V O L U M E 25, NO. 9, S E P T E M B E R 1 9 5 3 Compounds,” curYe 437, Kew York, John Wiley & Sons. 1951. (3) H a m m e t t , L. P., Dingwall, Andrew, a n d Flexser, L. d.,J . Am. Chem. SCC.,57, 2103-15 (1935). (4) Meyer, K. H., “Organic Syntheses,’ p. 60, Coll. Vol. I, 2nd ed., New York, John Wiley Br Sons, 1944. (5) hlorton, R. A., and Earlam, W. T.,J . Chem. soc., 1941, 159-69.
1337 (6) Seshan. P. K., Proc. Indian Acad. Sci., 3A, 148-71 (1936). (7) Sielisch, J., Z. angew. Chem., 39,1248-9 (1926). ( 8 ) Sokolov, P. I., a n d Gurerich, L., Zhur. Khim. Prom., 5, 308-9 (1928). (9) Stone, K. G., private communication. (10) Vainshtein, Y. I., Zanodskaya Lab., 15,411-13 (1949). RECEIVED Sovember 25, 1952. Aocepted July 6, 1953.
Improved Spectrophotometric Procedure for Determination of Serum Iron Using 4,7-Diphenyl-1,lO-phenant hroline RALPH E. PETERSON‘ Department of Biochemistry, Army Medical Service Graduate School, Walter Reed Army Medical Center, Washington 12, D . C .
Published procedures generally used for the determination of serum or plasma iron use chromogenic reagents which lack the desired sensitivity for the concentrations of iron usually found in serum, and when larger volumes of sera are used poor recoveries are obtained. The compound 4,7-diphenyl-l,10phenanthroline has a molar absorbancy index of 22,400 for iron, and with the use of this reagent in isoamyl alcohol the serum iron may be readily extracted from a trichloroacetic-thioglycolic acid supernatant solution obtained from 1 to 2 ml. of serum, with a precisionof 3 ~ 5 %and with 95 to 100% recoveries. In addition, this procedure m a y be used to determine iron in wet-ash digests of other biological material.
I
N T H E past few years there has been an increasing interest in the nonhemoglobin or transport iron fraction of the serum. .41so, the recent introduction of procedures for iron turnover studies has necessitated the use of precise measurements of the serum or plasma iron. Most standard published procedures ( 1 , 3, 6, 7, 11, 12) have certain shortcomings-viz., alackof sensitivity with the small quantities of serum that can conveniently be used and inadequate recoveries of iron. The lack of sensitivity of the methods requires the use of several milliliters of serum and makes the procedure of limited value for routine clinical studies. Also, when large amounts of sera are used [except in wet-ashing and extraction procedures ( I S ) ] poor recoveries of the iron are obtained. Wet- (5, I S ) and dry-ashing procedures are too time consuming to be of practical value, and the latter gives poor and variable recoveries of iron (8). A recently published method using small quantities of sera is seriously handicapped by the fact that special equipment is required. It cannot be used on icteric sera, and requires various arbitrary correction factors (3). The author has been unable to reduplicate results with some other recently published methods ( 2 , 10). The introduction of the compound 4,7-diphenyl-l,lO-phenanthroline by Case (C), and its application to the determination of iron in water by Smith, McCurdy, and Diehl(16) have afforded a means of partially circumventing some of the troublesome aspects of standard serum iron methods. This reagent makes it possible to measure smaller amounts of iron than can be measured with thiocyanate and other of the ferroin reagents (Table I). I n addition, the ferrous complex can be extracted from aqueous solutions by immiscible solvents such as the amyl alcohols; by this simple extraction procedure the iron may be concentrated to a 1
Present address, Clinical Center, National Institutes of Health, Bethesda,
Md.
small volume and the sensitivity increased. Also, this extraction removes the ferrous phenanthroline from ions that interfere with color development, and further adds to the specificity by removing the colored complex from the aqueous media which often has some background color or turbidity that may contribute to falsely high values (7). Table I.
Relative Sensitivities of Various Chromogenic Reagents Used for Determination of Iron Wave Length Maximum .Ibsorption,
Molar Absorbancy Chromogenic Agent x Index Thiocyanate 474 7,0005 Dipyridine 522 8,600 552 11,000 Tripyridine 1,lo-Phenanthroline 512 11,000 4.7-Dimethyl-1,lO-phenanthroIine 510 14,000 4,7-Diphenyl-l,IO-phenanthroline 533 22,400 a Approximate. Determinations of wave length maximum absorption and molar absorbancy index made with Beckman DU quartz spectrophotometer.
It is the purpose of this paper to describe the application of this phenanthroline derivative to a procedure for the determination of the serum iron, and to present data regarding the recovery of iron from serum. REAGENTS
Trichloroacetic acid-thioglpcolic acid reagent. Make a 20% aqueous solution of redistilled trichloroacetic acid, and add 1% of thioglycolic acid. [The redistilled trichloroacetic acid may be prepared by using an apparatus similar to that described for redistilling antimony trichloride ( I S ) ] . This reagent keeps indefbiteG. Sodium acetate, saturated (reagent grade, Merck). If significant iron contamination is present in the sodium acetate
ANALYTICAL CHEMISTRY
1338 it may be purified by adding a few drops of thioglycolic acid and shaking with the amyl alcohol-phenanthroline reagent. 4,i-Diphenvl-l,lO-phenanthrolinc,0.0025 Af in isoam!,l alcohol. Thii may be purchased alreadv prepared from G. Frederick Smith Chemical Co., Columbus; Ohio. Isoamyl alcohol, good reagent grade, or redistilled. Ethyl alcohol, 95 to loo%, good reagent grade or redistilled. Standard iron solutions. Dissolve iron wire (99.8%) in mixture of a feiv milliliters of r w g m t grade concentrated nitric and hydrochloric acids, and dilute with iron-free water to give a concentration of 1 mg. per ml. From this stock solution dilute standards containing 1 to 2 miwograms pdr milliliter may be prepared. These should be prescmwi with a few milliliters of reagent grade (Merck) concentratpd sulfuric acid. -111 water used in the determinations and the reagents must be distilled from an all-glass distillation apparatus. All glassware must be rinsed in either strong nitric or hydrochloric acid and thC.11 again rinsed two or ttirw times nith iron-free distilled \vatr.
PROCEDURE
Add 1 to 2 ml. of serum or plasma to a 15-ml. centrifuge tube. fhc amount depending upon the anticipated concentration of iion. Make to a volume of 6 ml with iron-free water and mix n ith a thin stirring rod. Add 2 ml. of 2001, trichloroacetic-thioglycolic acid reagent, mix. :ind Irt stand 5 to 10 minutes Place in a mater bath a t 90" to 95' C. for 10 to 15 minutes, remove, and centrifuge. Decant the supernatant solution into a 20-ml. (19-mm. diameter) glass-stoppered test tuhe, or a 25-1113. glass-stoppered graduate cylinder. To the precipitate in the centrifuge tube add 2 ml. of watei 0.5 ml. of trichloroacetic acid-thioglycolic acid reagent, and mix. Return this tube to the watrr bath, 90" to 95' C., for 5 to 10 minutes. Remove, centrifuge, and add to the first supernatant solution. Add 2 ml. of saturated sodium acetate to the combined supernatant solutions. This should bring the resultant supernatant solutim to a pH of 4.0 to 5.0. Add 2 ml. of the phenanthrolinc reagent in isoamyl alcohol ( I t is necessary to use 0.5 ml. of this reagent for each microgram of iron.) .\dd isoamyl alcohol to a total volume of 6 ml. Stopper the tulle or cylinder and shake with 25 or more inversions. PiDet 5 ml. of the alcohol laver off and add to a Coleman cuvet. Add 0.5 ml. of ethyl alcbhol and mix. (This addition of ethyl alcohol is necessary to obtain a clear solution.) Reagent blanks and standards containing 2 to 4 micrograms of iion are carried through the above procedure along with the unknown sera. It is also advisable to carry a standard serum though with each group of unknown samples. Read a t a wave length of 535X in the Coleman Junior spectrophotometer (19-mm. cuvet) against the reagent blank set a t 100% transmit tance.
quartz spectrophotometer :It 533X. the Coleman Junior spectrophotometer a t 535X, and the Evelyn filter photometer a t 540X. Stability of Color Complex, and Optimum pH and Time for Color Development. The red color of the ferrous complex develops immediately and has been shown to be stable to diffuse light for a t least 8 months in this laboratory. With the method as described, at a pH of 4.0 to 7.0, maximum color of the ferrous complex is obtained. Effect of Presence of Foreign Ions. The use of the extraction process in the isolation of t'he colored complex from aqueous solution extends specificity so that no interference results with any of the common cations (calcium, magnesium, sodium, potassium) or anions (carbo:iate, chloride, sulfate, phosphate) present in biological materials. Cobalt in greater than microgram amounts produces a yellow color, but this is not extracted from the acid solution. Copper in microgram amounts forms an alcohol-soluble yellow complex with 4.7-diphenyl-1,lO-phenant,hroline in neutral or alkaline solution, but on acidification is transformed to a colorless complex (16). The specificity of this reagent is further increased by the fact that the ferrous complex is removed from the aqueous layer, whirh often shows some opalescence.
:+
EXPERIMENTAL
Sensitivity and Optimum Concentration Range. The colored ferrous 4,7-diphenyI-l,lO-phenar1throlinecomplex in isoamyl alcohol has a molar absorbancy irides of 22,400 a t its point of maximum absorption, 533X (Beckman DU spectrophotometer). This corresponds to a sensitivity of approximately 1 X (0.01 p.p.m.). Table I s h o m the differences in the absorption of 4,7-diphenyl-l,lO-phenantliroliiie and some other sensitive colorimetric reagents for iron. K i t h the method as described in this paper, a plot of the per ccvit absorbancy against the logarithm of the concentration s h o w t h inflection ~ of the curve indicating a concentration range for l w t accuracy to be in the region of about 1.5 to 8.0 micrograms of iron (Figure 1). This range may be extended a t the lower concentrations by using a smaller volume of solvent and, if feasible, a longer light path. Conformity to Beer's Law. This ferrous complex in amyl alcohol conforms to Beer's law at 535X in concentrations up to 9 micrograms with the described methotl. This holds true for essentially the same range of allwrtxmcy with the Beckman DU
MICROGRAMS
Figure 1.
IRON
Absorbancy us. Concentration
Effect of Concentration of Phenanthroline Reagent and Extraction. To ensure that an excess of 4,7-diphenyl-1,10phenanthroline is present to combine with the iron, 0.5 ml. of the 0.0025 M phenanthroline compound should be used for each microgram of iron. Under the conditions of this method, using one extraction (25 inversions), practically complete extraction of the iron is obtained. Smith et a/. ( 1 6 ) postulate a distribution coefficient of the ferrous complex in amyl alcohol of approximately 1500 to 1. Radioactive iron recovery studies have confirmed the rationale of using the above ratio of phenanthroline reagent to iron, and the completeness of recovery of the fei rous comclex 1%ith one alcohol extraction. Recovery of Iron from Serum. Table I1 lists the percentage iecoveries of iron from serum as carried out by various different procedures. Columns -4,€3, E , and F represent averages of studies on three different sera, and C and D are average values of five separate sera. These represent studies of both pooled sera and sera from a single individual. I n all cases (except E and F ) the final volume of the trichloroacetic acid filtrate was maintained a t 7.5 to 9.0 ml. Serum containing radioactive iron, adde 1 both in vitro and in vivo, and the proteins precipitated with the trichloroacetic-thioglvcolic acid reagent (column D ) give nearly quantitative recoveries of iron. [Radioactive iron analvsis was carried out by a procelure previously described ( I d ) ] . These results would suggest that nearly all of the iron is removed from the serum proteins, and also that very little of thp iron is coprecipitated with the proteins when 2 ml of sera or leare used. IYhen the proteins ?re precipitated n ith trichloroncr-
V O L U M E 2 5 , NO. 9, S E P T E M B E R 1 9 5 3
1339
tic acid alone (column A ) ,pieliminary treatment with dilute h j drochloric acid followed by trichloroacetic acid (column B ) , or first denaturation of the proteins with heat followed by trichloroacetic acid precipitation with heat (column C), slightly less of the iron is recovered. This is also true in the methods that rely on an initial treatment of the serum with 6 M hydrochloric acid followed by trichloroacetic acid precipitation, without heat (6). The loss of iron is only noticeably less in the procedure that uses trirhloroacetic acid alone without heat (column A ) . These results with trichloroacetic acid precipitation confirm previously reported studies ( 5 , l Z . I ; .19). The slightly better recoveries of iron obtained by carryirig out the precipitation of the proteins in the presence of a strong reducing agent (thioglycolir aci~l)has been previously suggested (16, 17, 1 8 ) . \Vhen recoveries of iron are determined spectrophotometricallv n ith 1,lO-phenanthroline, lower recoveries are obtained than with the radioactive iron method of analysis. This is also true when inpyridine or tripyridine are substituted for the 1,lO-phenanthroline. These results would suggest that not all of the iron is unnccounted for through stable protein binding, and copreripitation. Incomplete recoveries of iron are also obtained when a wetasheti digest (hydrolyzed t o rpinove pyrophosphates) is made and color is developed with 1,IO-plie~iarithroline(column E ) . Also, recoveries of decreasing magiiitude result when iron is added directly to trichloroacetic acid supernatant solutions obtained From 1, 2, 3, and 4 ml. of sera, and the color is developed with 1,10-phenanthroline (column F ) . K i t h a trichloroacetic acid supernatant solution equivalciit to l e s than 0 5 ml. of wra. qu:iiitit:ttive recoveries may be obt,iineti.
Table 11. Percentage Recoveries of Iron from Serum as Determined b y \-arious Different Procedures Serum, 111.
1.00 1.0: 1.0
?.Oa
2.0: 2.0
A 90.0
r ...
100
91.0 95.0
100 91,s 100
101
86.3 77..7
89.0
97.2
83.0
i9.i
65.0
64.0
84.5 74.2 83.0
89.5 86.6
76.0
... ...
4.0;
70.5
A.
E 100 94.5 99 0
84.5
77.0 68.0
59.5
4.OC
D 98.0
92.0 ...
3 .O C
4.0
C 96.0
...
88.0
3.0a
3.0°
B 94.5
,..
...
85.5
81.3
.
..
, . .
9% 0
91 0 87.0
... ...
...
95.0 97.5
93.0
85.0 88:0
... ...
93.3
..
91
...
89.0
98.0
99.2
. . , . .
extraction of the ferroin complex formed with 4,i-dipheny1-1,10phenanthroline in amyl alcohol, 95 to 100% recoveries are obtained. This compares favorably with recoveries as measured with radioactive iron analysis. Also, when 1 to 2 ml. of sera are wet ashed, or when iron is added directly to the trichloroacetic acid supernatant solution, and the iron is determined by this procedure, quantitative recoveries are obtained. In these studies, where the final volume of the trichloroacetic acid supernatant solutions were kept constant a t 7.5 to 9.0 ml., decreasingrecoveries of iron were obtained with increasing amounts of sera used. However, if the ratio of sera to final volume of t I ichloroacetic acid supernatant was kept constant, then recoveries obtained with 4 ml. of sera were approximately the same as those with 1 ml., providing an amount of trichloroacetic acid supernatant solution containing the equivalent of 1 ml. of sera were used. In addition, with a constant volume of trichloroncrtic arid supernatant solution, better recoveries of iron were obtained from a serum containing 3 or 4 grams % of protein than from one rontaining i to 8 grams %. These findings suggest that i n the presence of a constant volume of trichloroacetic acid supernatant solution there is an inverse relationship of the recovery of iron to the concentration of protein. The above findings seem to indicate that the following factors may play a role in the incomplete recoveries of iron as obtained by commonly used serum iron prodedures: ( a ) incomplete removal of protein-bound iron (column A ) ; ( b ) coprecipitation of the iron nith the proteins when too large a volume of serum is used (column D); and (c) interference x i t h the color reaction in aqueous trichloroacetic acid supernatant solution, or wet-ash digest (column E and F ) . The method as described in this paper is rapid and simple. I t has no greater precision or reproducibility than some of the other published methods-an error of *5% under favorable conditions. However, it is more sensitive, and more accurate (gives more nearlv quantitative recoveries). No significant interference fioin hemoglobin occurs in concentrations less than 20 mg. %. This compares favorably M ith other trichloroacetic precipitation procedures, and is preferable to methods requiring wet ashing in which small amounts of hemoglobin produce a significant error. Ho\-ierer, this colorimetric extraction procedure may easily be adapted to the determination of iron in wet-ash digests of biological materials ( E , Table 11)such as tissues and hemoglobin.
...
86.0 99.0
Proteins precipitated with trichloroacetic acid, without heat.
B. Proteins precipitated by 0.1 .If HC1 and trichloroacetic a r i d Rnrkan
a n d Walker ( 1 ) :
C.
Probins precipitated by method of Kitzes et al. ( 1 1 ) .
E. F.
Wet-ash digests. 0.7 ml. of HsSO4, 1.0 ml. of HXOs, 0.5 ml. of I120?. Trichloroacetic acid supernatant solutions only.
D. Proteins precipitated by method described in this paper.
These results are in line xith the studies of Jackson (9). who found that there were a t least two substances present in a digest of biological material after wet ashing that interfere with the colorimrtric methods used for the determination of iron in an aqueou* medi;i-pyrophosphates n-hirh can be removed by hydrolysis, :ind an unident,ified component that he was able to remove through precipitation of the iron with hydrogen sulfide. Therefore, eit,her some “substance” in the trich1oro:wetic acid supernatant solution i; inhibiting color, or a portion of the iron is not being releaseti from stable organic or inorganic complexes, and riot reacting with the chromogenic reagent. When recoveries of iron are determined by the method described in this paper. using l to 2 ml. of sera with precipitation of the proteins and release of protein-bound iron with trivhloroacetic-thioglyrolic acid and heat. and development of the color by
LITER.ATURE CITED
(1) Barkan, G., a n d Walker, R. W.,J. B i d . Cliern., 135, 3 - 4 2 (1940). (2) Budtz-Olson, 0. E., J . W r i . Fatiiol., 4, 92-8 (1951). (3) Burch, H. B., Lowry, 0. H., Bessey, 0. .I.,and Rerson, B. %., J . B i d . Chem., 174, 791--802 (1948). (4) Case, F. H., J . Org. C h m z . , 16, 1541-5 (1951). (5) Fon-eather, F. S., Rioclirm. ,J., 28, 1160-4 (1934). (6) Heilmeyer, L., and Plotner, K.. “ D a s Perumeison uiid die Eisenmangelkrankheit,” J e n a , Fischer, 193i. 8:s) Hover. G., Scand. J . Lab. C l i n . M d ,2 , 1 X - 3 2 (1950). (8) Hummel, F. C., a n d Willard, €1. €1.. I s u . Exc. CHEM.,.\NIL. ED.,10, 13-16 (1938). (9) Jackson. 6. H., Ibid.. 10, 302 (1938). (10) Jones, Frances, ASAL. CHEM.,21, 1216-17 11949). (11) Kitzes, G . , E l r e h j e m , C. A , and Schuette, R. .I,, J . B i d . Churn , 165, 653-60 (1944). (12) >loore, C.V.,Arrowsmith, W.R., Quilligtrii, $1. .J.. arid R e a d , J. T., J . Clin. Incest., 16, 613-26 11937). ~.. 22, I570 -1 (1930).. (13) lIueller, A,, a n d Fox, G. H., A 4 s . ~CHEX. (14) Peterson, R . E., Ibid., 24, 1850-2 (1952). (15) Ruegamen. W.R., Llichael, L., and Ellvehjem, C . -4.. J , Bid. Chem., 158, 575-7 (1945). (16) S m i t h , G. F., RlcCurdy, W. H., and Diehl, €1.. I r ~ c d y . s / ,77, 418-22 (1952). (17) T o m p s e t t , S.L., Biochem. J . , 28, 1837-43 (1934). (18) Ibid.. pp. 1802-6. (19) Tompsett, S. L., and LIcAllistri.. O., d r t a l y s t . 74, 315-16 (1949). RECEIVED for review February 21. 19.33,
Accepted .J~ine10, 1953.
