The following in vitro methods have been used todetermine the total iron-binding capacity of serum:(A) Excess iron is added to serum to saturate theiron-binding capacity. The unbound iron is removedby using phenanthroline ( 1,2 ) or iron-exchangeresin (3) or magnesium carbonate (4), and thenthe bound iron is extracted and determined colonmetrically. (B ) The amount of serum-iron (SI)(2,5—9) and the unsaturated iron-binding capacityof serum (UIBC) ( 10—18) are determined, respectively, and then the two values are added (1 9) . (c)A method to measure transfernin quantitatively byusing an antigen-antibody reaction has been devisedto calculate TIBC ( 19—21) . These methods for theroutine determination of TIBC are relatively difficult to perform and are rather time consuming. Thispaper describes a simple method to remove serumiron without the loss of iron-binding capacity oftransfernin which makes possible direct and rapiddetermination of TIBC.

METHOD OF INVESTIGATION

Effect of desferrioxamine on removal of serumiron. Attempts were made to remove iron from transfernin at neutral pH by using desfernioxamine (DFO)(22 ) , an iron-chelating agent. The DFO failed toremove iron from transfernin, and complete removalof DFO from serum was extremely difficult. Even ifiron is removed by an iron-chelating agent withoutdamaging transfernin, removal of the chelating agentwill be difficult. These attempts were therefore abandoned.

Effect of various acids. Below pH 5.5 iron doesnot combine with transfennin. The iron-binding Capacity of transfernin was not always recovered whenit was treated in a too strongly acidic solution. Somereducing acids were conceived to be more effectivethan ordinary acids in terms of ability to release ironfrom transfennin. Reagents such as thioglycolic acid

and hydnoxylamine hydrochloride were tested, buttheir use resulted in a decrease of TIBC. A 2%ascorbic acid solution, with a pH of approximately3, was effective. The fresh ascorbic acid solution wasadded to serum in equivolume. The pH of this mixture was approximately 5.5. For these studies ahighly soluble ascorbic acid in granular form wasused*.

Ammonium sulfate solution to remove iron wasstudied. Saturated ammonium sulfate solution wasadded to the acidified serum with ascorbic acid, andtransferrin was precipitated. The supernatant wasdecanted, and the precipitate was dissolved in water.The percent of iron removal effectiveness using thetwo cycles of precipitation and dissolution was over90% . This procedure,however,is too troublesometo carry out routinely. Calcium carbonate was alsotested, but it failed to remove iron from the serumsolution, acidified with ascorbic acid.

Ambenlite IR-120 granules were evaluated fortheir ability to remove iron ion in acidified serum.IR-120 was soaked in N hydrochloric acid andwashed repeatedly for days. However, when 0.2 gmof this washed IR-120 was added to serum, it becamestrongly acidic, and a decrease in TIBC was foundalthough iron was removed. This phenomenon maybe caused by the denaturation of transferrin. Toavoid a decreased TIBC, resins were tested withoutsoaking in hydrochloric acid. Because powder possesses a greater active surface area than granule,Amberlite CG-120 Type-3 was added to serumwithout any pretreatment.

Received June 26, 1970; revision accepted Jan. 22, 1971.For reprints contact: Hiroshi Saito, Nagoya University

School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya,Japan.

* “Hicee― manufactured by Takeda Pharmaceutical Co.,

Ltd., Osaka, Japan. One gram of “Hicee―contains 250 mgof ascorbic acid, 5 mg of calcium pantothenate, and 745 mgof excipient.

Volume 12, Number 7 489

NEW METHOD FOR DETERMINING TOTAL IRON

BINDING CAPACITY OF SERUM (TIBC) WITH RADIOIRON

BY ELIMINATING IRON FROM TRANSFERRIN

Hiroshi Saito

Radioisotope Laboratory, Nagoya University School of Medicine, Nagoya, Japan

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TABLE 1. EFFECTONOF

UIICTIBC VALSATURATIONUES.

Conditions@g/1OO

mlSerum

ASerum BSerumCUIBC

saturatedwlihiron234392273Notsaturated230385273Not

saturated234393283

SIUIBCHemato.TlBC(@&g/lOOml)(/Lg/100(@g/1OOlogicallyml).ml).normal@FeColon

@FefemalemethodSI+ UIBCmetricmethod

SAITO

at 3,000 rpm for 5 mm by a centrifuge with a radiusof 12 cm or longer. Two milliliters of the supernatant solution is obtained.

Determination of TIBC. One milliliter of 59Feferric ammonium citrate containing 6 @gof iron isadded to 2 ml of the above supernatant solution(equivalent to 1 ml of serum), and then 1 ml of2% sodium bicarbonate solution is added. Mix well.Incubate at 37°Cfor over 15 mm. One-fourth grameach of dry Amberlite CG-120 Type-3 and wetIRP-67 powder which have been thoroughly swollenwith water and from which excess water has beenremoved by centrifugation in a filtration funnel isadded to the mixture. The mixture is stirred withan automatic mixer*, let stand for 5 mlii, and centrifuged. After centrifugation for 5 mm, 2 ml of thesupernatant solution is transferred to a countingtube and then the radioactivity is counted in a wellscintillation counter.

The TIBC is calculated as follows:

Fe content of std. Fe solution (@g/100 ml)flBC —@ cpm in 2-ml supernant solution X 2@

— cpm in 2 ml of standard iron solution

To make 2 ml of standard solution, 1 ml of waterand 1 ml of ferric ammonium citrate solution areadded. Counting the standard iron solution is necessary, but pre-counting of each sample is not required.

Experimental results and discussion. Using thismethod, the TIBC was determined. No significantdifferences were observed between the TIBC value

a Model TB-I, Taiyo-Bussan K.K., Kanda, Tokyo, Japan.

For the percent removal of iron, transferrin waslabeled with 59Fe and the decrease of the radioactivity was used as an indicator. The removal ofiron was 95 % in 10—30mm at 2 1°C,and it was96% in10 mm, 97% in20 mm, and 98% in30 mmat 37 °C.From these experiments it was shown thatAmberlite CG-120 Type-3 was effective in removingiron from transferrin without the loss of its ironbinding capacity. No significant decrease in theamount of transferrin was observed by immunoassay* in the serum after the treatment with 1.8%ascorbic acid and 2% sodium bicarbonate solution.The use of charcoal or florisil was not effective inremoving iron.

TIBC detennination. Radioactive ferric ammomum citratet and sodium bicarbonate solution wereadded to iron-free serum to saturate TIBC. Theunbound iron was removed by 0.25 gm each of wetAmberlite IRP-67 and CG-120 Type-3 powderlIRP-67 removed 96% , CG-120 Type-3 removed74%, and the mixture of IRP-67 and CG-120Type-3 powder removed more than 98% of 5-@tgexcess iron in iron-saturated serum solution at roomtemperature in 5 mm. Magnesium carbonate wasnot effective as the mixture of IRP-67 and CG-120Type-3 in the elimination of unbound iron.

The method described below for the determination of TIBC was established from results obtainedin the preceding studies.

METHOD

Removal of serum iron. While the test tube isshaken, 2 ml of 1.8% fresh ascorbic acid solution areadded to 2 ml of serum. (Do not add serum intoascorbic acid solution.) One-half gram dry CG-120Type-3 powder is added to the acidified serum. Mixwell. The mixture is incubated at 37°Cfor 15 mlii,stirred occasionally, and then the mixture is spun

a Partigen-Transferrin, Bebringwerke, A.G., MarburgLahn, Germany.

t Prepared in our laboratory using @‘Fepurchased at OakRidge,Tenn. (16).

t Rohm and HansCo., Philadelphia,Penna.

TABLE 2. COMPARISON OF DIRECTDETERMINATION OF TIBC VERSUS

CALCULATiON(SI + UIBC)

1 271 271 61 2102 265 264 87 @773 277 276 70 2064 281 272 60 2125 259 252 62 1906 258 255 98 1577 291 285 67 2188 282 288 63 2259 267 268 62 20610 334 358 107 251

Averagevalues 279±27 279±29 74±16 205±20

490 JOURNAL OF NUCLEAR MEDICINE

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SIUIBCTIBC(pig/100ml)(@g/100(,t&g/100ml)ml)“FeColon

mFeDiseasesmethodSI + UIBCmetricmethod

TOTAL IRON-BINDING CAPACITYOF TIBC

The iron-free serum solution was obtained by centrifugation.

After iron removal, excess radioactive ferric ammonium citrate and then sodium bicarbonate solution are added to the serum solution. Excess iron isremoved with Amberlite IRP-67 and CG-120Type-3 powder. By this method, TIBC can be rapidly and direcfly determined.

REFERENCES

1. BRENDSTRUPP: Serum iron, total iron-binding capacity of serum, and serum copper in normals. Scand I ClinLab Invest5: 312—320,1953

2. LAURELL CB: Studies on the transportation and metabolism of iron in the body. Acta Physiol Scand 14 (Suppl46): 1-29,1947.

3. PETERST, GIOVANNIELLOTJ, Air L, et al: A newmethod for the determination of serum iron-binding capacity.I LabClinMed 48:274,1956

4. RAMSAYWNM: The determinationof total ironbinding capacity of serum. Clin Chim Ada 2: 221—226,1957

5. BOTHWELLTH, MALLETr B: The determination ofiron in plasma or serum. Biochem I 59: 599-602, 1955

6. FEINSTEINAR, BEmARDWE, MCCARTHYJD: A newmethod, using radioiron, for determining the iron-bindingcapacity of human serum. I Lab Clin Med 42: 907—914,1952

7. HERBERT V, GOTFLIEB CW, LAU K-S, et al: Coatedcharcoal assay of plasma iron-binding capacity and ironusing radioisotope dilution and hemoglobin-coated charcoal.I Nuci Med 8: 529—541,1967

8. RAMSAYWNM: The determination of iron in bloodplasma or serum. Biochem I 53 : 227—231,1953

9. MATSUBARAT: Studies on the method for determination of iron in biological materials, especially serum andwhole blood—A new proposal for the standardization of themethod (in Japanese). Ada Haemat lap 24: 434—452,1961

10. BOThWELLTH, JACOBSP, KAMENERR: Determination of unsaturated iron-binding capacity of serum usingradioactive iron. S Air I Med Sci 24: 93—98,1959

11. CARTWRIGHTGE, WINTROBEMM: Chemical, dm1-cal and immunological studies on products of human plasmafractionation. 39 anemia of infection studies on iron-bindingcapacity of serum. I Clin Invest 28: 86—98,1949

12. R@m CE, FINCH CA: Chemical, clinical and immunological studies on products of human plasma fractionation. 38. Serum iron transport. Measurement of iron-bindingcapacity of serum in man. I Clin invest 28: 79—85,1949

13. RESSLERN, Z@x B : Serum unsaturated iron-bindingcapacity. Amer I Clin Path 30: 87—90, 1958

14. SAITOH: A method for the determination of unsaturated iron-binding capacity of serum with ion exchangeresin strips (in Japanese). Radioisotopes 19: 179—183,1970

15. SCHADEAL, et al: Bound iron and unsaturated ironbinding capacity of serum, rapid and reliable quantitativedetermination. Proc Soc Exp Biol Med 87: 443—448,1954

16. TAUXEWN : A rapid radioactive method for the determination of the serum iron-binding capacity. Amer IClin Pathol 35: 403—406,1961

17. TINGUELYCR, LOEFFERRK: Method for determiningiron-binding capacity of serum. Proc Soc Erp Biol Med 92:241—247,1956

18. YAMADAH: Studies on the radioisotopic determina

244 244 220 24269 267 60 207

216 216 70 146221 215 30 185

364 364 41 323

HemochromatosisMyeloftbrosisAcute myelo

cytic leukemaLymphosarcomaHemolytic

anemiaHypoplastic

anemiaHypoplastic

anemiaHypoplastic

anemiaIron deficiency

anemiaIron deficiency

anemiaIron deficiencyanemia

Liver cirrhosisNephrosis

289 281 87 194

248 242 148 94

216

309

214 105 109

309 21 288

393 400 6 384

12 294160 7742 49

310 306230 23793 91

in the group in which UIBC was saturated with ironand that in the unsaturated group. Experimentalresults are shown in Table 1.

The TIBC was determined in normal persons andpatients suffering from various diseases. No significant difference was observed between the TIBCvalue of the group determined by the present methodand that of the group in which TIBC was obtainedby adding SI to UIBC. SI was determined by themethod of Matsubara (9), and UIBC was determined by the slight modification of Tauxe (16).The experimental results are shown in Tables 2and 3.

To measure TIBC by colorimetric methods, manyreagents are necessary and the procedures are entirely different from the routine UIBC method. Although the determination of TIBC by immunereaction can be performed using a small amount ofserum, it takes 48 hr to obtain the results. Individualdetermination of both SI and UIBC is also troublesome to carry out. The present method is as simpleas the routine UIBC method and can be determineddirectly after simply removing iron.

SUMMARY

A simple method for removing serum iron is described. To dissociate iron from transferrin, freshascorbic acid solution is used and ionized iron isremoved with Amberlite CG-120 Type-3 powder.

491Volume 12, Number 7

TABLE 3. TIBC DETERMINATION INVARIOUSDISEASESTATES

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SAITO

tion of the unsaturated iron-binding capacity of serum (inJapanese).lapI NuciMed 2: 146—156,1965

19. HILLMAN RS, MORGANEH, FINCH CA: Comparisonof total iron binding capacity methods in the iron deficientstate. I. Lab C/in Med 69: 874—878,1968

20. GOODMANM, NEWMAN HS, RAMSAYDS: Use ofchicken antiserum for the rapid determination of plasma

protein components. III. Assay of human serum transferrin.JLabC!inMedSl: 814—823,1958

21. SCHULTZHE, HEREMANSJE: Molecular biology ofhuman proteins with special reference to plasma proteins.vol 1. Amsterdam, Elsevier Publishing Co, 1966

22. GRoss R: iron Metabolism. Berlin-Gottingen-Heidelberg, Springer-Verlag, 1964

I

CALLFOR ABSTRACTS

Twelfth Annual Meeting SoutheasternChapterThe Societyof Nuclear Medicine

The scientific program committee welcomes submissionof abstracts on Nuclear Medicine from members andnonmembers. A limited number of papers will be presented at the meeting, as it is designed primarily as areview sessionin basic science for clinicians and non-clinicians associated with Nuclear Medicine.

A. ScientificProgram:Submitted papers on all aspects of Nuclear Medicine

B. Education Sessions:A comprehensivereviewof the basicand clinical sciencesof NuclearMedicine

Each abstract should contain the following information:1. Purpose2. Methods used3. Results with pertinent supporting data4. Conclusions

Abstracts that are accepted will be published in the Southeastern Medical Journal.Please send the abstract and three copies to:

J. C. Hewitt, M.D.Department of Nuclear MedicineTampa General HospitalDavis Islands,Tampa, Florida 33606

Deadline for acceptance of abstracts is July 31, 1971.

MEETING DATE: November 4—6,1971

MEETINGPLACE:Sheraton-FourAmbassadorsHotel, Miami, Florida

492 JOURNAL OF NUCLEAR MEDICINE

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1971;12:489-492.J Nucl Med.   Hiroshi Saito  Radioiron by Eliminating Iron from TransferrinNew Method for Determining Total Iron-Binding Capacity of Serum (TIBC) with

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