Method for the determination of unsaturated

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1 J. clin. Pth. (1968), 21, Method for the determintion of unsturted iron-binding cpcity of serum using rdioctive iron nd mgnesium crbonte B. BROZOVICH From the Deprtment of Hemtology, St. Brtholomew's Hospitl, London, nd M.R.C. Group for Reserch into Megloblstic nd Sideroblstic Anemis SYNOPSIS A method for mesuring the unsturted iron-binding cpcity of serum using rdioctive iron with mgnesium crbonte s the dsorbing substnce is described. The rdioctive ironmgnesium crbonte method, compred with the methods of Crwy(1963), Herbert, Gottlieb, Lu, Fisher, Grevirtz, nd Wssermn (1966), nd Bothwell, Jcobs, nd Kmener (1959), ws shorter nd simpler nd eqully reproducible. The specific binding protein for iron in serum is bet-l-globulin, siderophilin, or trnsferrin. The trnsferrin concentrtion in helthy persons is in the rnge of 2 to 32 mg/1 ml of serum, which corresponds to totl iron-binding cpcity (TIBC) of 25 to 4,tg Fe/1 ml (Bothwell nd Finch, 1962). Approximtely one third of the trnsferrin is sturted with iron nd the reminder represents the unsturted iron-binding cpcity (UIBC). A chnge in the concentrtion nd sturtion of trnsferrin in vrious diseses is well recognized nd its significnce hs been reviewed in detil by Bothwell nd Finch (1962). It is now generlly cknowledged tht the determintion of totl or unsturted iron-binding cpcity, together with the serum iron estimtion, should be vilble s routine procedure in hospitl lbortories. The totl or unsturted iron-binding cpcity is usully mesured by using colorimetric or rdioctive technique. Other techniques for the estimtion of TIBC or UIBC hve lso been described, for exmple the immunodiffusion method (Jger nd Gubler, 1952; Soothill, 1962), the gel filtrtion method (Brber, Dempster, nd Anderson, 1963), nd the tomic bsorption spectroscopy method (Rodgerson nd Helfer, 1966; Zettner, Sylvi, nd Cpcho- Delgdo, 1966), but they re infrequently used in routine work. The colorimetric determintion of TIBC is crried out by sturting trnsferrin with iron nd mesuring the colour of the iron-trnsferrin complex (Rth Received for publiction 13 November nd Finch, 1949; Ventur, 1952; Kldor, 1955), or fter removl of unbound iron, by estimting the totl mount of iron left in the serum (Lurell, 1947; Rmsy, 1957; Peters, Giovnniello, Apt, nd Ross, 1956). It is lso possible to estimte UIBC, fter sturtion with iron, by seprting nd mesuring the iron not bound to trnsferrin (Holmberg nd Lurell, 1945; Schde, Oym, Reinhrt, nd Miller, 1954; Ressler nd Zk, 1958). The successful use of rdioctive iron for estimting UIBC ws n elegnt improvement which simplified the procedure. Trnsferrin ws sturted with rdioctive iron, excess iron ws removed, nd rdioctivity of the superntnt counted. Unsturted iron-binding cpcity ws clculted from rdioctivity of smple nd stndrd (Bothwell et l., 1959; Tuxe, 1961). Different dsorbing substnces hve been used in colorimetric nd rdioctive techniques for the removl of excess iron fter sturtion of trnsferrin. Mgnesium crbonte (Rmsy, 1957; Morgn nd Crter, 196; Crwy, 1963; Fischer nd Price, 1964) nd ion exchnge resin (Peters et l., 1956) hve been used in colorimetric methods. In rdioctive methods n ion exchnge resin hs been used (Bothwell et l., 1959; Tuxe, 1961). More recently, Herbert et l. (1966) used hemoglobin-coted chrcol for the sme purpose. All these dsorbing substnces hve some disdvntges. Mgnesium crbonte is powder, nd fter centrifugtion the light sediment nd number of prticles on the sides of tube might.a ,. - J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by

2 184 B. Brozovich esily be stirred up by creless pipetting. The presence of prticles in the superntnt considerbly increses the opticl density of smple nd ffects the colorimetric mesurement. A number of investigtors hve observed tht the use of the methods involving ion exchnge resin give flsely high results for UIBC (Chrlton, Hrdie, nd Bothwell, 1965; Stojceski, Mlps, nd Witts, 1965; Birdsll, Kok, nd Wild, 1965). Chrlton et l. (1965) suggested tht the bsorption of wter by the dry resin, with proportionl increse in the concentrtion of ll substnces in the superntnt, results in incresed UIBC, nd tht this cn be overcome by counting the rdioctivity in the superntnt. However, this mens wshing the resin which prolongs the procedure. The other modifictions of the originl method of Bothwell et l. (1959) by Stojceski et l. (1965) nd Birdsll et l. (1965) proposed chnge in the order of dding the regents. These workers clim to hve obtined good results in comprison with the colorimetric method. The hemoglobin-coted chrcol is good dsorbing substnce but only removes pproximtely 98% iron from solution. It is sid tht this cn be overcome by the ppliction of correction fctor to the results. However, one disdvntge is tht the coted chrcol hs to be prepred every month. It ppered possible tht mgnesium crbonte, which, in spite of the disdvntges referred to bove, hd been used successfully in some colorimetric methods for determintion of UIBC, might be used in rdioctive method to which these disdvntges would not pply. Its use in such method would overcome the difficulties experienced with resin nd hemoglobin-coted chrcol. The purpose of this pper is to present simple method for the determintion of UIBC using rdioctive iron with mgnesium crbonte s the dsorbing substnce, nd to compre it with three other methods for the determintion of unsturted iron-binding cpcity. MATERIAL AND METHODS The determintion of UIBC ws crried out on ser from 3 ptients suffering from vrious diseses. COLLECTION AND STORAGE OF SAMPLES Using plstic syringes smples of venous blood were tken from the ptients (Johnson's Ethicl Plstics Ltd.) nd plced in plstic tubes (Luckhm Ltd.) mde iron free by stndrd procedures. After seprtion of serum, smples were ssyed immeditely or deep frozen until the mesurements were crried out. All mesurements on one smple were done on the sme dy. Iron-free glsswre ws used throughout the experiment. SERUM IRON Serum iron ws estimted on duplicte smples by the method of Crwy (1963) s modified by Hrriss (1963). UNSATURATED IRON-BINDING CAPACITY This ws estimted on duplicte smples using the method described in this pper nd lso by three other well known methods, detils of which follow. Using rdioctive iron nd mgnesium crbonte To mke up the stock solution, 9-6 g ferric chloride (Anlr, Hopkin & Willims Ltd.) ws dissolved in 1 ml 1 NHC1 nd -5 ml of this solution ws diluted to 1 ml with 1 N HCI; for the working solution, to 5 ml of stock solution pproximtely 5 juc of rdioctive iron (rdioctive ferric chlorite in 1 N HCI, Rdiochemicl Centre, Amershm) ws dded nd then diluted to 1 ml with distilled wter. The solution contined pproximtely 5 ug Fe/ml in 5 N HCI, nd the exct mount of iron ws ccurtely mesured by one of the existing methods for iron determintion. The other regent required is mgnesium crbonte, nhydrous (Hopkin & Willims Ltd.) Adsorptive properties were checked before routine use. ASSAY PROTOCOL Duplicte tubes were set up contining 5 ml of serum nd 1 ml of working rdioctive iron solution. The tubes were shken nd left to stnd for 5 min t room temperture. Approximtely 125 mg of mgnesium crbonte ws then dded, the tubes were covered with prfilm, shken vigorously for 1 to 15 sec, nd reshken four to five times during the next minute. After centrifugtion (2, rpm for 1 min) 1 ml of superntnt ws tken for rdioctive counting. Duplicte stndrds were lso mde contining 5 ml of wter nd 1 ml of the working rdioctive iron solution nd 1- ml ws tken for rdioctive counting. The UIBC in ug Fe/1 ml of serum ws clculted from: Rdioctivity of smple Rdioctivity of snddx 2 x iron content of work- Rdioctivity of stndrd ing solution (pg/ml x 1). The colorimetric determintion of TIBC using tripyridyltrizine nd mgnesium crbonte (Crwy, 1963) The method used ws modifiction by Hrriss (1963). The volume of serum ws reduced from 2- to 5 ml with corresponding reduction of the mounts of the regents used. Unsturted iron-binding cpcity ws obtined by subtrction of serum iron from the totl iron-binding cpcity. Coted chrcol ssy of UIBC using rdioctive iron nd hemoglobin-coted chrcol (Herbert et l., 1966) The method ws used s described by the uthors. The determintion of UIBC using rdioctive iron nd n ion exchnge resin (Bothwell et l., 1959) The method ws used s modified by Chrlton et l. (1965). RECOVERY OF TRANSFERRIN The recovery of trnsferrin ws estimted using humn trnsferrin (Behringwerke AG.) shown to be 1% pure on cette cellulose electrophoresis. The trnsferrin ws dissolved in sline to mke concentrtion of 6- mg/mi nd 2- ml of this solution ws dded to 5 ml of serum. The control smple ws mde by dding 2- ml of sline to 5 ml of J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by

3 Determintion of UICB ofserum using rdioctive iron nd mgnesium crbonte the sme serum. Unsturted iron-binding cpcity ws mesured on four smples of trnsferrin nd serum mixture nd four control smples by ech method under investigtion. The recovery ws represented by the difference between UIBC vlues of smples with nd without dded trnsferrin, nd expressed s percentge of the expected increse of UIBC, clculted on the bsis tht 1 mg of trnsferrin ws cpble of binding 1-25 jzg of iron. APPARATUS A Unicm SP 6 spectrophotometer ws used for colorimetric mesurements nd Philips gmm utomtic counter for rdioctive counting. RESULTS SUITABILITY OF MAGNESIUM CARBONATE FOR USE IN A RADIOACTIVE METHOD FOR UIBC DETERMINATION The use of mgnesium crbonte in colorimetric method for UIBC determintion hs two disdvntges. First, the recommended time for shking mgnesium crbonte suspension is t lest 3 min (Rmsy, 1957; Morgn nd Crter, 196; Crwy, 1963). Secondly, if the prticles of mgnesium crbonte re left in the superntnt fter centrifugtion they my increse opticl density of smples nd thus give flsely high vlues for unsturted iron-binding cpcity. In order to see whether these disdvntges of mgnesium crbonte ffect rdioctive iron method for UIBC determintion, the following experiments were crried out. Mgnesium crbonte ws dded to 12 tubes with 1-5 ml of rdioctive ferric chloride in queous solution (3 jig of iron) nd duplicte smples were in turn shken for 3 sec, 1, 5, 1, 2, nd 3 min respectively. After 1 U' iz 99.8 ui : z SHAKING TIME MIN. FIG. 1. Shking time nd percentge of iron removed by mgnesium crbonte from n queous solution contining 3,ug of iron. centrifugtion, liquot smples of superntnt were tken for rdioctive counting. Although most of the iron ws removed fter 3 min the rdioctivity removed from the superntnt of the smples tken from 1 to 2 min ws higher thn 99-8 % (Fig. 1). The possible effect of the trces of mgnesium crbonte in the superntnt on the ccurcy of UIBC determintion is illustrted in the following experiment. Mgnesium crbonte ws dded to 1 smples of rdioctive ferric chloride (3,ug of iron) in queous solution. Tubes were shken for 1 min nd then centrifuged for 1 minutes. After centrifugtion tubes in duplicte were gently shken so tht different mounts of mgnesium crbonte were suspended in the superntnt. Aliquots of these suspensions were pipetted, the opticl density ws determined in spectrophotometer, nd the rdioctivity ws mesured. There ws good correltion when the rdioctivity ws plotted ginst opticl density (Fig. 2). It cn be seen tht OPTICAL DENSITY FIG. 2. The reltion between the opticl density nd the rdioctivity of mgnesium crbonte suspensions. Ech circle represents the men of duplicte mesurement. 185 mounts of mgnesium crbonte which would give n enormous increse of opticl density did not ffect the results of the rdioctive ssy. COMPARISON OF THE ABILITY OF DIFFERENT ADSORBING SUBSTANCES TO REMOVE IRON FROM AQUEOUS SOLUrIONS The bility of mgnesium crbonte (nhydrous, Anlr, Hopkin & Willims Ltd.), hemoglobin-coted chrcol (Chrcol, Norit A Neutrl, phrmceuticl grde, Amend Drug nd Chemicl Co., Inc. New York, N.Y.), nd ion exchnge resin (Amberlite IRA 41 (HCI), Hopkin & Willims Ltd.) to remove 3 pg of iron trom queous solutions of ferric chloride, ferrous mmonium sulphte, nd ferric citrte, respectively, ws compred. In ll, 1 mesurements in duplicte were mde for ech J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by

4 186 B. Brozovich dsorbent (Tble I). Mgnesium crbonte nd resin removed more thn 99 % of iron from solution in every instnce, while the mount ofironremoved by hemoglobincoted chrcol ws slightly less nd in the rnge from 95 to 99%. TABLE I COMPARISON OF ADSORBING SUBSTANCES IN THEIR ABILITY TO REMOVE 3 Jug IRON FROM AN AQUEOUS SOLUTION (1 DUPLICATE MEASUREMENTS) Adsorbing Substnce Iron Slt Iron Removed (Y.) Mgnesium crbonte Hemoglobin-coted chrcol Ion exchnge resin Amberlite IRA-41 (Cl) Men Ferric chloride 99 8 Ferrous 97*4 mmonium sulphte Ferric citrte 99-2 Rnge REPRODUCIBILITY OF METHODS USED FOR UIBC DETERMINATION The reproducibility of ll methods for UIBC determintion ws tested by mens of (1) the coefficient of vrition obtined on 1 estimtions on one smple; (2) the combined coefficient of vrition between number of duplicte smples with UIBC rnging from 5 to 55,tg Fe/1 ml; nd (3) the recovery of trnsferrin dded to serum smples. These prmeters, obtined for ech one of four methods, were compred (Tble II). It ws shown tht there ws no substntil difference in the reproducibility of ll four methods. TABLE II REPRODUCIBILITY OF THE RADIOACTIVE IRON-MAGNESIUM CARBONATE METHOD AND THE METHODS OF CARAWAY (1963), HERBERT et l. (1966), AND BOTHWELL et l. (1959) Method Rdioctive Crwy Herbert Bothwell Iron-mgnesium (1963) et l. et l. Crbonte (1966) (1959) Coefficient of 3-1 (1) 4-7 (1) 3 (1) 56 (1) vrition (7)1 3-2 (9) 1-9 (6) Combined co- 3 5 (27) 4-5 (19) 54 (19) 2-4 (8) efficient of vrition between duplicte estimtions (%)' Trnsferrin recovery ('%)' 'In brckets number of estimtions on one smple. 2In brckets number of duplicte estimtions. 3Estimted on qudruplicte smples. CORRELATION OF UIBC MEASURED IN DIFFERENT SERUM SAMPLES BY FOUR METHODS The correltion of UIBC mesured in different serum smples using the rdioctive iron-mgnesium crbonte method, the method of Crwy (1963), nd the method of Herbert et l. (1966) is shown in Figure 3, b, nd c. There is n excellent correltion between mesurements of UIBC done in duplicte, obtined by ll three methods. However, when the method of Herbert et l. (1966) ws correlted with the rdioctive iron-mgnesium crbonte method nd the method of Crwy (1963) slight displcement of the regression line from the idel one ws found. There were insufficient smples mesured by the method of Bothwell et l. (1959) to be included in the generl sttisticl nlysis. However, s cn be seen from Tble III, there is no obvious difference between UIBC determined by the rdioctive iron-mgnesium method nd the method of Bothwell et l. (1959). TABLE III COMPARISON OF UIBC OBTAINED BY THE RADIOACTIVE IRON- MAGNESIUM CARBONATE METHOD AND BY THE METHOD OF BOTHWELL et l. (1959) Unsturted Iron-binding Cpcity (gg Fe.1 ml) Rdioctive Iron-mgnesium Method of Bothwell et l. Crbonte Method (1959) DISCUSSION There ws no striking difference in the bility of mgnesium crbonte, hemoglobin-coted chrcol, nd ion exchnge resin to remove iron from solution. However, mgnesiom crbonte hd certin dvntges over other dsorbing substnces (Tble IV), TABLE IV COMPARISON OF PROPERTIES OF MAGNESIUM CARBONATE, HAEMOGLOBIN-COATED CHARCOAL, AND ION EXCHANGE RESIN Mgnesium Crbonte Hemoglobincoted Chrcol Ion Exchnge Resin Preprtion Quntity dded Unnecessry Approximte Shking time 1 min. Correction for Unnecessry rdioctivity remining in superntnt Storge Indefinite t room temperture Necessry Unnecessry Exct volume of Approximte suspension 1 min. I min. Necessry Unnecessry I month t +4 C Indefinite t room temperture J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by

5 E ) 3 I 1- Mm.4 Determintion of UIBC ofserum using rdioctive iron nd mgnesium crbonte N: 22 r :-994 Y: X RADIOACTIVE IRON- MAGNESIUM CARBONATE FIG. 3. METHOD UIBC (pg /looml) -- 6 E CD - U 4 2 w I" RADIOACTIVE E cm - I Iw go w *t N: 15 r :.991 v Y :.98X FIG. 3c. CARAWAY/1963j UIBC (pg/loo ml) N:1I r :.994 y:.9sx 7* IRON- MAGNESIUM CARBONATE FIG. 3b. METHOD UIBC (pg/ioomi) viz., the fct tht it ws not necessry to dd n exct mount of substnce, the short shking time required, nd no need for the correction of results, no preprtion nd the indefinite shelf-life t room temperture mke mgnesium crbonte superior to hemoglobin-coted chrcol. In comprison to resin, mgnesium crbonte ppers to hve n dvntge becuse of the simplicity of the whole procedure. The sttisticl nlysis on results obtined by ll four methods for determintion of UIBC in serum (rdioctive iron-mgnesium crbonte, Crwy (1963), Herbert et l. (1966), Bothwell et l. (1959)) FIG. 3, b, nd c, The correltion of UIBC vlues mesured by the rdioctive ironmgnesium crbonte method, the method of Crwy (1963), nd the method of Herbert et l. (1966). N=number of duplicte estimtions, r=coefficient of correltion, y=regression line showed them to be eqully reproducible (Tble II). The coefficient of correltion lso showed n excellent greement between vlues for UIBC of different serum smples mesured by three methods (rdioctive iron-mgnesium crbonte method, Crwy (1963) Herbert et l. 1966)). However, the position of the regression line indicted tht the method of Herbert et l. (1966) gve slightly lower vlues for UIBC in the rnge from 4 to 6 jug Fe/1 ml when compred with the rdioctive ironmgnesium crbonte method nd the method of Crwy (1963). This could be the result of pplying one correction fctor s described by Herbert et l. J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by

6 188 w I= z 1 O/ 99 /1 98. I 1i IRON IN SAMPLE (pg) FIG. 4. The reltion between the mount of iron nd the percentge of iron removed by hemoglobin-coted chrcol from n queous solution. Ech circle represents the men of duplicte mesurement. (1966) for correction of ll vlues of UIBC. This is illustrted in the following experiment (Fig. 4). Different concentrtions of n queous solution of ferrous mmonium sulphte were prepred in duplicte nd fter the ddition of hemoglobin-coted chrcol nd centrifugtion, the rdioctivity of the liquots of superntnt ws counted. It ws shown tht the rdioctivity remining in the superntnt ws dependent on the mount of free iron, nd, therefore, single fctor could not be pplied uniformly for correction of ll UIBC vlues. Needless to sy, this smll error does not ffect the vlidity of results in routine procedure. In smll number of ser, UIBC ws mesured by the rdioctive iron nd mgnesium crbonte method nd by the method of Bothwell et l. (1959), nd no substntil difference ws found. This is in greement with reports in the literture tht there is prcticlly no difference in UIBC mesured by rdioctive iron method using ion exchnge resin nd by colorimetric or immunodiffusion technique (Birdsll et l., 1965; Chrlton et l., 1965; Hillmn, Morgn, nd Finch, 1967; Stojceski et l., 1965; B. Brozovich Tuxe, 1961). On the other hnd, Burrows (1967), Herbert et l. (1966), nd Koepke (1965) found tht UIBC vlues mesured by the rdioctive iron method with resin were pproximtely 1,ug Fe/1 ml higher thn mesured by other methods. However, these uthors used resin sponges (Irosorb- 59, Abbott Lbortories, North Chicgo, Ill.) insted of resin beds in their investigtions. The discrepncy needs to be clrified. It is plesure to thnk Professor D. L. Mollin for much help nd vluble dvice nd Dr. V. Herbert, who kindly supplied the chrcol used in this study. I would lso like to thnk Miss A. M. Unwin for help in prepring the mnuscript. REFERENCES Brber, A. A., Dempster, C., nd Anderson, N. G. (1963). Clin. chim. Act, 8, 143. Birdsll, N. J. M., Kok, D'A., Wild, F. (1965). J. clin. Pth., 18, 453. Bothwell, T. H., nd Finch, C. A. (1962). Iron Metbolism. Churchill, London., Jcobs, P., nd Kmener, R. (1959). S. Afr. J. med. Sci., 24, 93. Burrows, S. (1967). Amer. J. clin. Pth., 47, 326. Crwy, W. T. (1963). Clin. Chem., 9, 188. Chrlton, R. W., Hrdie, N., nd Bothwell, T. H. (1965). S. Afr. J. med. Sci., 3, 71. Fischer, D. S., nd Price, D. C. (1964). Clin. Chem., 1, 21. Hrris, E. B. (1963). Unpublished dt. Herbert, V., Gottlieb, C. W., Lu, K.-S., Fischer, M., Gevirtz, N. R., nd Wssermn, L. R. (1966). J. Lb. clin. Med., 67, 855. Hillmn, R. S., Morgn, E. H., nd Finch, C. A. (1967). Ibid., 69, 874. Holmberg, C. G., nd Lurell, C. B. (1945). Act physiol. scnd., 1,37. Jger, B. V., nd Gubler, C. J. (1952). J. Immunol., 69, 311. Kldor, I. (1955). Med. J. Aust., 2, 886. Koepke, J. A. (1965). Amer. J. clin. Pth., 44, 77. Lurell, C. B. (1947). Act physiol. scnd., 14, suppl. 46. Morgn, E. H., nd Crter, G. (196). Aust. Ann. Med., 9, 29. Peters, T., Giovnniello, T. J., Apt, L., nd Ross, J. F. (1956). J. Lb. clin. Med., 48, 274. Rmsy, W. N. M. (1957). Clin. chim. Act, 2, 221. Rth, C. E., nd Finch C. A. (1949). J. clin. Invest., 28, 79. Ressler, N., nd Zk, B. (1958). Amer. J. clin. Pth., 3, 87. Rodgerson, D. O., nd Helfer, R. E. (1966). Clin. Chem., 12, 338. Schde, A. L., Oym, J., Reinhrt, R. W., nd Miller, J. R. (1954). Proc. Soc. exp. Biol. (N.Y.), 87, 443. Soothill, J. F. (1962). J. Lb. clin. Med., 59, 859. Stojceski, T. K., Mlps, J. S., nd Witts, L. J. (1965). J. clin. Pth., 18, 446. Tuxe, W. N. (1961). Amer. J. clin. Pth., 35, 43. Ventur, S. (1952). J. clin. Pth., 5, 271. Zettner, A., Sylvi, L. C., nd Cpcho-Delgdo, L. (1966). Amer. J. clin. Pth., 45, 533. J Clin Pthol: first published s /jcp on 1 Mrch Downloded from on 22 December 218 by guest. Protected by