THE DETERMINATION OF IRON IN BIOLOGICAL MATERIALS

BY THEODORE G. KLUMPP

(From the Department of Internal Medicine of Yale University, and the Medical Service of the New Haven Hospital, New Haven)

(Received for publication, April 11, 1934)

In an attempt to find a method for the determination of iron that could be used for the clinical study of iron metabolism, certain calorimetric procedures (1, 2) were studied. Some difficulties were encountered with these techniques and especially in their adaptation to the measurement of minute amounts of iron in such diverse materials as blood, food, feces, and urine. This led to the investigation of volumetric methods, with the final adoption of the titanium method, which proved eminently satisfactory.

In 1903 Knecht (3) and Knecht and Hibbert (4) described an oxidation-reduction reaction for the titration of ferric iron, involv- ing the use of a dilute solution of titanium chloride. Thornton and Chapman (5) in 1921 studied the chemistry of the reaction, confirmed its accuracy, speed, and relative simplicity, and defined more precisely its sphere of usefulness and the factors which modi- fied it. The titanium method was first adapted to the deter- mination of iron in biological materials by Jahn (6) in 1911. Peters (7) in 1912 used the Jahn modification, after changing the method of ashing, for his studies on the iron content of hemoglobin. Brukl (8) utilized titanium for the microestimation of iron, but his procedure was essentially the same as those previously described. Pincussen and Roman (9) in 1931 found the method satisfactory for the determination of iron in blood and organic materials, but L&szl6 (10) in the same year decided that the Jahn modification was less accurate for blood iron than Willstatter’s calorimetric estimation of hemoglobin.

The procedure which has finally been adopted contains certain technical details which contribute to convenience and accuracy

213

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214 Iron in Biological Materials

and a method for the preparation for analysis of die&, feces, blood, and urine. Diets and feces are sampled without drying after they have been stirred at high speed. All materials are ashed, after preliminary digestion with sulfuric acid, in a muffle furnace equipped with a thermopile which permits accurate control of t,emperature. Peters (7) found dry ashing superior to the Neuman wet digestion which had been employed by Jahn (6) and Pincussen and Roman (9), because it saved time and assured destruction of oxidizing agents and a neutral ash. Preliminary digestion with sulfuric acid eliminates chlorides, converting the ash entirely to sulfate and phosphate, and avoids spattering and creeping during the subsequent muffling. By the combination of this procedure with exact temperature regulation of the muffle the preparation of materials is simplified.

The error caused by the presence of copper was recognized by Knecht and Hibbert (11) who recommended for the removal of copper the standard procedure (12, 13) which has been adopted. Although it is possible that the separation is not complete, no appreciable error due to copper contamination could be detected in control experiments. Jahn (6) recognized the interference of copper but made no provision for its elimination. Pincussen and Roman (9), L&se16 (lo), and Peters (7) made no reference to copper. In agreement with Thornton and Chapman (5) KMn04 has been preferred to HzOz for reoxidizing iron to the ferric state after the removal of copper. Potassium permanganate, however, cannot be used with the trichloride of titanium as reducing agent.

The tendency for high concentrations of phosphates to suppress the color of ferric thiocyanate is less serious a problem in the volumetric procedure than it is in calorimetric methods; it cannot, however, be neglected and has been provided against. This point has been generally ignored in the titanium method, probably because it is a source of difficulty primarily in the analysis of diets, and no detailed application of the titanium method to foods has yet appeared. Jahn (6) showed that an artificial mixture of elec- trolytes resembling the ash of animal and plant tissues caused no aberration in the titration with titanium. The concentration of phosphates in certain foods, however, particularly milk, is signifi- cantly higher, and definit.ely interferes with the color development, a fact which has been recognized by others (14, 15).

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T. G. Klumpp 215

The necessity of excluding oxygen during the titration has been generally recognized. Both Knecht and Hibbert (11) and Jahn (6) protected the titanium reagent from oxidation by an atmos- phere of hydrogen; subsequent workers have universally employed COz (5, 9, 10). Both Jahn (6) and Thornton and Chapman (5) also provided an atmosphere of COZ over the surface of the solutions during titration. Further errors can be obviated by bubbling CO, through the solutions in advance of the titration to eliminate dissolved oxygen. By certain modifications of the apparatus previously employed, and especially by the intro- duction of immersion titration and the utilization of CO2 for stirring, as well as for the elimination of oxygen, and by ridding the solutions of oxygen in advance of titration, the sensitivity of the method has been greatly increased. The present procedure is sensitive to less than 0.005 mg. of iron.

Apparatus

The set-up illustrated in Fig. 1 is a modification of the apparatus described by Knecht and Hibbert (11) and Thornton and Chap- man (5).

It consists essentially of a storage bottle A for the titanium trichloride or sulfate which is kept under an atmosphere of carbon dioxide. This is provided by the Kipp generator B. C is a cali- brated 2 cc. burette, the top of which is connected by tubing with the top of the storage bottle A, in order to maintain the solution in the burette under an atmosphere of carbon dioxide. The tip of the burette is drawn out to capillary bore for immersion titra- tion. By means of a 3-way stop-cock D, a stream of carbon dioxide can be led to the two glass nozzles E, from which carbon dioxide is bubbled through the solutions for titration.

The side arm F, connected by means of a 3-way stop-cock to the pipe-line bearing the titanium solution, is utilized for two purposes: (a) to flush the pipe-line and burette clean of titanium solution, since it has been found that the slow oxidation of stag- nant titanium trichloride or sulfate leaves an obscuring white deposit of titanic hydrate; and (b) to admit freshly prepared or additional titanium solution into the storage bottle A with minimal exposure to the air. This is effected by suction at D.

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216

Reagents

Iron in Biological Materials

All water must be distilled from glass. Titanium trichloride or sulfate. Because of the small amounts

used, the ultimate expense of these reagents is not great. Either the 15 Fer cent or 20 per cent commercial solution is satisfactory.

FIG. 1. Apparatus for immersion titration in an atmosphere of CO2

The stock solution is prepared as follows: (a) 1000 cc. of boiled water, which has been distilled in glass, are sucked up through F into the storage bottle A. (b) A liberal stream of carbon dioxide is bubbled through the distilled water via F for about 10 minutes. (c) 15 cc. of a. 15 per cent solution of titanium trichloride, or 11 cc. of a 20 per cent solution of titanium sulfate are added to an

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T. G. Klumpp 217

equal amount of concentrated iron-free hydrochloric acid or sul- furic acid respectively, and boiled for 5 minutes to drive off possible traces of hydrogen sulfide. Boiling may be omitted with certain high grade titanium solutions now available. (d) The boiled solution, followed by three small rinsings of boiled water, is then sucked up into the storage bottle.

Potassium thiocyanate. A 3 N solution is prepared by dissolving 146 gm. of a high grade iron-free product in 500 cc. of distilled water. To this 20 cc. of acetone are added for preservative purposes.

Standard iron solutions. Dissolve 7.0226 gm. of ferrous ammo- nium sulfate of the highest purity (such as Kahlbaum), and pref- erably from a freshly opened bottle, in about 200 cc. of distilled water. Add 20 cc. of iron-free concentrated HzS04, warm slightly, and oxidize with 1 N potassium permanganate to the first perma- nent pink. Cool the solution to room temperature and dilute it to exactly 1 liter. This standard contains 1 mg. of iron per cc. Although the accuracy of the standard prepared in the above manner had been assured by both Knecht and Hibbert (Il.) and Thornton and Chapman (5), it was checked against standards pre- pared from (a) ferric ammonium sulfate made up according to Peters and Van Slyke (16), and (b) iron wire according to estab- lished technique.

Hydrogen sulfide. Used for the elimination of copper and other heavy metals, delivered from a Kipp generator in the usual manner.

Potassium permanganate. Approximately 0.05 N solution. In order to pursue iron investigations successfully it is quite

essential to have the exclusive use of all apparatus and glassware which, after scrupulous cleaning, should be rinsed with water dis- tilled from glass. Both reagents and glassware should be repeat- edly tested with a few drops of potassium thiocyanate and acid as a check against iron contamination.

Analytical Procedure

Technique for Determination of Blood Iron-l to 5 cc. of oxalated or defibrinated blood, drawn by means of either a platinum or a rustless alloy needle, are accurately measured with a pipette calibrated to deliver between two marks into a porcelain evaporat-

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218 Iron in Biological Materials

ing dish. The procedure to be described is equally applicable to the analysis of blood serum except that at least 10 cc. must be used. 0.1 cc. of iron-free concentrated sulfuric acid for each cc. of blood or for each 2 cc. of serum is added. The combination is dried for about 3 hours on the steam bath and then heated in the muffle furnace below 550” until it has been reduced to a fine white or pinkish white ash. At 500” this ordinarily consumes about 8 hours. Although it has been previously shown that no loss of iron or base occurs at temperatures below 600” (17), this fact was corroborated by recovery experiments. We found that loss of iron begins at about 700”. The ash is taken up with a few cc. of 20 per cent iron-free sulfuric acid. If there is much pink ferric oxide present, heating on the steam bath is generally necessary to effect solution. The iron solution is then quantitatively trans- ferred to a 25 cc. Erlenmeyer flask. A few drops of potassium thiocyanate and acid are admitted into the evaporating dish as a check upon the completeness of the transfer. The concentration of copper in blood is so small that the error introduced by its presence is negligible (18, 19). No significant alteration of iron values could be detected when the procedure for the elimination of copper was applied to blood.

Titration of Iron Solutions-In order to drive oxygen from the solution carbon dioxide is bubbled through it for at least 5 minutes by means of the nozzle E, shown in Fig. 1. Approximately 3 cc. of potassium thiocyanate are then added and bubbling continued for exactly 2 minutes more. With the tip of the burette C immersed in the solution, titration with titanium is then com- menced, the continued stream of carbon dioxide serving to stir the iron solution as well as to prevent absorption of oxygen at the surface. With each batch of determinations two standard solu- tions ordinarily containing 1 or 2 mg. each of iron are titrated, in order to establish the titer of the titanium. Two carbon dioxide nozzles are provided as shown at E in Fig. 1, so that the next solution to be tested can be saturated while the preceding titration is going on. Spontaneous reoxidation of the iron solutions does not occur while carbon dioxide is being admitted during titration, but takes place slowly afterward, becoming visible to a very slight extent after the titrated solutions have been exposed to the air for from 3 to 3 hours.

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T. G. Klumpp 219

In a series of duplicate analyses of thirty-four normal bloods the mean difference between duplicates was 0.003 mg., ranging from 0.000 to 0.010 mg. The data of recovery experiments are given in Table I.

Determination of Iron in Food-Food is particularly liable to significant contamination through iron-containing utensils. This

TABLE I

Recovery Experiments

Experiment No. Material Fe content

1

2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

FeNHr(SO& “ “ “ “

Blood “ ‘I “ “ “ ‘I “

Food “ “ “ ‘I

Stool “ “

Urine “

m!J.

05 0.5 1.0 2.0 2.0 0.761 1.255 1.434 0.607 0.868 0.506 0.410 1.242 0.905 0.890 1.306 1.510 1.358 7.842 8.770 9.332 0.513 0.428

Fe added

WT.

1.0 1.0 1.0 1.0 1.0 1.0 2.0 1.0 1.0 2.0 2.0 2.0 2.0 2.0 2.0 20 2.0 2.0

Fe recovered

m!7.

0.498

0.495

0.999

1.987 1.996 1.782 2.268 2.439 1.604 1.875 1.512 2.400 2.240 2.030 2.868 3.290 3.498 3.352 7.770 8.740 9.343 2.505 2.396

can be avoided to a large extent by the use of aluminum or monel metal containers and table utensils. If it is necessary to cut meat with a steel knife this should be done by cutting in a similar man- ner both the meat for the subject and the parallel portion for analysis. We prefer to use diets containing meat that can be readily subdivided without the use of a sharp edge. For metabo-

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220 Iron in Biological Materials

lism experiments two or three standard daily diets are prepared in duplicate, one of which is given to the subject, the other used for analysis.

For the purpose of iron determination, the meat is separated from the rest of the day’s diet, further subdivided by means of a glass knife, ground in a mortar, and digested on the steam bath by means of from 10 to 25 cc. of concentrated iron-free nitric acid. The digested meat with the remainder of the diet which has been previously triturated in a large mortar, is transferred to a weighed wide mouthed 2 liter bottle. After weighing the diet and bottle, the diet is stirred at high speed for 1 hour by means of a propeller of monel metal driven by an electric motor. About 80 gm. of an ordinary day’s diet are quickly transferred to previously weighed evaporating dishes with cover-glasses and weighed. During the stirring there is a loss due to evaporation which amounts to about 0.2 per cent. A correction for this is made. This method of mix- ing and sampling has been regularly used in this laboratory for nitrogen determinations on food and feces, and aliquots have been found to be in agreement consistently within a fraction of 1 per cent.

During the course of evaporation on the steam bath, a few cc. of 20 per cent iron-free sulfuric acid are added in order to form the sulfate of iron. The residue is reduced to ash in the muffle furnace below 550”. The ash is then taken up with 20 per cent sulfuric acid and treated to remove copper as follows: (a) Hydrogen sulfide is bubbled through the solution for about 10 minutes. This pre- cipitates the copper. (b) Th e copper precipitate is removed by filtration through a filter of fritted Jena glass followed by ten washings with 0.5 cc. each of 20 per cent H,S04 which has been saturated with H,S. (c) Hydrogen sulfide is driven off by boiling the filtrate for 5 minutes or longer. (d) The iron in the filtrate is reoxidized with 0.05 N KIMnO to the first faint permanent pink if titanium sulfate is to be used for the titration. The presence of hydrochloric acid with titanium trichloride makes it necessary to use an oxidizing agent other than potassium permanganate. In this case hydrogen peroxide may be used as described by Knecht and Hibbert (11).

Titration is carried out as described for blood iron. By means of this technique analyses of duplicate samples differ by less than

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T. G. Klumpp 221

2 per cent. Thus, for instance, a day’s diet was found to contain between 38.16 and 39.13 mg. of iron. Data on recovery experi- ments are given in Table I. When copper was eliminated from a diet sample, the titration value for iron fell from 1.056 to 0.822 mg. Because of the possibility that traces of copper escaped precipitation, tests were made to ascertain whether such losses, if present, were sufficiently great to impair the sensitivity of the method. A sample of blood was divided into six aliquots and treated in duplicate as follows: (1) iron determined; (2) iron determined after elimination of copper; (3) iron determined after addition of 0.3 cc. of 8 per cent CuS04 solution and elimination of copper. The values for iron in all three groups were found to be in agreement within the limits of error of the method. As an additional control 0.5 cc. of 8 per cent CuS04 was added to acidulated distilled water and carried through the procedure for eliminating copper and determining iron. A faint trace of color was produced when KCN was added, but it was not sufficient to permit titration.

Phosphates, especially pyrophosphates, are troublesome when they are present in concentrations large enough to cause decoloriza- tion of ferric thiooyanate. If this occurs, it is necessary to elimi- nate pyrophosphates by one of the usual methods (14, 15). Hor- witt (20) has suggested dry ashing at 500” with a sodium carbonate mixture to avoid formation of pyrophosphates.

Determination of Iron in Feces--Feces for a given period, col- lected in an iron-free container, are accurately transferred to a large, weighed, narrow mouthed bottle and, with an appropriate amount of water distilled from glass, weighed, and subjected to stirring at high speed for 1 hour. In the manner described for food, aliquots are removed, and, in the presence of sulfuric acid, evapo- rated to dryness. The size of the aliquot is governed roughly by the anticipated concentration of iron present. In general, whereas about 80 gm. of food are taken in the sample, half that amount of feces is ordinarily sufficient. The further procedure for muffling, transfer, and titration is the same as for food. The degree of accuracy is likewise approximately the same. Thus two samples of the same stool were found to contain 3.785 and 3.760 mg. of iron per 100 gm. of stool. In Table I recovery experiments are outlined.

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222 Iron in Biological Materials

Determination of Iron in Urine-The 24 hour output, of urine is transferred to a beaker of appropriate size and subjected to evaporation on the steam bath. When the volume has been reduced to approximately 200 cc., it is treated with a few CC. of dilute sulfuric and concentrated nitric acids until the residue has been completely dissolved. The solution is then accurately trans- ferred with the necessary washings to a porcelain evaporating dish or casserole and evaporated to dryness. Ashing in the muffle furnace and titration for iron are carried out in the same manner as for food and feces. The amount of iron in urine is small, but duplicate samples can be analyzed with an error of less than 2 per cent. Thus 2 liters of normal urine were found to contain exactly 0.271 mg. of iron each. Recovery experiments are given in Table I.

SUMMARY

A procedure for the determination of iron in blood, food, feces, and urine is given. The method of Knecht and Hibbert for the determination of iron by means of oxidation-reduction titration with titanium trichloride or sulfate has been modified and adapted to biological materials.

The guidance and generous cooperation of Dr. John P. Peters are gratefully acknowledged.

BIBLIOGRAPHY

1. Kennedy, R. P., J. Biol. Chem., 74, 385 (1927). 2. Wong, S. Y., J. Biol. Chem., 77, 409 (1928).

.3. Knecht, E., Ber. chem. Ges., 36, 166 (1903). 4. Knecht, E., and Hibbert, E., Ber. them. Ges., 36, 1549 (1903). 5. Thornton, W. M., Jr., and Chapman, J. E., J. Am. Chem. SOL, 43, 91

(1921). 6. Jahn, F., 2. physiol. Chem., 76, 308 (1911). 7. Peters, R. A., J. Physiol., 44, 131 (1912). 8. Brukl, A., Mikrochemie, 1, 54 (1923). 9. Pincussen, L., and Roman, W., Biochem. Z., 231, 54 (1931).

10. L&szM, T., Biochem. Z., 237, 483 (1931). 11. Knecht, E., and Hibbert, E., New reduction methods in volumetric

analysis, New York (1918). 12. Treadwell, F. P., Analytical chemistry, Quantitative analysis, translated

by Hall, W. T., New York, 7th edition, 192 (1928).

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13. Noyes, A. A., Qualitative chemical analysis, New York, 9th edition (1922).

14. Elvehjem, C. A., and Hart, E. B., J. Biol. Chem., 67, 43 (1926). 15. Leeper, G. W., Analyst, 66, 370 (1930). 16. Peters, J. P., and Van Slyke, D. D., Quantitative clinical chemistry,

Methods, Baltimore, 671 (1932). 17. Hald, P. M., J. Biol. Chem., 103, 471 (1933). 18. Schiinheimer, R., and Oshima, F., 2. physiol. Chem., 180, 249 (1929). 19. McFarlane, W. D., Biochem. J., 26, 1022 (1932). 20. Horwitt, M. K., Proc. Am. Sot. BioZ. Chem., 8, xli (1934); J. BioZ. Chem.,

106, xli (1934).

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Theodore G. KlumppBIOLOGICAL MATERIALS

THE DETERMINATION OF IRON IN

1934, 107:213-223.J. Biol. Chem. 

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