Daniel C. Harris University of California Davis, California 95616
Serum Iron Determination: A Sensitive Colorimetric Experiment
Nearly three quarters of the iron in an adult man is found in the oxygen hinding proteins, hemoglobin and myoglohin. One quarter of the iron is in the storaae forms. ferritin and hemosiderin. Only traces of the metal are found in all of the remainina forms in the hods, notably metalloenzvmes such as the cyiochromes. All of the iron "sed for biosinthesis is transported through the circulatory system hy way of the serum iron transport protein, trnnqferrin.' Harh molwule consists of a single peptide chain of molerular weight 81,400. containing two Fe(111) binding sites. Only ahout one third of the total sites are occupied in a healthy person. Since the transferrin concentration in the serum of an average adult is about 1.8-2.9 gfi, the total iron hinding capacity is in the range 2.5-4.0 pg Felml and the serum iron concentration is about 1pg Felml. We have modified an existing colorimetric procedure2 for the determination of serum iron for use in our freshman and sophomore level quantitative analysis laboratories. Our modification makes use of the ~ e ( f 1chelate, ) ferrozine, whose molar absorptivity is about 25% greater than that of the more commonly used hathophenanthroline." T h e procedure involves the following steps 1) Fe(II1) is reduced to FP(II) by hydroxylamine hydrochloride, NH30H+CI-, and thereby released from transferrin. 2) Addition of triehloroacetie acid (C13CC02H)causes d proteins to precipitate, but leaves the Fe(I1) in solution. The protein, which would interfere with the iron determination,is removed by centrifugation. 3) The liquid phase is treated with an excess of ferrozine in acetate-buffered solution to form a purple complex whose absorbance is measured. Fe(I0
+ 3 fenaine a
The mode of attachment of the ferrozine shown
not
been established. The molar absorptivity of this complex at 562 nm is 28,000 M-' cm-' and the overall formation constant for the reaction is 10'5s%.4 T h e only metal commonly found in serum which can interfere with the measurement of Fe bv ferronine is Cu. The results of the present procedure could he around 10%high hecause of such interference. hut a ~ ~ .technique ~ is ~ available for more accurate Other ions'which can he analyzed with ferrozine, besides Fe(I1) and Cu(II), include Co(II), Ru(III), and OS(VIII).~Sensitive tests for Fe using ferrozine have heen developed for natural waters. plant materials, and reagent chemical^.^ Human blood typically consists of 45% cells and 55% plasma (by volume). The plasma obtained from a blood hank contains
the anticoagulant, citrate, which interferes with the iron determination described below. If whole blood is collected without anticoamlant. the cells clot and can he removed. The resulting liquid,ldevoid of clotting proteins and anticoagulant, is called serum. I t is serum which is used in this exoeriment.
Experimental Materials Serum was obtained from humans or horses. Analysis of the in,tro&r3s or students2sem created additionalinterest.~ l ~ ~ only be drawn by qualified medical personnel. Serum is alsoavailable in small quantities from blood banks. (CAUTION! Any sample of human or animal blood should he treated as a potential carrier of disease, notably hepatitis and equine encephalitis. It should not be allowed to make contact with an open wound. ~ n ~ tsplashed h i on the skin should he washed immediately.) Serum was prepared from whole hlood by the instructor. After aelotting time of 3-6 hr at rmm temperature (overnight for horse serum), the was de. canted from the were removedby mass. Suspended centrifugation in a desk top centrifuge. Serum should be stored frmen to nrevent bacterial erowth. F ~ ( I I I )stock soluiion containing 1.00 mg Felml is prepared hy dissolving 1.00g of iron wire in 40 ml of 12M HCI and diluting to 1.00 1. Hydroxylaminehydrochloridesolution should contain 16gfiin 0.10 M HCI. Trichloroacetic acid solution should contain 300 gfi in water. Ferrozine (3-(2-pyridyl)-5.6-bis(4-penylsuffonic acid)-1,2,4-triazine, sodium salt from the Sigma Chemical Co.) is prepared by dissolving 1.0 g of the sodium salt in 500 ml of H20,adding and dissolving 3.0 moles of sodium acetate, and diluting to 1.0 1.
Procedure Because the procedure is so sensitive, dirty glassware is a prime source of error in the experiment. Therefore all glassware,including pipets, was soaked in 5 M nitric acid for 1-2 hr followed by several Gses with distilled or deionized water to remove traces of metal. The entire cleaning procedure was done in batches in the stockroom. From the Fe stock solution, students prepare working standards containing 1.00, 2.00, or 3.00 *g Felml using distilled water for all dilutions. Studentsanalyze one of each stsndard, ss well as two blanks (distilled water),and two or three replicate serum samples. If enough glassware is available, it is advised that duplicate standards be analyzed. In student hands, the absorbance of the blanks is nearly 4Wb of the absorbance of the most dilute standard, so it is important to have an accuratelv known value. T
