthesia alone or used with surgical procedures is fre- quently employed to facilitate the study of renal function. Corcoran and Page (1943) found that sodium pentobarbital anesthesia (30 mg./ kg.) produced no consistent change in inulin and diodrast clearances or tubular excretion of diodrast in dogs. Glauser and Selkurt (1952) reported that pentobarbital or barbital anesthesia suitable for sur- gery of five to six hours' duration has no effect on glomerular fil- tration but did reduce renal plasma flow and tubular excretion of PAH. Habif SLE' (1951) compared the renal clearances of creatin- ine, PAH, sodium, potassium, and chloride before and during anesthesia with ether, cyclopropane, or thiopental; and during major surgery. Anesthesia depressed all clearances but the surgical pro- cedures had no additional effect. METHODS I. Routes of Chromium Excretion The objectives of experiments discussed in this section were twofold: to investigate the quantity of chromium excreted in urine, bile. and feces, and to‘ compare the excretion of trivalent and hexa— valent chromium by each of these routes. Acute measurements on anesthetized dogs gave information on the relative rates of chro- mium excretion in urine and bile, while prolonged experiments on dogs with bile fistulas were used in order to evaluate chromium excretion in feces as well as urine and bile. General procedure Seven healthy male and female mongrel dogs weighing be— tween 7.5 and 15 kg. were used in these experiments. Each animal was housed in an individual cage with a constant supply of food (Borden's Chunx) and water. Trivalent chromium solutions for administration to these dogs were prepared from stock Cr51C13 that ranged in specific activity from 38 to 47 curies per gram. For dogs receiving small quanti- ties of chromium the stock solution was diluted with five parts 22 a.“ A” Z3 physiological saline and the acidity adjusted to pH 3 to 4 at least one day before use. If a large dose of chromium was to be given 51 . . 52 . . Cr C13 was diluted w1th stock Cr Cl3 solutlon and made isotonic by adding the proper quantity of sodium chloride. These solutions were also prepared a day in advance of use and always had a pH of 3 to 4. The reason for aging trivalent dosing solutions before 5 . use was to allow time for the Cr lCl3 to reach ionic stability. Concentration, pH, and other ions present all affect complex forma- . . 51 52 tion of trivalent chromium so that Cr Cl3 added to Cr C13 does not immediately have the same ionic composition as the latter. 51 5 Isotopic Na Cr 04 was prepared by oxidizing Cr 1Cl3 with hydro- 2 gen peroxide in alkaline solution (see Appendix B). The method of preparation of hexavalent chromium solutions for injection was the 52 . same as for trivalent solutions except that Na Cr 04 was used in 2 place of Cr52Cl3 for large doses and the pH was adjusted to 7. The activity of Cr51 in the dosing solutions described above was between 1000 and 1500 pc. per dose. The isotope-counting'apparatus consisted of Nuclear Corpora- tion's scintillation crystal detector (model D—55), scintillation counter (model 183), and radiation analyzer (model 1810). The minimum de- 51 . . tectable activity of Cr with this apparatus in terms of microcurles required to give a count equivalent to background (approximately 35 I‘ll (If Ill-(fig Z4 c.p.m.) was 4.4 x 10.3 IJ.C. for a five-volt window width setting counted on the middle shelf. Urine and bile samples for counting were pipetted in 4 or 5 ml. quantities into disposable aluminum culture dishes and counted without additional preparation. Fecal samples were well mixed with water to a thin paste consistency and 4 or 5 gram quantities placed in the culture dishes for counting. No correction for self absorption was used or necessary, since all samples and standards from any given dog were counted at the same volume and geometry. The counting of a weight rather than 3 vol- ume of feces would result in a small counting error but this was ignored. Standards were prepared in duplicate by making a dilution of the injection solution whose concentration was known and counting an aliquot. From the activity and the chromium concentration of the standards the chromium content of urine, bile, and feces could be calculated. Acute experiments on anesthetized dogs Five dogs were used in this section of the experiment. Food was withdrawn from each dog the day prior to use. Each animal was anesthetized with 30 mg. sodium pentobarbital per kg. body weight and the ureters cannulated with the proper size polyethylene tubing through a midline abdominal incision. The common bile duct 25 was similarly cannulated and a serrefine clamp placed on the cystic bile duct. Radioactive chromium was injected into one of the brachial veins, the time recorded, and urine and bile quantitatively collected in 10 ml. graduates at one-half-hour intervals for four hours. Small urine and bile samples were diluted to 6 ml. to provide an adequate volume for counting. Prolonged experiments on bile fistula dogs During the course of a year eleven dogs were operated on to establish functional bile fistulas. Each animal was anesthetized with 30 mg. per kg. sodium pentobarbital and prepared for aseptic sur- gery. The duodenum was located and the rostral end packed off through a ventral midline incision. A small cut was made in the duodenum about one inch posterior to the pylorus, the ampula of Vater located, and either polyethylene or rubber tubing inserted ap- proximately two inches into the common bile duct. The duodenal incision was closed with interrupted Lambert stitches and the can— nula imbedded in the outside surface of the duodenum caudal to the wound with three of four additional stitches. Where the cannula left the gut a purse-string suture was placed for additional insurance against slipping. The free end of the tubing was brought to the 26 outside through a stab wound and sutured to the skin. The operation was completed by closing the midline incision and taping the cannula to the abdomen. When not on experiment, the bile fistula was closed so that bile could flow around the tubing and into the duodenum. Two of the eleven dogs died within twelve hours after the operation but the remainder recovered rapidly. Polyethylene tubing was used as cannula material in six of the surviving dogs with poor results. These cannulae invariably pulled away several days to two weeks after surgery. Number 8 Fr. nasal catheters were used in the three remaining animals with excellent results. Two of these dogs were placed on experiment about two weeks after surgery but the third one had a severe biliary infection when operated on (due to a previous unsuccessful cannulation) and was not used. Just before intravenous chromium injection the bladder of each dog was catheterized, the catheter taped securely to the animal, and a balloon of 200 ml. capacity tied in place. The bile fistula was opened and a similar balloon tied to it. Bile and urine were col- lected at two, four, eight, sixteen, and twenty-four hours and for the next three days after injection. Because of its size the balloon on the urine catheter had to be changed several times each day for the daily collection periods. Feces were collected after volitional 27 evacuation by the dog and each day's excrement added to separate bags containing a solution of phenol in water. 11. Mechanisms Involved in the Renal Excretion of Chromium The purpose of experiments discussed in this section were to (1) evaluate the effects of chromium on kidney function, (2) in- vestigate which aspect of nephron function was most prominent in excreting chromium, and (3) establish a renal clearance for chro- mium. Renal clearance procedure Eleven mongrel dogs weighing 14.5 to 25 kg. were used in the renal clearance measurements. Food was withdrawn from each dog at least twelve hours prior to the experiment. Water was ad- ministered to some of the animals by stomach tube to insure ade- quate urine flow. Sufficient sodium pentobarbital was given intra- venously to produce light anesthesia. At no time was more than 25 mg. per kg. of anesthetic necessary to produce the desired ef- fect. In experiments of long duration additional doses of 3 to 5 mg. per kg. pentobarbital were given as necessary when the dog began to show excessive movement. The corneal, palpebral, and paw- pinch reflexes were present at all times in all dogs under this 2.8 anesthesia. Sodium pentobarbital anesthesia is known to cause a lowering of body temperature. Furthermore, a change in body tem- perature affects renal function (Grant and Medes, 1935; Smith, 1939- 40); for this reason two heating pads were placed under each dog on experiment and the body temperature, determined rectally, was maintained at 101° to 102.5° F. Creatinine (0.5 per cent) and 0.25 per cent PAH (para-amino- hippuric acid) were infused through a brachial vein with a Sigmamotor infusion pump. The rate of infusion (1.5 to 3.0 ml./min.) varied with the size of the dog but was maintained constant in any one ani- mal. After the infusion was started a priming dose of creatinine (5.0 per cent) and PAH (1.2 per cent), calculated to establish the appropriate plasma level of each was administered intravenously. Preparation of the chromium solutions for dogs receiving a single injection was the same as in the previous section. When chromium was administered by infusion it was simply added to the creatinine-PAH infusion solution. Urine samples were collected by an indwelling catheter. The bladder was rinsed by instilling and then withdrawing and discarding 2.0 cc. water with the aid of a 50 cc. syringe attached to the cathe- ter. The rinsing process was repeated a total of three times. After the last rinse was withdrawn. 30 cc. of air was introduced {filial-III ill- ‘llll’lll’ llllllll’lllllllllll 29 into the bladder and aspirated in order to remove the last wash-out fluid. Urine collection periods usually lasted about thirty minutes but this was variable. Two minutes before the end of a collection period the bladder was emptied and the process of rinsing and flushing the bladder repeated. These rinse fluids were collected and added to the urine sample. Urine from dogs Nos. 8 to 13 was col— lected in 200 ml. volumetric flasks. The volume of urine plus wash was then determined by measuring the amount of water necessary to fill the flask to the mark. For the last five dogs urine was collected and measured in a graduated cylinder. Blood samples were drawn from a jugular vein into a hepa- rinized syringe. The total number of blood samples drawn from any one dog was usually equal to one more than the number of urine col- lection periods. These were drawn at approximately equal time intervals throughout the experimental period. Blood samples were centrifuged and the concentration of total chromium, dialyzable chro- mium, creatinine, and PAH measured on the plasma. Dialysis procedure An equilibrium dialysis procedure was used to determine the plasma level of dialyzable chromium. This method was applicable because Gray and Sterling (1950) had shown that binding of 30 chromium to plasma proteins was essentially irreversible; that is, once chromium became bound it could not be dialyzed off the plasma proteins. The dialysis procedure is described below. Visking di- alysis tubing (20/32 in. inflated diameter) was wetted and two knots tied in one end. Five ml. of plasma whose radioactive count was known were placed in the sack and the other end tied with two knots. The sack was placed in a test tube and covered with 7 m1. physio- logical saline. Sacks were prepared for each blood sample withdrawn from the dog. Diffusion equilibrium was allowed for two days at 4° C.; then 5 m1. of the dialyzate was counted. This count was cor- rected for background, decay, and to 1 ml. volume, then multiplied by 12/5 to give the count equivalent to 1 ml. dialyzable chromium in the original sample. The factor 12/5 represents the correction nec- essary because 5 m1. of dialyzable chromium in the plasma was diluted to a total volume of 12 ml. Creatinine and PAH procedures Creatinine and PAH clearances measured glomerular filtration rates (GFR) and effective renal plasma flows (ERPF) respectively. Plasma creatinine and PAH levels were determined on a trichloro- acetic acid filtrate (Greenwalt, as modified by Kennedy _e_t__al., 1952). Creatinine determinations on diluted urine and plasma filtrates were 31 done by the alkaline picrate method (as modified by Brod and Sirota, 1948). PAH was determined by the method of Smith 313.1: (1945). Creatinine and PAH analysis were done in duplicate but only one protein-free filtrate was prepared for each plasma sample. Methods for these chemical analyses are given in detail in Appendix B. The plasma concentration in‘ the clearance formula (UV/ P) was determined by plotting the plasma level of total chromium, di- alyzable chromium, creatinine, or PAH versus time on coordinate graph paper and selecting the plasma value interpolated to coincide with a point three minutes before the midpoint of the urine collection period (Figure l). The choice of a plasma value three minutes be- fore the midpoint of the urine collection period is arbitrary and compensates for the time required for urine to flow from the nephron to the bladder. Renal clearances in dogs are almost universally expressed per one square meter surface area. The nomogram found in Smith (1956) was used to estimate surface area of dogs in this experiment. This nomogram is based upon body weight in kg. and length from nose to anus measured over the belly in centimeters. 32 603mg cofioozoo oer“: mo EBQEE wagon $558 wont to cofimficoocoo gamma oEEnmwoo 09 com: pogo—Z .H muzmfim :ofiomfis 5380.30 new? mowssflz 5 egg. 03 one a: 8m 3m 3: on” 3 o J a q q d 4 q u m s o m. w m N H x. Till 1+1 Tali .1111 ‘f’ ‘/ I l ‘3. mm. I¢IITO/ lmfio. D . - 5 .58. lmNo. lvoo. loco. 1 do. loll; 0'..T_o . . 18. J0 _ _ A (g 50. .«EOQEE 98qu .58 moEHH cornea cofioofioo o:ED* Creatinine PAH (mg/ml) (mg/mm Dialyz able Cr (Hg/m1.) Total Cr. (Hg/ m1-) 33 111. Some Physical and Chemical Properties of Chromium in Biological Fluids Experimental procedures presented here were intended to compare some of the physical and chemical properties of chromium in biological fluids to its characteristics before injection. The pos- sibility that chromium could be excreted in organic combination as Edmunds and Gunn ( 1928) report was a directing influence in the choice of the following experimental methods. Dialysis procedures The equilibrium dialysis procedure, already described for use on plasma, was used without change on urine samples from dogs 14 and 15 to measure percentage of dialyzable chromium. A rate dialysis procedure was used to compare the diffusion properties of Cr51 excreted in urine to that of Cr51(+6) added to normal urine. Twenty ml. of a sample whose rate of diffusion was to be measured was placed in a sack made from dialysis tubing (Cenco, one inch diameter) and dialyzed against 2000 ml. sodium chloride solution made isotonic with the sample as determined by freezing point depression measurements. Two stirrers, one in the sample and one in the dialyzate, assured constant agitation of both liquids. At frequent intervals 2 ml. samples were removed, counted, N ’ a ‘< ' ° "$1 "’m".. I . {III II} ‘Ilul'llll llcllllw 34 and returned to the dialysis chamber. Counts per minute per 2 m1. corrected for background were plotted on the ordinate of semiloga- rithmic graph paper versus time on the abscissa and a line drawn through the points. The half-time (tl/Z) was measured from the line and the diffusion rate expressed as the constant k (k = .693/ t1/2)' Diffusion rates as defined above were made on the following samples: (1) urine from a dog injected intravenously with 8.70 pg. Cr51(+ 6); (2) Cr51 (+6) added to salt solution; (3) urine from a dog injected with 9.81 pg. Cr51 (+6); (4) Cr51(+ 6) added to normal dog urine and dialyzed immediately; and (5) Cr51(+ 6) added to normal dog urine and dialyzed after five hours. Measurements were made in the order listed and the same dialyzing bag employed so that re- sults could be compared. Reduction of hexavalent chromium The ease with which chromates are reduced in vitro suggests that reduction also may occur in vivo. In order to determine if re- duction took place a 10 kg. male dog was injected intravenously with 40 mg. NaZCr5 1O4 and urine samples immediately analyzed for hexa- valent and trivalent chromium. The Saltzman (1952) reaction (see Appendix B), but without previous ashing of samples, was used for \ 35 qualitative analysis for hexavalent chromium While total chromium was determined by radioassay for Crsl. Schiffman (1957) showed that ashed and nonashed samples of chromate in water gave essen- tially the same results in this reaction. To test the reducing action of urine in vitro, 100 or 200 pg. quantities of sodium chromate were added to 100 ml. of stored or fresh urine and 5 m1. samples ana- lyzed at intervals for the presence of hexavalent chromium. Ion exchange procedure Ion exchange columns were prepared by drawing out one end of 8 mm. I.D. glass tubing, placing a disk of glass wool in the bot- tom. and washing in sufficient resin to form a cylinder 8 cm. high. The resins used were Amberlite IR-120 (cation exchange) and Dowex 1-X100 (anion exchange). Urine, a plasma dialyzate. and Cr51 infu- sion solution from dog 18 were counted. run through the resin at a rate less than 0.5 ml. per minute and recounted. The fluids men- tioned above plus bile from dog 4 were also subject to ion exchange separation. Chromatographic procedure 5 Urine, Cr 1(+ 3) infusion solution, and a plasma dialyzate from dog 12 were used in the following procedure. Capillary tubing drawn to a fine point was used to apply each of the chromium D . 'v ,_ ""31 IMWMJ‘TF : .'.' 1- . u .- . . - 36 solutions in a fine line to strips of Whatman No. 3 mm. filter paper (3.8 by 46 cm.) then the strips were dried under a heat lamp. It was necessary to repeat this process several times in order to have sufficient Cr51 present to count. The solvent used to obtain separa- tion consisted of n-butanol: water: glacial acetic acid: acetoacetic acid ester in the proportions 50:35:10:5 respectively. The ends of the paper strips were dipped in the butanol phase of the solvent, while the aqueous phase was used to saturate the chromatographic chamber. Descending separation was allowed for twenty-four hours, and the strips quickly dried in an air oven at 70° C. The chromatograms were then analyzed to locate the position of Cr“, ninhydrin-positive substances, creatinine, and PAH. The migration of Cr51 on the strips was determined by means of the scintillation detector and radiation analyzer previously used connected to a count rate meter (model 1620) and Esterline Angus recorder (model AW). The recorder was used to drive an actograph (model C-100) which moved the strips under the scintillation detector. To restrict scanning to a narrow band of the paper a lead shield 13 mm. thick with a slot in the middle (3.5 by 3.8 mm.) was placed between the paper and detector. The filter paper strips were moved past the detector at a rate of 0.75 inch per minute, and the peak ac- tivity used in calculating Rf values. 37 Ninhydrin color was developed by spraying the strips with 0.25 per cent ninhydrin in butanol and heating for 15 minutes in an oven at 70° C. This reagent also produced a pink color with PAH. An orange color indicative of creatinine was developed by spraying the filter paper first with 0.75N NaOH, then with 0.4M picric acid. Chromium precipitation procedure Normal urine and heparinized blood were collected from a 19 kg. dog; then 13.4 mg. Cr5 lCl3 was injected intravenously and urine and blood again collected. The blood samples were centrifuged and the plasma dialyzed as previously described. To the normal urine and dialyzate sufficient Cr51Cl3 injection solution in anionic form was added so that their activity was approximately equal to the samples collected after the dog received the radioisotope. To 7 ml. portions of the two urine and two dialyzate samples an equal quan- tity of CrszCl3 in anionic form was added. Chromium in CrCl3 solutions is present almost entirely in cationic form. Conversion to the anionic form was done by adjusting the acidity of such solutions to pH 5 or above which favors hydroly- sis, allowing them to stand overnight and absorbing any cationic chromium remaining with amberlite IR-120. The reason for adding CrfizCI3 to the urine and dialyzate samples was to provide sufficient 38 chromium so that a visible precipitate would form upon the addition of ammonium hydroxide. A brief discussion of precipitation of metals with ammonium hydroxide is given in Willard and Diehl (1950). Two ml. 1 N ammonium hydroxide were added to each preparation, samples were removed for counting, and the remainder centrifuged at 3000 r.p.m. for one-half hour in an International centrifuge (model SBV). The supernatant was then counted and the quantity of precipitated Cr5 1C13 in each sample calculated. The or- ganically bound chromium, if present, would not precipitate with am- monium hydroxide, while free chromium would. Chromium added to urine and plasma dialyzate served as controls to assure that non- bound chromium would precipitate under these conditions. RESULTS I. Routes of Chromium Excretion The data used to compile Figures 2 through 8, expressed as total micrograms chromium excreted for each collection period, are presented in Appendix A. Figures 2 through 6 show the accumulated . 51 , . . . excretion of Cr in urine and bile for a period up to four hours . . . . 51 51 after intravenous injection of NaZCr 04 or Cr C13. These dOgs were anesthetized and the common bile duct and ureters cannulated to obtain urine and bile samples. Additional experiments (summar- ized in Figures 7 and 8) extending over a four-day period were es- sential to evaluate the amount of chromium excreted in feces as well as bile and urine after intravenous injection. Feces were collected after volitional defecation by the animal, while a biliary fistula and indwelling catheter provided quantitative collection of urine and bile. , 51 51 . . . The data for excretion of Cr after Cr Cl3 administration were 51 obtained from one dog while two dogs were used with NazCr 04. Since the bile fistula Operations resulted in only two dogs suitable 51 for experiment, one of these (dog 6) was used first with Cr C13, 51 . . . . then three weeks later with NazCr 0 At this time an ihSignificant 4. 39 40 Figure 2. Accumulated excretion of hexavalent chromium in urine. 4 m 10 - U) 8 Dog 3 U 8-Dose = 4000 pg./kg\‘ *3 .2” 6 _ O H E 3 4 .. '\ g Dog 1 g: 2.- Dose = 0.395 [lg/kg. Dog 2 Dose = 7.28 pg./kg. 0 I l l 1 1 2 3 4 Time in Hours after Chromium Injection Figure 3. Accumulated excretion of hexavalent chromium in bile. Per Cent Injected Dose Time in Hours after Chromium Injection 41 Figure 4. Accumulated excretion of trivalent chromium in urine. 30 .7 o a: 25 n 3 Dog 4 0 C1 Dose 2 3200 pg./kg. . . 0 'o 2.0 - . ' ,9 . 8 I '815 - :/ H / Dog 5 i. o g /. Dose = 9.4 (lg/kg. 0 10 - s. 3 0) (3.. 5 I- / 0 l I l l 2 3 4 Time in Hours after Chromium Injection Figure 5. Accumulated excretion of trivalent chromium in bile. .15 9 — O o . o 8 o ’ ’ '0 10+ Dog 5 ' Q3. \ O . w . o . .93., ’ ‘3 o t: 0 \Dog 4 E, o 0.05I- . u o a. O /o 0 L l . l l 1 2. 3 4 Time in Hours after Chromium Injection Figure 8. <3 N w o 0 Per Cent Injected Dose g—a O \Feces Cr (+3) Feces Bile Cr (+ 3) Comparison of four day excretion values of hexavalent and trivalent chromium in bile and feces. Time in Days after Chromium Injection 44 quantity of chromium from the previous injection was found in urine and bile samples. II. Mechanisms of Renal Excretion of Chromium Tablel shows the renal clearance determinations on eleven dogs used to evaluate renal mechanisms involved in chromium ex- cretion. Creatinine and PAH clearances were measured to establish the normal range of renal function in each dog as well as to detect any depression in this function induced by intravenous injection of chromium solutions. Data used for calculating chromium clearances ) by formula 2 (C r ) and dialyzable chromium clearances (C ch d-chr. of Appendix C are given in Appendix A. In addition the ratios of clearances of creatinine to PAH (also called filtration fraction or /C ), and F.F.), dialyzable chromium to creatinine (C creat. /c d—chr. dialyzable chromium to PAH (C ) are presented. Chro- d- chr. PAH mium clearances in the first nine dogs were measured after a single . . . . . 51 _ intravenous injection of a solution of Cr C13. In the last Six of these, dialyzable chromium clearances were also measured. In dogs 17 and 18 both C and C were determined over a period of sev- chr. d-chr. 51 eral hours during intravenous infusion of Cr C13 in solution. Figures 9 and 10 graphically illustrate the decrease in chro- mium clearances with time after injection. Figure 9 gives individual TABLE 1 RE NAL C LE ARANC E VALUES 45 Clear- Time Description ance after C C Inj. creat PAH chr d-chr. No. , (m1n.) Dog No. 8 13.5 kg. female 1 78 109.8 233.9 2.86 - 75 ml./kg. waterb 2 98 104.1 196.0 2 50 _ 0.53 sq. meter 3 122 122.6 194.8 2.21 - 5.03 pg./kg. Cr 4 141 107.2 229.1 2.08 - 1 CPM=35.09 upgfi 5 166 110.2 223.1 1.84 - Dog No. 9 15 kg. female ' 1 66 98.9 240.6 2.91 - 75 ml./kg. water 2 82 97.5 244.2 2.37 - 0.66 sq. meter 3 106 97.2 238.0 2.36 - 294 pg./kg. Cr 4 127 119.3 273.8 2.25 - 1 CPM=551.0 pug. 5 148 119.7 297.2 2.32 - Dog No. 10 22 kg. male 1 117 72.3 275.0 2.60 - No water 2 147 77.8 243.8 2.09 - 0.87 sq. meter 3 218 78.2 202.5 1.50 - 250 pg./kg. Cr 4 240 69.7 187.2 1.37 - 1 CPM=440.0 pug. 5 321 77.0 205.2 0.86 - 6 350 69.4 167.8 0.86 - 7 430 94.4 287.3 0.70 - 8 450 98.4 291.0 0.63 - a . Units are milliliters per minute per square meter. bWater given orally. cl CPM = one count per minute. TABLE 1 (Continued) Li ‘—:-—‘ 46 Time Clear— after Descri tion anc p Noe Inj. creat. CPAH Cchr. Cd-chr. ' (min.) Dog No. 11 18 kg. male 1 60 108.3 285.4 3.11 37.9 No water 2 72 110.8 336.2 2.75 38.6 0.74 sq. meter 3 183 123.9 248.3 2.10 37.1 5.22 jig/kg. Cr 4 206 134.1 270.1 2.12 47.2 1 CPM=13.2 p.p.g. 5 297 164.1 307.2 1.74 44.3 6 312 161.7 330.5 1.65 43.4 7 457 174.4 357.7 1.21 45.9 8 482 172.8 360.7 1.18 50.0 Dog No. 12 14.5 kg. female 1 122 115.6 217.7 2.23 25.6 50 m1./kg. water 2 148 118.9 231.3 2.21 25.7 0.62 sq. meter 3 342 134.4 380.0 1.60 30.5 6.50 pg./kg. Cr 4 2880 141.4 - 0.28 34.7 1 CPM=15.5 ppg. 5 2940 114.2 — 0.25 31.3 DogNo. 13 19 kg. male 1 152 85.8 244.9 2.48 28.3 50 mg./kg. water 2 266 79.5 279.0 2.07 28.0 0.88 sq. meter 3 385 78.4 279.4 1.37 26.7 4.94 pg./kg. Cr 4 512 77.4 269.7 0.98 26.4 1 CPM=17.5 pug. 5 633 76.2 243.0 0.98 28.7 Dog No. 14 22 kg. male 1 63 84.0 291.8 3.05 30.0 50 m1./kg. water 2 135 80.3 222.8 2.28 30.6 0.87 sq. meter 3 222 98.0 286.3 2.14 37.7 91.0 pg./kg. Cr 4 305 105.8 314.1 1.20 25.6 1 CPM=80.5 p.p.g 5 393 105.3 344.0 1.15 32.8 6 453 102.6 337.9 0.99 40.0 1. 1.1.1.11 1 J1 ll 47 TABLE 1 (Continued) Clear— $1,152: Descri tion anc C p e Inj. creat. CPAH chr d-chr. (min.) Dog No. 15 23 kg. female 1 74 88.4 299.3 2.72 29.2 50 m1./kg. water 2 194 92.1 302.4 2.11 34.3 0.97 sq. meter 3 281 96.8 286.6 1.66 33.7 86.2 (lg/kg. Cr 4 374 99.0 325.9 1.24 35.1 1 CPM=221 (mg. 5 496 98.3 314.6 0.86 35.5 Dog No. 16 19 kg. male 1 79 58.3 164.9 1.69 33.4 50 m1./kg. water 2 169 56.3 185.9 1.18 36.6 0.86 sq. meter 3 259 62.9 172.1 0.80 36.6 1900 ug./kg. Cr 4 377 69.4 191.7 0.59 31.8 1 CPM=303 pug. ‘5 445 63.7 200.2 0.49 32.2 25 kg. male 1 103 111.3 246.0 11.24 50.8 50 ml./kg. water 2 133 118.3 265.4 11.67 54.9 1.09 sq. meter 3 258 136.3 262.0 7.65 67.1 97.0 pg./m1. at 4 350 137.2 173.9 6.65 67.5 2.43 ml./min. 5 409 136.0 290.7 3.72 67.0 1 CPM=502 p.p.g. 6 444 132.9 319.9 3.32 69.7 Dog No. 18 22 kg. male 1 71 70.4 203.6 2.98 24.3 50 ml./kg. water 2 185 77.1 188.0 1.80 25.8 .087 eg./m1. at 3 309 96.8 228.2 1.32 30.0 1.95 m1./min. 4 385 98.5 228.4 1.20 33.8 1 CPM=3.2 pug. 5 468 116.7 233.8 0.84 34.1 Number(:1 11 11 8 Mean 101.7 259.9 36.6 Standard deviation 24.34 40.13 11.58 Coefficient of variation 0.239 0.154 0.316 dEach observation is average of five to eight determinations. . . Z Cchr. in m1./m1n./M 48 3.0j. Figure 9. The decrease of chro- 0 mium clearance with time after injection. 0 2.0- 1.0- 0.9- 0.8. 6 Dog 8 . Dog 9 0.71- 0 Dog 10 .3. 0.4. c l I A I I I. l I 1 2 3 4 5 6 7 8 Time in Hours after Chromium Injection 49 Figure 10. The decrease in chromium clearances of Dogs 8 through 17 with time after Cr51 injection. 3.0 _ o 0 N2 2.0 ., o q, \. CI W4 8 \. E «j- .E 0 E ., U U . T MT 1, Two Standard 0'9 1" Deviations J 0.8 p 0 J. 0.7 j n n 1 I— 100 200 300 400 500 Time in Minutes after Chromium Injection 50 clearances for the first three dogs, while in Figure 10 chromium clearances measured in a given hour after the Cr5lCl3 injection were averaged for all dogs receiving a single dose of chromium chloride. Trivalent chromium in plasma is largely bound to-plasma proteins but some exists in the "free'' state (the term "free" in quotation marks is used only to indicate chromium that is not bound to plasma proteins). Since the "free" form should be more readily excreted by the kidney, its measurement could clarify some of the renal processes involved in chromium excretion. Figure 11 shows the per cent dialyzable“ ("free") chromium in the plasma of an 18.5 kg. dog after the intravenous injection of 650 pg. chromium chloride per kg. body weight. The per cent dialyzable chromium in the plasma of six dogs each given a single injection of trivalent chromium is in- dicated in Figure 12. 111. Some Physical and Chemical Properties of Chromium in Biological Fluids Equilibrium dialysis of urine samples gave the results shown in Table 2. Duplicate determinations were made on five urine sam- ples collected from each of two dogs after the intravenous injection of chromic chloride. The results are expressed as per cent of total chromium that is dialyzable. ' 51 Figure 11. The decrease in per cent dialyzable chromium in plasma Per Cent Dialyzable Chromium in Plasma with time after injection. I n n 1 100 200 300 400 Time in Minutes after Chromium Injection O\ U1 Per Cent Dialyzable Chromium in Plasma w as 52 Figure 12. The decrease in per cent dialyzable chromium in plasma of six dogs with time after injection. l O 100 200 300 I l I 400 500 Time in Minutes after Chromium Injection " t. .1 » . 5 WV" 3‘9”}. I ‘_-F"~. er .. TABLE 2 PER CENT DIALYZABLE CHROMIUM IN URINE Urine Collection Dog 14 Dog 15 Period 1 98.5 86.2 92.5 76.6 2 105.1 69.3 - 80.2 3 103.2 85.6 — 86.6 4 104.3 89.9 - 88.2 5 98.5 81.2 99.3 83.2 Number 5 5 Mean 101.4 82.7 Standard deviation 4.1 5.4 Coefficient of variation 0.04 0.06 54 Figures 13 and 14 graphically present results of the rate dialysis procedure. In these figures the rate of diffusion through a 51 dialysis bag of Cr excreted in urine after intravenous injection of 51 51 NaZCr O4 is compared to that of NaZCr 04 added to saline or urine- Table 3 shows the results of chemical reduction when hexa- valent chromium was added to stored (previously frozen) or fresh urine. Urine of one dog injected with hexavalent chromium. was also tested qualitatively for presence of chromate ion and quantitatively for total chromium content. Ion exchange resins were used in investigating the ionic charge of chromium in various fluids from two dogs (Table 4). Samples from dOg 4 were run through anion exchange resin after passage through cation exchange resin, while in dog 18 fresh sam- ples were used for each resin. Results are expressed as per cent 51 Cr J~"enioved by the resin. The last two experiments in this group were designed Spe- Cifically to gain information on possible binding of chromium to a dialyzable component of urine and plasma. Table 5 shows the chro- matogr‘11)hic Rf values of chromium and ninhydrin-positive substances 51 present in urine and a plasma dialyzate. While the presence of Cr and rlirll'lydrin-positive substances at the same place on paper Counts per Min. per M1. Sample 55 5 7000 Figure 13. Dialysis of Cr 1 in saline and urine of a 10.5 kg. dog after intravenous injection of NaZCr5104. Cr51(+ 6) added to Saline tl/Z = 63 min. k -.= 1.1.03110‘3 min.'1 N o ‘2 o 1:: - - o 51 80 Cr excreted by 7 0 10.5 kg. female . e_DOS€ = 0.33 pg./kg. 60 tl/Z = 380 min. k = 1.83x10"3 min."1 50 o 40 O o 3 0 2? W 4175 61575 M Time Dialyzed in Minutes Figure 14. Counts per Min. per Ml. Sample 4000 - 3000 '- 2000 1 000 Zon 56 51 Dialysis of Cr added to urine in vitro and urine after intravenous injection of NazCr 1O4. 51 Cr (+6) added to urine (_—and dialyzed immediately. tl/Z = 52 min. _1 k = 13.035110“3 min. Cr51(+ 6) added to urine and dialyzed after 5 hours. tl/Z = 56 min. k = 12.04x10‘3 min.'1 Cr51 excreted by 7.5 kg. female. Dose = 1.31 jig/kg. tl/Z = 426 min. k = 1.63xio‘3 min.’1 0 I l L L 4 100 200 300 400 500 Time Dialyzed in Minutes 57 TABLE 3 REDUCTION OF HEXAVALENT CHROMIUM IN VIVO AND IN VITRO Time Total Hexavalent Procedure (minutes) Chromium Chromium (pg/5 ml.) (qual. test) 100 pg. hexavalent 2 5 +++ chromium added to 10 5 +++ stored urine 60 5 +++ 120 5 +++ 360 5 +++ 100 pig- hexavalent 5 5 - chromium added to 10 5 - fresh urine 15 5 - 200 Kg- hexavalent 5 10 + chromium added to 10 10 :1: fresh urine 30 10 - 60 10 - CTSI excreted by dog 0-6a 125 - after injection of 40 mg. 6-18 168 - NazCrs 104 18-30 119 - aIndicates time interval in which sample was collected. TABLE 4 58 IONIC PROPERTIES OF CHROMIUM IN BIOLOGICAL FLUIDS Dog 18 Dog 4 Sample Per Cent Avg. Pct. Per Cent Avg. Pct. Removed Removed Removed Removed Cation Exchange Resin Cr infusion or 48.42 48.25 99.85 99.90 injection solutiona 48.09 99.95 _ Pooled urine 0.56 0.14 6.74 5.99 -0.28 5.24 Plasma dialyzate 0.73 -0.79 5.86 4.07 -2.30 2.29 Pooled bile — -2.41 4.13 _ 10.67 Anion Exchange Resin Cr infLIsion or 40.56 40.56 87.48 88.33 injectic>r1 solution - 89.20 Pooled urine 98.64 98.64 95.97 96.01 - 96.06 Plasma dialyzate 97.81 97.81 89.71 89.61 _ 89.52 Pooled bile - 27.59 27.40 - 27.22 a _ , Dog 18 was infused with Cr51C13; dog 4 1n3e°ti°n of Cr51C13. received a single THE Rf VALUES OF CrSl, NINHYDRIN-POSITIVE SUBSTANCE, TABLE 5 CREATININE. AND PAH IN SAMPLES TAKEN FROM DOG 12 59 Creat— . h . _ . . Sample ' . PAH Cr51 Nin ydrin pOSitive inine Substance Infusion solution . .16 .38 .06 .17 .37 .07 Plasma dialyzate. .13 _a .05 .06 .39 .15. - .04 .07 .35 Urine ........ .23 .34 .06 .03 .06 .09 .27 .24 .05 .03 .06 .08 PAH - . ....... .41 Creati nine ..... .22 aIndicates none could be found in sample. 60 chromatograms does not prove that they are bound together, their location at the same spot would be expected if binding occurred. 5 Per cents of Cr precipitated from urine and a plasma dialyzate by ammonium hydroxide are shown in Table 6. The addition of Cr5 1 C13 to normal urine and plasma dialyzate in vitro served as 51 controls to demonstrate that Cr in these fluids would precipitate with ammonium hydroxide . TABLE 6 61 PRECIPITATION OF CHROMIUM FROM BIOLOGICAL FLUIDS USING AMMONIUM HYDROXIDE Per Cent Average Sample Preci itated Per Cent p Precipitated Uri he in vivoa .................. 28.23 26.93 25.63 Uri rue in vitro .................. 77.47 77.82 78.18 Plasma dialyzate in vivo ........... 30.34 31.73 33.13 Plasma dialyzate in vitro .......... 97.11 97.16 97.21 ‘1) 51 a19 kg. Dog injected intravenously, with 705 pg Cr C13 and urin e used. DI SC USSION I. Routes of Chromium Excretion Data summarized in Figures 2 through 6 compare excretion of chromium in urine and bile over a four-hour period after intra- venous injection of chromium. 51 Over 94 per cent of Cr excreted 51 afte r administration of NaZCr O4 appeared in urine, while 99.5 per cent of Cr51Cl3 was excreted by this route. These Figures also show that about three times more trivalent than hexavalent chromium was exc reted four hours after injection. Three different dosage levels of hexa.valent and two of trivalent chromium were administered to these dOgS - In all cases the larger doses resulted in a greater per cent 0f the injected dose being excreted. These findings were also con- firmed with NaZCrSlO4 over a four-day period. A possible explana- tioh for these findings'is that a smaller per cent of large doses is bound to erythrocytes or plasma proteins (as Gray and Sterling, 1950, Shc“Ned in vitro), leaving more "free" for urinary excretion. How- ever, biliary excretion of either valence state showed no such dosing effect _ 62 5 ‘. an“ *M' vv-z-c..- ‘ , 63 Bile fistula dogs were used to measure chromium excretion in feces as well as urine and bile over a four-day period (Figures 7 . . . 51 51 51 and 8). Figure 7 indicates that Cr of Cr Cl3 or NazCr O was exc reted rapidly immediately following injection but that the rate of exc: retion decreased with time. With both valences, over 80 per cent of t he four-day excretion value was found in urine collected the first 51 day - The liver is one of the major organs of uptake of Cr 5 l 51 . . . . Cr Cl3 or NaZCr O4 injection (Visek et al., 1953), so that a rela- afier tive 13 large excretion of chromium in bile would perhaps be expected. 51 . . However, less than 5 per cent of excreted Cr was present in bile. Afte r NaZCrfilO4 injection, the amount of Cr51 found in feces was 3180 negligible and only slightly greater after dosing with Cr51C13. Data on Cr excretion, after administration of Cr51C13, are in good agreement with those of Kraintz and Talmage (1952). They found 40 per cent of Cr51 in urine and none in feces of rats one _ 51 day after the intravenous injection of Cr C13 in acetate buffer. The one-day excretion values in dog 6 (Figures 7 and 8) were 41.5 and 51 2-5 per cent of the injected Cr C13 in urine and feces, respectively. 51 On the other hand, the four-day excretion values of Cr after intra- 51 venous administration of Cr Cl3 to rats reported by Visek et a1. (1953) do not agree with those found here. They reported 15 per cent in urine and 20 per cent in feces, while the corresponding values 64 here in dog 6 were 49.3 per cent in urine and 4.4 per cent in feces . . . . . 51 . , 51 plus bile. Discrepanc1es in excretion of Cr after givmg NaZCr 04 we re also large. Their four—day excretion values for this valence we re 35 and 17 per cent in urine and feces, while the average re- sults obtained here for dogs 6 and 7 were 20.5 per cent in urine and 2.0 per cent in feces plus bile. The results reported by Sutherland and McCall (1955) are intermediate between those of Visek et a1. and the data reported here. They found that urine contained 35 per cent 51 and feces a negligible amount of Cr after the intravenous adminis— - 51 tratlon of NaZCr O4 to humans. The inconsistencies in results reported for chromium excre- tion can probably be attributed to differences in concentration, pH, and presence of other substances in the dosing solutions as well as Species differences in the animals used. II. Mechanisms of Renal Excretion of Chromium The concept of the renal excretion of plasma dialyzable chro- mium did not originate until after chromium clearances (CC r) were h measured in several dogs. Chromium clearances on the first two dogs (Table 1, dogs 8 and 9), measured on consecutive urine sam- ples Over a relatively short period of time, varied frOm 2.91 to 1.84 1171- per minute per sq. meter surface area. This range of variation u i I ‘. i l 65 might be expected in Cchrf however, a tendency for the clearances to decrease systematically with time after chromium dosing was un- expected. Clearances on a third dog, No. 10, were determined at several time intervals over a six-hour period. Data from this dog (from 2.60 to 0.63 ml. per minute per sq. meter surface area) decreased with time after the CrSICl3 clearly showed that C . _ chr. inj ection. A plot of Cchr. in Figure 9 established that chromium Clearances decrease exponentially with time after Cr51C13 adminis- tration. Since GFR and ERPF determinations indicated normal renal function, some aSpect of chromium excretion was producing a reduc- tion of C ; in any case, chromium clearances alone did not clarify c . hr renal mechanisms employed in chromium excretion. Trivalent chromium in plasma is largely bound to the plasma PrOt eins, but some exists in the "free" state. Since the "free" form would be filterable at the glomerulus, its measurement should Shed some light on renal mechanisms involved in chromium excre- t'101'1. Figure 11 shows the per cent dialyzable ("free") chromium in the plasma of an 18.5 kg. dog after the intravenous injection of 650 pg. per kg. body weight of trivalent chromium. The finding that the per cent dialyzable chromium in plasma also decreased exponen- tially with time after the CrSlCl3 injection offered an explanation of the reduction of Cchr observed with time. If each of the chromium 66 clearances were multiplied by the reciprocal of the decimal equiva- lent of the per cent plasma dialyzable chromium at a time midway in the urine collection period, the answer would be in terms of a clearance of plasma dialyzable chromium (Cd-chr.)' For example, the mid-point of the urine collection period for clearance 1 of dog 8 (Table 1) was 78 minutes after the CrSICl3 injection. At this time, Figure 11 shows that 9.7 per cent of the plasma chromium was dialyzable. The factor used to multiply CC by was 1/0.097, or hr. 10- 3 - Then 10.3 times the Cc of 2.86 gives a dialyzable chro- hr. mi L111) clearance of 29.5 ml. per minute per sq. meter surface area. Thi— 8 means that 29.5 ml. of plasma are completely cleared of dia- 1YZ able chromium each minute for each square meter surface area 0f the dog. The dialyzable chromium clearances calculated in this mal‘tuier for the first three dogs are shown in Table 7. This method of determining Cd-chr. depends on the supposi- t101:1 that per cent plasma dialyzable chromium at any one time after CPS lC13 injection is the same in different dogs and at different doses. Figure 12, showing the per cent plasma dialyzable chromium of dogs 11 through 16, demonstrates that this is not the case. There is wide individual variation in the per cent chromium unbound in plasma of different dogs at the same time after injection, although the per cent dialyzable chromium in any one dog a short time after injection DIALYZABLE CHROMIUM CLEARANCES IN MILLILITERS PER MINUTE PER SQUARE METER SURFACE AREA AS CALCULATED FROM PER CENT "FREE" TABLE 7 CHROMIUM IN PLAS MA 67 Dog Num be r Clearance 8 9 10 1 29.5 28.5 31.3 2 27.8 24.9 28.4 3 27.4 27.4 27.3 4 27.7 29.5 28.5 5 27.1 31.8 23.7 6 26.6 7 29.0 8 30.5 68 shows a uniform exponential decrease. The relation between dose of chromium and the rate constant, k (where k = .693/t1/Z), for de- creasing plasma dialyzable chromium is shown in Table 8. The coefficient of linear correlation, r, between dose and k is 0.75, which is significant at the 1 per cent level. This statistic means 2 that 56 (0.75 ) per cent of the variation in k can be attributed to the dose. With respect to the dose—k relationship, it was surprising that larger doses resulted in more rapid decline in per cent plasma di alyzable chromium. Figure 12 (dog 16) shows that the per cent plasma dialyzable Chromium decreases more rapidly immediately after the chromium inIlecztion than at a later time. In three other dogs a rapid compo- nent was also observed. The cause for this rapid fall is not clear. In determining percentage of plasma dialyzable chromium, the amount dialyzable is divided by total chromium content of plasma. Any fac- tor decreasing the amount dialyzable without greatly affecting the total will result in a greater per cent bound. The rapid rate of deC rease in per cent plasma dialyzable chromium is probably due to "free" chromium diffusing out of the plasma space, leaving a larger part of that remaining in the bound form. In vitro tagging 1 . 0f dog plasma proteins with Cr5 indicates that only two-thirds of trivalent chromium becomes bound (Gray and Sterling, 1950, found 69 TABLE 8 RELATIONSHIP BETWEEN THE DOSE OF CHROMIUM INJECTED AND THE RATE CONSTANT (k) FOR THE DECREASE IN PER CENT DIALYZABLE CHROMIUM 1N PLASMAa Dose tl/z k (pg/kg.) (min.) (per min.) 1900 178 .00395 650 170 .00408 91.0 ._ 235 .00295 82.2 225 .00308 6.50 295 .00245 5.22 242 .00287 4.94 330 .00211 aCoefficient of linear correlation (r) = 0.75. 70 about 66 per cent bound with bovine albumin in vitro), while in vivo tagging of dog plasma shows 85 to 90 per cent bound a few min- utes after injection. The difference between these two figures rep- resents "free" chromium that left the plasma. The slow rate of decrease in per cent dialyzable chromium extending over many hours after injection is visualized as a net rate equal to the decrease caused by renal excretion minus the return of dialyzable chromium to the plasma space from extravascular sites. Another factor affecting the rate of decrease would be chromium freed from plasma proteins by elution or degradation. This aspect of chromium metabolism deserves further study. Cd-chr. in dogs 10 through 18 was determined by the stand- ard clearance formula using the plasma concentration of dialyzable chromium. The value for Cd-chr. in eight dogs was 36.6 :I: 11.6 ml. per minute per sq. meter surface area. Five to eight measurements were made on each animal at periods of 1 to 10-1/2 hours after the Chromium injection. Two clearances measured on one dog at 48 and 49 hours after the chromium injection were within the normal range but the Cr51 activity of the plasma dialyzate was so low (an average of 0.95 count above background for four determinations) that the re- sults are questionable. The last two animals used in this study re- ceived chromium by'infusion over a 7 to 8 hour period instead of 71 a single injection. One of these dogs had a high Cd-chr. but other- wise the clearances were similar to those of other dogs. The mean and standard deviation for creatinine clearances in the eleven dogs in this study was 101.7 :L- 24.3 ml. per minute per sq. meter surface area. This value is somewhat higher than that of Houck (84 :t 19.1 ml. per min.) or Russo (95 ml. per min.) possibly because of saline contained in the creatinine-PAH infusion solution. Saline "has been shown to increase GFR and ERPF (Baldwin 9131., 1949; Hare 9131., 1944), and at the infusion rates employed (1-3 ml. per min.) some dogs received as much as 1.5 liters of 0.85 per cent sodium chloride. The value for PAH clearances of 259.9 :h 40.1 was in good agreement with those of Houck (266 :1: 66) and Russo (238), all expressed as ml. per minute per sq. meter surface area. A small quantity of blood proteins is excreted by the normal kidney (Waterhouse and Holler, 1948; McGeachin and Hargan, 1957), and there is evidence that hemoglobin reabsorption from the glomer- ular filtrate amounts to about 3 per cent of GFR (Monke and Yuile, 1940). These processes could be involved in chromium excretion; however, the amount of protein (as measured by Albumtest tablets) in clearance urine samples was too small to account for more than a few per cent of the urine chromium. Also, the finding that nearly 72 all of the Cr51 excreted in urine was dialyzable (Table 2) rules out excretion of protein-bound chromium. The alternate possibility, that plasma proteins containing bound chromium are filtered and the chro- mium eluted into glomerular fluid prior to protein reabsorption, is extremely unlikely because of the firmness of the chromium-protein bond (Gray and Sterling, 1950). The finding that CC is dependent hr. on time after the chromium injection (Figure 10), while Cd-chr. is not (Figure 15; F values not significant), argues against excretion of chromium in this manner. On the other hand, plasma dialyzable chromium is certainly filterable at the glomerulus (the average pore diameter of the dialy- sis tubing was 24 A, while the pore size of the glomerulus [lamina densa] may be on the order of 100 A [Hall, 1954]) and must exist in glomerular filtrate in the same concentration as in plasma water. The amount of dialyzable chromium filtered is equal to glomerular filtration rate times plasma concentration. If only glomerular fil- tration were involved in chromium excretion this quantity should appear in the urine. The finding that less chromium is present in urine than is filtered indicates some chromium is reabsorbed from tubular fluid. Tubular reabsorption amounts to 63 per cent (1 - d-Chr./Ccreat. X 100) 0f the amount filtered in the eight dogs in which it was measured. - men." 13'“ _.~. 73 .sofiooflcfi 5350.50 swan 653. 515 moflmu mundane? 5 nofimzmkw .2 0.5m?“ sofiomfig Engofio head mesom 5 95B P4 “1 Ccreat./CPAH ._. creat. . n "1 M. / Cd—chr./C PAH Cd—chr./C \om 74 Unequivocal proof that a substanceis excreted by the tubules is extremely difficult unless its clearance is greater than GFR. How— ever, tubular excretion in every instance where it has been adequately examined is limited by a maximal rate of tubular transport (Smith, 1951). At plasma levels above those necessary to saturate the tubular excretory mechanisms the clearance declines. Figure 16 shows the dialyzable chromium clearance ratios plotted against the plasma level of chromium. There is over an 800 fold difference in the dialyzable chromium concentration in plasma without a reduction in the clearance ratios. Either the peak plasma level of dialyzable chromium was not high enough to saturate tubular mechanisms or tubular excretion of dialyzable chromium plays little part in chro- mium excretion. Tubular maximum (TM) measurements for chro- mium are necessary to clarify the role of the tubules in chromium excretion. and C for dialyzable The ratios, C d—chr./CPAH d-chr./Ccreat.’ chromium at high plasma levels increased significantly (”t" tests) above those at medium or low plasma levels. Reasons for this are not known. 75 853.853 mo coflmficm00=o0 manna 5E» mosh.” 0000.820 5 ”833.85 .3 0.53m .2: con. .mi 5 “8305:0280 mammam odes 9m o.~ o; m. 4. m. N. a. 8.3. 8. .8. 8. u q d _ u a u u - u _ . 4 d _ . 12. II I IE I . Hflmmulnol -3 o I I 3. mm. IDI do 10.000| :33?on m. II .OIOI I Unmesmem II loI Iolmmmlaodlo o I I obloboI. -em I oo I.I4 I I .OI IEI 1 . I? B qul 3. IoIdm I o deuowguI 13.. low. 76 III. Some Physical and Chemical Properties of Chromium in Biological Fluids Rate dialysis (Figures 13 and 14) of Cr51 excreted in urine conclusively demonstrated that this chromium diffused more slowly and presumably existed as larger molecules than the NaZCrE‘lO4 in- jected into the animal. The finding that intravenously injected hexa- valent chromium was excreted in the trivalent state (Table 3) offers an explanation to this decreased diffusion rate of excreted Cr51. Since the diffusion rate of Cr51C13 was not measured, it is not known if reduction of NaZCrSIO4 would account for the large decreases in diffusion rates observed. The finding by ion exchange absorption that trivalent chro— mium excreted in urine or present in a plasma dialyzate existed in the anionic form could be interpreted as being due to the binding of this chromium to organic material to form negatively charged com- plexes. However, in interpreting these results it is essential to remember that hydrolysis of CrCl3 readily occurs to form anionic complexes with water when no organic material is present. This is well illustrated in the infusion solutiOn of dog 18. A small quantity of CrSICl3 was added to 1300 ml. of creatinine-PAH infusion solution at a pH of 5.2, and only 48.2 per cent of the Cr51 was removed by the cation exchange resin, while 40.6 per cent was absorbed by the 77 anion exchange resin. The difference of 11.2 per cent was presum- ably uncharged. In contrast, the injection solution for dog 4 had a pH of 3.8, and 99.9 per cent of the Cr51 existed in the cation form as shown by absorption with Amberlite 120. One factor in favor of the organic complexing concept is that chromium in urine or a plasma dialyzate is soluble, while CrC13 in a solution adjusted to the same pH precipitates. Paper chromatograms of chromium excreted in urine or present in plasma dialyzate resulted in a small but consistent (Rf = 0.04 to 0.06) movement of the Cr51 from the origin. In the solvent mixture used, cationic chromium was reported to have an Rf value of 0.65 (Pollard and McOmie, 1953). The difference in the Rf value found here and those reported previously may have been due to the use of acetate complexed chromium in determining the value reported by Pollard and McOmie. By developing the chromatograms with nin- hydrin, definitive proof that Cry.>1 was not bound to amino-containing substances could result since this reaction gives a positive test with proteins, peptones, peptides, amino acids, and other primary amines including ammonia (Hawk 9131., 1954). The finding of ninhydrin- positive substances on the paper strips with Rf values abJut the same as those of Cr51 supports but does not prove that chromium is ex- creted in the bound form. 78 The best evidence that dialyzable chromium in plasma and urine is at least in part in organic combination was provided by the precipitation procedure. In this experiment the greater part of an- ionic CrSICl3 added to normal urine and plasma dialyzate was pre- cipitated by ammonium hydroxide, while less than one-third of intravenously injected chromium present in these fluids was pre- cipitated. xiv/“J ‘ l\/ SUMMARY AND CONC LUSIONS 1. In the dog the major route of excretion of intravenously injected chromium is in the urine; excretion in bile and feces is negligible. In acute experiments on anesthetized dogs about 9 per cent of the injected dose of hexavalent and 25 per cent of trivalent chromium were excreted in the urine in four hours. Less than 0.5 per cent of either valence was present in bile collected over a four- . . . . . 51 51 hour period after intravenous injectlon of Cr Cl3 or NaZCr 04. The four-day excretion values in urine, bile, and feces in one bile fistula dog were 50, 0.5, and 3.7 per cent, respectively, of injected 51 . . Cr C13. In two dogs hexavalent chromium average excretion values were 20, 0.9, and 1.2 per cent of the injected dose in urine, bile, and feces, respectively. 2. After a single intravenous injection renal clearances of 51 . . . . Cr C13 decrease exponentially w1th time from 2.5 or 3 ml. per min. at one hour to less than 1 ml. per min. at eight hours after injection. . ' 51 , 3. Measurement of dialyzable Cr 1n plasma showed that the per cent of chromium not bound to plasma proteins also decreased exponentially with time after injection and at a rate about equal to the decline in chromium clearance. Large doses of chromium were 79 ,.\_/‘—' /~\\. / 80 found to result in a more rapid decline in the percentage of chro- mium not bound to plasma proteins. Possible reasons for the re- duction in per cent dialyzable chromium in plasma are discussed. 4. A dialyzable chromium clearance with a mean and stand- ard deviation of 36.6 :1: 11.6 ml. per minute per sq. meter surface area was determined. This clearance showed that glomerular fil- tration and tubular reabsorption are two mechanisms involved in the renal handling of unbound chromium. Tubular excretion of dialyzable chromium may occur. However, no reduction in the clearance at high plasma chromium levels favors the conclusion that excretion by the tubules is of minor importance. 5. Simultaneous creatinine and PAH clearances were normal, demonstrating that renal function was not impaired over a large range of plasma chromium concentration. 6. No hexavalent chromium could be found in urine after intravenous injection of sodium chromate, an indication that in vivo reduction occurs. Freshly collected urine was found to reduce hexa- valent chromium while stored urine did not. 7. Chromium in urine, bile, and plasma dialyzate is anionic, as shown by ion exchange absorption after the intravenous adminis- tration of cationic chromic chloride. 81 8. Results of dialysis, ion exchange absorption, paper chro- matography, and precipitation studies indicate, but do not prove con- conclusively, that chromium is excreted at least in part in organic combination. REFERENCES CITED Baldwin, D., E. M. Kahana, and R. W. Clarke. 1949. The Renal Excretion of Sodium and Potassium in the Dog. Med. Dept. Field Res. Lab., Fort Knox, Kentucky. Project no. 6-64-12- 06-(17). Referred to in Smith, H. W. 1951. The Kidney, Structure and Function in Health and Disease. New York: Oxford University Press. p. 331. Bowes, J. H., and R. H. Kenten. 1949. The Effect of Deamination and Esterification on the Reactivity of Collagen. Biochem. J .. 44:142-52. Braidich, M. M., and F. H. Emery. 1935. The Spectrographic De- termination of Minor Chemical Constituents in Various Water Supplies in the United States. J. Amer. Water Works Assoc, 272557-80. Brard, D. 1935. Toxicologie du Chrome. Actualities Scientifiques et Industrielles. No. 228. Paris: Hermann et Cie. Brauer, R. W., R. J. Holloway, and G. F. Leong. 1957. Temperature Effects on Radio-Colloid Uptake by the Isolated Rat Liver. Amer. J. PhysioL, 189:24-30. Brinton, H. P., E. S. Frasier, and A. I... Koven. 1952.. Morbidity and Morality Experience among Chromate Workers. Pub. Health Repts., 69:835-47. Brod, J.. and J. H. Sirota. 1948. The Renal Clearance of Endoge- nous "Creatinine" in Man. J. Clin. Invest., 27:645-54. Conn, L. W., H. L. Webster, and A. H. Johnson. 1932. Chromium Toxicolog. Absorption of Chromium by the Rat When Milk Containing Chromium Was Fed. Amer. J. Hygiene, 15:760-65. Corcoran, A. C., and I. H. Page. 1943. Effects of Anesthetic Dos- age of Pentobarbital Sodium on Renal Function and Blood Pressure in Dogs. Amer. J. PhysioL, 140:234-39. 82 83 Cunningham, T. A., E. M. McGirr, and W. E. Clement. 1957. The Effect of Prior Contact between Acid Citrate Dextrose and Sodium Radiochromate Solutions on the Efficiency With Which Cr51 Labels Red Cells. J. Lab. and Clin. Med., 50:778-87. David, H., and M. Lieber. 1951. Underground Water Contamination by Chromium Wastes. Water and Sewage Works, 982528—34. Davidson, A. M. M., and R. L. Mitchell. 1940. The Spectrographic Determination of Trace Elements in Soils. II. The Variable Internal Standard Method, Applied to the Determination of Chromium in the Cathode Layer Arc. J. Soc. Chem. Ind., 59:213-32. Edmunds, C. W., and J. A. Gunn. 1928. Cushny's Pharmacology and Therapeutics. Philadelphia: Lea and Febiger. 9th Ed. p. 169. Fields, T., and Seed. 1957. Clinical Use of Radioisotopes. Chicago: The Year Book Publishers. p. 163. Frank, H., and S. J. Gray. 1953. The Determination of Plasma Volume in Man with Radioactive Chromic Chloride. J. Clin. Invest., 322991-99. Gabrieli, E. R. 1951. Effect of Antihistamine on the Reticulo- endothelial System; Investigation of Reticulo-endothelial Func- tion with the Aid of Radioactive Chromium Phosphate. Nature, 168:467-68. Glauser, K. E., andI'E. E. Selkurt. 1952. Effects of Barbiturates on Renal Function in the Dog. Amer. J. Physiol., 168:469-79. Goldring, W., R. W. Clarke, and H. W. Smith. 1936. The Phenol Red Clearance in Normal Man. J. Clin. Invest., 152221-28. Grant, W. H., and G. Medes. 1935. Creatinine Clearances during the Hyperthermia of Diathermy and Fevers. J. Clin. and Lab. Med., 202345-49. Gray, S. J., and K. Sterling. 1950. The Tagging of Red Cells and Plasma Proteins with Cr“. J. Clin. Invest., 29:1604-13. 84 Green, R. W. 1953. The Role of the Amino and Hydroxyl of Col- lagen in Chrome Tanning. Biochem. J., 54:187-91. Grogan, C. H., and H. Oppenheimer. 1955. Experimental Studies in Metal Cancerigenesis. V. Interaction of Cr111 and Cr Compounds with Proteins. Archives of Biochem. and Bio- physics, 56:204-21. Grushko. Ya. M. 1948. Chromium as a Bioelement. Biokhimiya, 13:124-26. Chem. Abs., 42:8302i. Gustavson, K. H. 1949. Advances in Protein Chemistry. Vol. V, Edited by Anson, N. L., and J. T. Edsall. New York: Academic Press Inc. pp. 354-421. Gustavson, K. H. 1956. The Chemistry of Tanning Processes. New York: Academic Press Inc. Chapters 1 and 4. Habif, D. V., E. M. Papper, H. F. Fitzpatrick, P. Lowrance. C. McC. Smythe, and S. E. Bradley. 1951. The Renal and Hepatic Blood Flow, Glomerular Filtration Rate and Urinary Output of Electrolytes During Cyclopropane, Ether and T10- pentyl Anesthesia, Operation, and the Immediate Postopera- tive Period. Surgery, 30:241-55. Hall, B. V. 1954. Further Studies of the Normal Structure of the Renal Glomerulus. Proc. VIth Annual Conf. on the Nephrotic Syndrome, Nov. 5-6, National Nephrosis Foundation, New York. Referred to in Smith, H. W. 1956. Principles of Renal Physiology. New York: Oxford University Press. p. 182. Hare, R. S., K. Hare, and D. M. Phillips. 1944. The Renal Excre- tion of Chloride by the Normal and by the Diabetes Insipidus Dog. Amer. J. Physiol., 1402334—48. Hawk, P. B., B. L. Oser. and W. H. Summerson. 1954. Practical Physiological Chemistry. New York: The Blakiston Co. Inc. 13th Ed. p. 172. Hopkins, B. S. 1942. General Chemistry for Colleges. New York: D. C. Heath and Co. p. 679. 85 Houck, C. H. 1948. Statistical Analysis of Filtration Rate and Ef- fective Renal Plasma Flow Related to Weight and Surface Area in Dogs. Amer. J. Physiol., 153:169-75. Jolliffe, N., and H. W. Smith. 1931. The Excretion of Urine in the Dog. I. The Urea and Creatinine Clearances on a Mixed Diet. Amer. J. Physiol., 98:572-77. Kennedy, T. J., Jr.', J. G. Hilton, and R. W. Berliner. 1952. Com- parison of Inulin and Creatinine Clearance in the Normal Dog. Amer. J. Physiol., 171:164-68. Koch, H. J., Jr., E. R. Smith, N. F. Shimp, and J. Connor. 1956. Analysis of Trace Elements in Human Tissues. 1. Normal Tissues. Cancer, 9:499-511. Korst. D. R., D. V. Clatanoff, and R. R. Schilling. 1955. External Scintillation Counting over the Liver and Spleen after the Transfusion of Radioactive Erythrocytes. Clin. Res. Proc., 32195-201. Kraintz, L., and R. V. Talmage. 1952. Distribution of Radioactivity following Intravenous Administration of Trivalent Chromium 51 in the Rat and Rabbit. Proc. Soc. Exper. Biol. and Med., 81:490-92. MacKenzie, R. D. 1957. A Study of the Toxicity of Chromium in the Albino Rat. Ph.D. Thesis. Mich. State Univ. Mancuso, T. F., and W. C. Hueper. 1951. Occupational Cancer and Other Health Hazards in a Chromate Plant: A Medical Ap- praisal. Industrial Med. and Surg., 20:358-63. McCall, M. S., D. A. Sutherland, A. M. Eisentraut, and H. Lantz. 1955. The Tagging of Leukemic Leukocytes with Radioactive Chromium and Measurement of the In Vivo Cell Survival. J. Lab. and Clin. Med., 45:717-24. McGeachin, R. L., and L. A. Hargan. 1957. Amylase Clearance during Water Diuresis. Proc. Soc. Exp. Biol. and Med., 95:341-43. 86 Moller, E., J. F. McIntosh, and D. D. VanSlyke. 1929. Studies of Urea Excretion. II. Relationship between Urine Volume and Rate of Urea Excretion by Normal Adults. J. Clin. Invest., 6:427-65. Monier-Williams, G. W. 1949. Trace Elements in Food. New York: John Wiley and Sons Inc. p. 440. Monke, J. V., and C. L. Yuile. 1940. The Renal Clearance of Hemoglobin in the Dog. J. Exper. Med., 72:149-65. Nechels, T. F., I. M. Weinstein, and G. V. LeRoy. 1953. Radio- active Sodium Chromate for the Study of Survival of Red Blood Cells. I. The Effect of Radioactive Sodium Chromate on Red Cells. J. Lab. Clin. Med., 42:358-67. Owen. C. 8., Jr.. J. L. Bollman, and J. H. Grindlay. 1954. Radio- chromium-labeled Erythrocytes for the Detection of Gastro- intestinal Hemorrhage. J. Lab. and Clin. Med., 442238-45. Pollard, F. H., and J. F. W. McOmie. 1953. Chromatographic Methods of Inorganic Analysis. London: Butterworths Scien- tific Publications. p. 91. Read, R. C., G. W. Wilson, and F. H. Gardner. 1954. The Use of Radioactive Sodium Chromate to Evaluate the Life Span of the Red Cell in Health and Certain Hematologic Disorders. Amer. J. Med. Sc., 228:40-52. Rehberg, P. B. 1926. Studies on Kidney Function. 1. The Rate of Filtration and Reabsorption in the Human Kidney. Biochem. J., 20:446-60. Russo, H. F., J. L. Ciminera, S. R. Gass, and K. H. Beyer. 1952. Statistical Analysis of Renal Clearance by the Dog. Proc. Soc. Exper. Biol. and Med., 80:736-40. Saltzman, B. E. 1952. Microdetermination of Chromium with Diphenylcarbazide by Permanganate Oxidation. Anal. Chem.. 24:1016-24. Schiffman, R. H. 1957. Normal Values and Chromium-Induced Blood Changes in Blood Physiology of Rainbow Trout, Salmchairdnerii. Ph.D. Thesis. Mich. State Univ. Shannon, J. A. 1935. Excretion of Phenol Red by the Dog. Amer. J. Physiol. 113:602-10. Shuttleworth, S. G. 1950. The Theory of Chrome Tanning. J. Soc. Leather Trades Chemists. 34:410-37. Smith. H. W. 1939-40. The Physiology of the Renal Circulation. Harvey Lectures, 166-222. Smith, H. W. 1951. The Kidney, Structure and Function in Health and Disease. New York: Oxford University Press. Chapter 9, p. 146. Smith, H. W. 1956. Principles of Renal Physiolog. New York: Oxford University Press. pp. 197, 182. Smith, H. W., N. Finkelstein, L. Aliminosa, B. Crawford, and M. Graber. 1945. The Renal Clearances of Substituted Hippuric Acid Derivatives and Other Aromatic Acids in Dog and Man. J. Clin. Invest., 24:388-404. Smith, H. W., W. Goldring, and H. Chasis. 1938. The Measurement of the Tubular Excretory Mass, Effective Blood Flow and Filtration Rate in the Normal Human Kidney. J. Clin. Invest., 17:263—78. Sollman, T. 1957. A Manual of Pharmacology. Philadelphia: W. B. Saunders Co. p. 1281. Sterling, K., and S. J. Gray. 1950. Determination of the Circulating Red Cell Volume in Man with Radiochromium. J. Clin. In- vest., 29:1614-19. Strakhov, I. P. 1951. The Character of the Reaction of Chromium Complexes with the Most Important Groups of Proteins. Zhur. Priklad. Khim. USSR. 24:142-47. Referred to in Chem. Abst. 46:106li, 1952. 88 Sutherland, D. A., and M. S. McCall. 1955. The Measurement of the Survival of Human Erythrocytes by In Vivo Tagging with Cr51. Blood, 10:646-49. Udy, M. J. 1956. Chromium. Vol. 1. Chemistry of Chromium and Its Compounds. New York: Reinhold Publishing Corp. Chapters 5 and 6. US. Public Health Service. 1952. Division of Occupational Health. "Health of Workers in Chromate-Producing Industry." Public Health Service Publication No. 192. Washington. D.C.: Government Printing Office. Van der Walt, C. F. J.. and A. J. Van der Merwe. 1938. Colori- metric Determination of Chromium in Plant Ash. Soil. Water and Rocks. Analyst. 63:809-11. Visek. W. J., I. B. Whitney. U. S. G. Kuhn III. and C. L. Comar. 1953. Metabolism of Cr51 by Animals as Influenced by Chemical State. Proc. Soc. Exp. Biol. and Med., 84:610—15. Walker, H. M., and J. Lev. 1953. Statistical Inference. New York: Henry Holt and Co. Walsh, E. N. 1953. Chromate Hazards in Industry. J. Amer. Med. Assoc., 15321305. Waterhouse, C., and J. Holler. 1948. Metabolic Studies on Protein Depleted Patients Receiving a Large Part of Their Nitrogen Intake from Human Serum Albumin Administered Intravenously. J. Clin. Invest., 27:560—61. White, R. P. 1934. The Dermatergoses or Occupational Afflications of the Skin. London: H. K. Lewis and Co. 4th Ed. p. 155. Willard, H. H., and H. Diehl. 1950. Advanced Quantitative Analysis. New York: D. Van Nostrand Co., Inc. pp. 44-48. APPENDIXES 89 APPENDIX A FOUR-HOUR EXCRETION VALUES OF CHRONIIUM IN ACUTE ANESTHETIZED DOGS, FOUR-DAY EXCRETION VALUES OF CHROMIUM IN BILE FISTULA DOGS, AND DATA USED TO CALCULATE CHROMIUM CLEARANCES 90 91 FOUR-HOUR EXCRETION VALUES OF CHROMIUM IN ACUTE ANESTHETIZED DOGS (in pg. per collection period) _: r a Collection Dog Period 1 2 3 4 5 Urine 0-30 minutes .0275 2.797 1276.0 2499 8.924 30-60 minutes .0358 1.104 692.0 1243 4.858 60—90 minutes .0370 0.975 668.0 1011 2.846 90-120 minutes . . .0340 1.325 412.0 1108 2.076 120-150 minutes . .0201 0.672 272.0 962.8 1.787 150-180 minutes . .0186 0.754 236.0 995.0 1.530 180-210 minutes . .0231 0.580 288.0 940.2 0.888 210-240 minutes . .0278 0.598 296.0 579.6 0.674 Bile 0-30 minutes .0007 0.0442 3.200 9.016 0.0171 30-60 minutes .0027 0.0736 8.800 9.016 0.0342 60-90 minutes . .0012 0.0524 7.600 3.542 0.0278 90-120 minutes . . .0013 0.1113 25.60 5.796 0.0235 120-150 minutes . .0017 0.0442 29.60 3.864 0.0193 150-180 minutes . .0018 0.0414 28.80 2.898 0.0171 180-210 minutes . .0017 0.0883 27.60 2.899 0.0128 210-240 minutes . .0018 0.1049 29.60 2.897 0.0096 aDog 1 weight, 7.5 kg.; dose, .395 [lg/kg.; valence, +6. Dog weight, 12.5 kg.; dose, 7.28 (lg/kg.; valence, +6. Dog 3 weight, 10.0 kg.; dose, 4,000 (lg/kg.; valence, +6. Dog 4 weight, 10.0 kg.; dose, 3,200 [lg/kg.; valence, +3. Dog 5 weight, 11.0 kg.; dose, 9.40 pig/kg.; valence, +3. 92 FOUR-DAY EXCRETION VALUES OF CHROMIUM IN BILE FISTULA DOGS (in pg. per collection period) r I r a Collection Dog Period 6 7 6 Urine 0-2 ,hours ................... 1375 1.665 6.214 2-4 hours ................... 939 3 0.6773 2.312 4-8 hours ................... 937 5 0.6365 2.910 in 8-16 hours .................... 505 8 0.5141 3.500 16-24 hours .................. 567 2 0.3754 3.229 0—1 day ..................... 4325 3.868 18.165 1-2 days .................... 417 3 0.4366 1.844 2-3 days .................... 390 2 0.2203 0.9046 3-4 days .................... 231 2 0.1306 0.6468 Bile 0-2 hours ................... 10.84 0.1306 0.0481 2-4 hours ................... 48.59 0.0290 0.0297 4-8 hours ................ . . . 35.40 0.0139 0.0546 8-16 hours ................... 50.22 0.0187 0.0336 16-24 hours .................. 7.948 0.0094 0.0315 0-1 day ..................... 1530 0.2016 0.1975 1-2 days .................... 59.43 0.1469 0.0367 2-3 days .................... 22.04 0.0261 0.0271 3-4 days .................... 16.44 0.0078 0.0236 Feces 0-1 day ..................... 52.20 0.1783 1.071 1-2 days .................... 62.50 0.1199 0.2827 2-3 days .................... 24.75 0.0620 0.1512 3-4 days .................... 11.74 0.0622 0.1084 Dog 6: weight, 11 kg.; dose, 1,640 pg./kg.; valence, +6. Dog 7: weight, 15 kg.; dose, 2.72 pg./kg.; valence, +6. Dog 6: weight, 11 kg.; dose, 4.02 gig/kg.; valence, +3. 93 DATA USED TO CALCULATE CHROMIUM CLEARANCES r __i— #— Urine Clear- Time Collec- Urine Urine Urine after , Flow Conc. ance Inj. tion Vol. (ml./ (ng No. , Period (m1.) , (min.) , min.) ml.) (min.) Dog No. 8 l 78 20.5 200 3.12 0.0099 2 98 24.5 200 4.92 0.0099 3 122 19.5 200 4.32 0.0067 4 141 24.7 200 2.93 0.0077 5 166 15.5 200 2.64 0.0041 Dog No. 9 1 66 17.5 200 5.27 0.2640 2 82 21.0 200 6.56 0.2450 3 106 19.0 200 7.09 0.2070 4 127 21.7 200 6.02 0.2150 5 148 20.5 200 3.63 0.1960 Dog No. 10 1 117 32.0 200 1.82 0.4530 2 147 28.0 200 3.00 0.3000 3 218 24.0 200 3.56 0.1580 4 240 20.5 200 3.60 0.1200 5 321 25.0 200 3.99 0.0863 6 350 33.0 200 3.39 0.1130 7 430 20.0 200 3.62 0.0529 8 450 66.5 200 3.50 0.0881 94 DATA USED TO CALCULATE CHROMIUM CLEARANCES (Continued) # r Dial. Plasma Conc. Plasma Cd-chr Cd-chr (Hg-/ COI‘IC. F.F. —'E-—-—- -C-—-—-- (pg./ PAH creat. ml.) ml.) Dog No. 8 0.0634 - .47 - - 0.0607 - ’ .53 - - 0.0585 - .63 - - 0.0560 - .47 - - 0.0538 - .49 - - Dog No. 9 1.570 - .41 - - 1.493 - .40 - - 1.396 - .41 - - 1.335 - .44 - - 1.250 - .40 - - DogNo. 10 1.250 — .26 - - 1.180 - .32 - - 1.010 - .39 - - 0.980 - .37 - - 0.920 - .38 - - 0.910 - .41 - - 0.870 - .33 - - 0.850 - .34 - - F "W "- ‘ ‘ 7 F‘ n,“ 4 ’m<;-‘z *nmm xvv-vx “WW ~ _ 95 DATA USED TO CALCULATE CHROMIUM CLEARANCES (Continued) I . Urine . . 1 Clear- Time Collec- Urine Urine Urine after , Flow Conc. ance In' tion Vol. (ml/ ( / No. .3' Period (m1.) , ‘ ”g' (m1n.) . m1n.) m1.) (m1n.) Dog No. 11 l 60 24.0 200 0.37 0.0185 2 72 21.0 200 0.43 0.0135 3 183 22.0 200 1.11 0.0080 4 206 24.5 200 1.56 0.0087 5 297 13.5 200 2.25 0.0035 6 312 18.0 200 2.34 0.0043 7 457 26.5 200 2.50 0.0038 8 482 23.5 200 2.59 0.0031 Dog No. 12 l 122 27.7 200 1.75 0.0161 2 148 23.7 200 2.85 0.0126 3 342 40.0 200 1.12 0.0104 4 2880 68.0 200 0.48 0.0009 5 2940 70.0 100 0.22 0.0017 Dog No. 13 1 152 40.5 200 2.67 0.0262 2 266 41.0 200 1.12 0.0178 3 385 42.5 200 1.07 0.0100 4 512 43.5 200 0.48 0.0061 5 633 52.5 200 0.48 0.0061 Dog No. 14 1 63 39 380 9.23 0.3149 2 135 31.5 95 1.75 0.6391 3 220 31.5 104 2.35 0.4492 4 305 29.0 112 2.48 0.1843 5 393 26.0 92 2.00 0.1812 6 453 43.0 101 1.42 0.2327 96 DATA USED TO CALCULATE CHROMIUM CLEARANCES (Continued) [$213.9 ._ : Plasma D1a1. 13.2: Plasma C C C!” COnC . d- Chr (1" Chr (08/ Cone. F.F. T—" 5;. (pg./ PAH creat. . m1.) IT...I ml.) Dog No. 11 W 0.0670 0.0055 .38 .133 .350 “jg 0.0631 0.0045 .33 .115 .348 “jg 0.0470 0.0023 .50 .149 .299 “jjj: 0.0452 0.0020 .50 .175 .352 “:1 0.0402 0.0017 .53 .144 .270 if“? 0.0394 0.0015 .49 .131 .268 “-“jj’ 0.0318 0.0008 .49 .128 .263 “-1”- 0.0304 0.0007 .48 .139 .289 0.005; Dog No. 12 ‘ 0.0840 0.0073 .53 .118 .221 0.0131 0.0780 0.0062 .51 .111 .216 W 0.0525 0.0028 .35 .083 .227 0.0104 0.0153 0.0001 - - .245 0.005"3 0.0153 0.0001 - - .274 .0017 0 Dog No. 13 0.0593 0.0052 .35 .116 .330 0.02:3 0.0475 0.0035 .28 .104 .352 0.01:5 0.0390 0.0020 .28 .096 .341 0,0190 0.0322 0.0012 .29 .098 .341 0.00.1 0.0270 0.0009 .31 .118 .377 “-“CU Dog No. 14 1.155 0.1175 .29 .103 .357 0310 0.973 0.0725 .36 .137 .381 0,30 0.795 0.0452 .34 .132 .385 0,423 0.680 0.0320 .34 .082 .242 0'1,” 0.640 0 0225 .31 .095 .311 0'1513 0.632 0.0152 .30 .118 .390 (12337 III III] 1'1”!!!“ III IIIII -‘ "-5—: u‘" m '- 97 DATA USED TO CALCULATE CHROMIUM CLEARANCES (Continued) Urine Clear- Time Collec- Urine Urine Urine after , Flow Conc. ance Inj. tion Vol. (ml./ (ug/ N0. . Period (m1.) , (m1n.) , min.) ml.) (m1n.) DogNo. 15 1 74 33.5 132 2.75 0.5480 2 194 35.0 . 147 3.05 0.2704 3 281 35.0 103 1.80 0.2489 4 374 34.0 91.5 0.93 0.1858 5 496 35.0 71.0 0.31 0.1442 Dog No. 16 1 79 35.0 186 3.60 4.052 2 169 39.0 102 1.08 4.936 3 259 39.0 114 1.38 2.769 4 377.. 36.0 96 1.00 1.847 5 445 55.0 96 0.65 ‘ 2.141 Dog No. 17 1 103 28.5 244 5.75 10.73 2 133 30.5 253 6.98 12.27 3 258 37.5 290 6.67 14.01 4 350 27.5 211 5.49 13.71 5 409 35.0 176 3.31 17.42 6 444 37.0 187 3.43 16.96 Dog No. 18 1 71 36.0 250 5.83 0.0250 2 185 44.0 162 2.32 0.0554 3 309 33.0 113 1.61 0.0636 4 385 33.5 164 3.10 0.0478 5 468 40.0 140 2.00 0.0543 Numbera Mean Standard deviation Coefficient of variation hi ——_‘——i L aEach observation is an average of five to eight determina- tions. 98 3““ DATA USED TO CALCULATE CHROMIUM CLEARANCES (Continued) 1.7:: Plasma P1122;a C C “*“4' Cone. d-chr d-chr ,5? (P-g-/ Conc. F.F. j T— -—C-—-——-- ~' (P-g/ PAH creat. ml.) ml.) Dog No. 15 Céef; 0.8170 0.0076 .30 .098 .330 0.214 0.5550 0.0023 .30 .113 .372 0.14:2 0.4540 0.0022 .34 .118 .348 0:5: 0.4170 0.0015 .30 .108 .355 0.14;: 0.3500 0.0009 .31 .113 .361 Do; No. 16 4,352 14.80 0.7500 .35 .203 .573 4.93: 12.70 0.4100 .30 .197 .650 27:: 11.70 0.2570 ' .37 .213 .582 164' 9.70 0.1800 .36 .166 .458 z 11 8.90 0.1350 .32 .161 .505 DogNo. 17 1.3.73 7.50 1.660 .45 - .207 .456 13.2; 8.00 1.700 .45 .207 .464 14.01 13.0 1.480 .52 .256 .492 13.71 14.5 1.430 .50 .246 .492 17 .3 21.6 1.200 .47 .230 .493 , '0. 23.7 1.130 .42 .218 .524 1“ Dog No. 18 00.3, 0.0670 0.0082 .35 .119 .345 "0;; 0.1300 0.0091 .41 .137 .335 “'15.“; 0.1890 0.0084 .42 .131 .310 ““33; 0.2240 0.0080 .43 .148 .343 ““1; 0.2600 0.0064 .50 .146 .292 0.0:: / 9 11 8 8 0.397 0.140 0.369 0.075 0.045 0.102 0.189 0.320 ' 0.276 '6. \\ | APPENDIX B DETAILED PROCEDURES FOR PAH, CREATININE, AND CHROMIUM ANALYSES, AND FOR OXIDATION 51 51 OF Cr Cl3 TO NaZCr O4 99 100 DETAILED PROCEDURES FOR PAH, CREATININE, AND CHROMIUM ANALYSES. AND FOR OXIDATION OF Cr51Cl3 TO NaZCr5104 Protein-free Plasma Filtrate 1. Centrifuge the heparinized blood to obtain plasma. 2. Pipette 2 m1. plasma into 20 ml. water in a 50 ml. centrifuge tube. 3. Add 8 ml. 11.2 per cent trichloro-acetic acid solution. 4. Stopper, shake well, and let stand 10 minutes, shaking at least once. 5. Centrifuge for 10 minutes at 2500 r.p.m. and use clear supernatant for chemical analysis. Urine Creatinine l. Pipette 3 ml. of the final diluted urine into a 10 ml. test tube in duplicate. 2. Add 1 ml. 0.04 M picric acid1 solution. 3. Add 1 ml. 0.75 N NaOH and mix. 4. Let stand exactly 20 minutes and read in a colorimeter at 540 mu. Reagent blank: Three ml. distilled water in place of diluted urine. Other reagents are the same. J'Made by diluting saturated picric acid solution with water and standardizing to 0.04 M with 0.1 N NaOH and phenolphthalein. Standards: Three tubes containing 20, 40, and 60 pg. creatinine in 3 ml. distilled water. This is done in duplicate. A stand- ard curve is then prepared by plotting the per cent transmis- sion (B and L Spectronic 20 colorimeter was used) against content of creatinine on semilogarithmic paper. Plasma Creatinine Pipette in duplicate 3 ml. of protein-free filtrate into two 10 ml. test tubes and proceed as for urine creatinine. The creat— inine standards and reagent blank are the same as for urine creat- inine. Urine PAH . Pipette 3 m1. of the final diluted urine into a 30 ml. test tube containing 7 ml. water. This is done in duplicate. . Add 2 ml. 1.2 N HCl and mix. . Add 1 ml. NaNOz and mix. The NaNOZ (100 mg. per cent) is stored in the refrigerator and prepared fresh every three days. . After standing not less than three nor more than five minutes add 1 ml. ammonium sulfamate and mix. Ammonium sulfamate (500 mg. per cent) is stored in the refrigerator and prepared fresh every three weeks. 102. After standing not less than two nor more than five minutes add 1 ml. N (1—napthyl) ethylenediamine dihydrochloride (100 mg. per cent) and mix well. This reagent will keep indefinitely if stored in a dark bottle in the refrigerator. Let stand 20 minutes and read in the colorimeter at 540 mu. The color is stable indefinitely. Reagent blank: Three tubes containing 10, 20, and 50 pg. PAH in 10 ml. Ten ml. distilled water is used. Standards: distilled water are used. This is done in duplicate. A stand— ard curve is prepared as described for creatinine determina- tion. Plasma PAH Pipette, in duplicate, 5 ml. of protein—free filtrate into two 30 ml. test tubes containing 5 ml. distilled water and proceed as for urine creatinine. Reagent blank and standards are the same as in the urine PAH determination. Saltzman Procedure for Chromium 1. Each sample prepared so as to contain 4 to 15 pg. chromium in l to 10 ml. is ashed in 125 ml. Phillips beakers by adding 0.5 ml. concentrated HNO3 (redistilled) and 0.25 ml. 40 per cent 103 sodium bisulfate, and evaporating on a Lindberg hotplate. If or- ganic material is present the ashing procedure should be repeated. Duplicates are run on all samples. 2. Ten ml. 0.5 N H2804 is added to redissolve the sample, 0.5 ml. 0.1 N KMnO4 added, and the beaker heated for 20 minutes at 100° C. 3. Five per cent sodium azide added to the hot samples at a rate of 1 drop every 10 seconds with swirling between drOps, is used to decolorize the sample. Three to 5 drops are usually sufficient. 4. The decolorized sample is immediately cooled in a tray of cold water and transferred quantitatively to a 25 ml. volumetric flask. 5. Color is deveIOped by adding 1 ml. s-diphenylcarbazide reagent.1 6. One minute after color deve10pment, 2.5 ml. 4 M NaHzPO4 is added as a buffer and the flask diluted to the mark with distilled water. 7. The pink color is read at 540 mu. within 30 minutes. Reagent blank: Water is used instead of a sample and carried through the entire procedure. 1Prepared by dissolving 10 gms. phthalic anhydride in 175 ml. redistilled 95 per cent ethanol (warmed for solution) and adding 0.625 gms. s-diphenylcarbazide dissolved in 50 ml. ethanol, the combina- tion being made up to 250 ml. with ethanol. 104 Standards: 5, 10, and 15 ug. quantities of NaZCrO‘4 prepared in duplicate are added to Phillips beakers and carried through with the samples. A standard curve is prepared as described for urine creatinine. Oxidation of CrSlCl3 to NaZCrSIO4 1. Place sample to be oxidized in 125 ml. Phillips beaker and make basic with 2 ml. 6 N NaOH. 2. Add 1 ml. 3 per cent hydrogen peroxide and boil for 1 hour. Add distilled water as necessary to keep from drying out. 3. Add 0.2 ml. more 6 N NaOH while still hot. If any bubbles ap- pear, boil for 15 min. longer and again add 0.2 ml. NaOH. 4. Make neutral with 2 ml. 6 N HCl. APPENDIX C RENAL CLEARANCE AND STATISTICAL FORMULAS 105 106 RENAL CLEARANC E FORMULAS The basic formula used in renal clearance calculations is cX = (Ux-V)/(Px) (1) where Cx is the renal clearance of substance x in milliliters per minute, Ux is the urine concentration of substance x in undiluted urine, Px is the average plasma level of the substance during the urine collection period, and V is the rate of urine flow in milliliters per minute. A more workable formula is C = (U-V)/(P'T-SA) (2) where C is the renal clearance in milliliters per minute per square meter surface area, U is the urine concentration including all wash- ings, V is the urine volume including all washings over the entire urine collection period, P is the plasma concentration at a point three minutes before the mid-point of the urine collection period, and SA is the animal's surface area in square meters. This for- mula is easier to use in calculations because less effort is required to get the values into the proper units for substitution. In addition, it is felt that this formula provides a more accurate estimate of renal clearance than formula 1. In clearance measurements the rate of urine flow (V in formula 1) is subject to the largest error 107 because it depends on the volume of fluid minus washings obtained during the urine collection period. If, for example, a small volume of the last wash fluid is'left in the bladder at low rates of urine flow during a creatinine measurement, a large error in V of formula 1 would result but only a small decrease in the value U-V in formula 2 would be noted because the last wash fluid would contain little cre- atinine. 108 CORRELATION COEFFICIENT (r) l l/ 2 2 r = ny/ 22x -Zy where: 2xy = EXY - [(2X)(2Y)]/N 2 2 2 2x = xx - (EX) /N 2 2 2 2y = EY - (2Y1 /N N = number of observations. Significance of r is determined by referring to a table of percentile values of r when p = 0. 1 Statistical formulas, pages 108-10, based on Walker and Lev. 1953. 109 F STATISTIC The F statistic is used to determine if the slope of a plotted line is significantly different from 0. 2 2 F = [r (N-2)]/[1-r ] where: F and r are the same as in the correlatiOn coefficient. The F statistic in this instance has 1 degree of freedom for the numerator and N-Z degrees of freedom in the denominator. where: 110 "t" TEST N1 = number of observations in X1 N2 = number of observations in X2 2 s -— - = variance of difference between the means. X -X 1 2 2 2 2 2 2X -(X)/N +2X -(2X)/N 1 1 1 2 2 2 (N +N )/N N N1 + N2 - 2 1 2 1 2 Degrees of freedom : N1 + N2 - 2. .‘v‘fl T 11111111” “'11111111111111