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The analysis of blood iron
The Analysis of Blood Iron’ R. C. Dickenman, From
the Department
of Pathology, Receiving
Wayne Hospital,
Received
B. Crafts and B. Zak University Detroit, April
College Michigan
of Medicine
and Detroit
1, 1954
INTRODUCTION
A number of procedures have appeared in the literature that involve technics used in standardizing the several routine hemoglobin methods now available in many clinical laboratories. These processes include the use of Van Slyke gas analysis equipment as the main tool for calibration purposes. Although nearly all laboratories have photometric instrumentation available, many do not have manometric apparatus. This necessitates the employment of methods which do not involve gas analysis. The determination of total blood iron is a useful means of standardizing the various hemoglobin procedures (1, 2). Since Wong (3) introduced a practical method for this type of analysis and gave impetus to several modifications (4, 5), the procedure has seen much use in the hands of those laboratory personnel who do not have manometric gas analysis devices for the standardization of hemoglobin. The percentage of iron in hemoglobin has been well established (6, 7), while it has been found that the relative percentage of total iron present in the plasma fraction of normal blood is small (8, 9). Therefore, it is convenient to take normal blood, determine its iron content accurately, and then set up a chemical procedure such as the one involving the conversion of hemoglobin present to oxyhemoglobin by means of dilute sodium carbonate, quantitating it finally by spectrophotometric means at the wavelength of maximum absorbance (10). Other spectrophotometric procedures for hemoglobin can be set up in the same manner (11-13). The technic presented in this article involves a simple and rapid pro1 Supported in part by the Receiving Detroit Institute of Cancer Research.
Hospital 381
Research
Corporation
and
the
382
DICKENMAN,
CRAFTS AND ZAK
cedure for the release of iron from its strong complex-binding in hemoglobin, isolation in the supernatant by virtue of the fact that the oxidizing solution is also a protein precipitant, and its final quantitation by filtration into a buffer which contains both reducing agent and color-forming complexing reagent. This enables one to accurately analyze normal blood for iron concentration and to use this latter value as a standard for the calibration of any kind of routine spectrophotometric hemoglobin determination. MATERIALS
AND METHODS
Reagents Buffered 2,2’-Bipyridyl or l,lO-Phenanthroline Solution. Three hundred grams of pure sodium acetate is dissolved in about 700 ml. of iron-free distilled water in a liter volumetric flask. To this is added 1 g. of 2,2’-bipyridyl or 1, lo-phenanthroline which has been dissolved previously in about 200 ml. of distilled water with warming. The solution is diluted to the mark and mixed by inversion. Ascorbic Acid Solution. One gram of ascorbic acid is dissolved in 100 ml. of distilled water and kept refrigerated except when using. This is freshly made for each standardization procedure. Sodium Chlorate-Perchloric Acid Solution. Weigh out 3.2 g. of C.P. sodium chlorate and dissolve in about 500 ml. of distilled water. Cool in an ice bath and then pour in slowly with mixing 85 ml. of 720/, HClO+ which has previously been cooled in an ice bath. Some decomposition of the chlorate may occur which will be evident in the appearance of chlorine. The perchloric acid can be added to the sodium chlorate solution at room temperature without danger, but there may be more decomposition of the sodium chlorate than is desirable. The final mixture contains 0.25% chloric acid and 10% perchloric acid. The former serves as an oxidizing agent while the latter precipitates the protein. Perchloric Acid Wash Solution. Pour 82 ml. of 72yo perchloric acid into a liter volumetric flask containing 700 ml. of distilled water and dilute to the mark with mixing. Transfer to a ground-glass-jointed wash bottle. Iron Stock Standard Solution. Weigh out 1.0 g. of electrolytic iron and transfer to a 11. volumetric flask. Add 10% sulfuric acid and shake until dissolved. Dilute to the mark with mixing. Iron salts may be used in the place of the electrolytic iron, but they must be standardized by some appropriate procedure (13). Standard Iron Working Solution. Pipet 10.0 ml. of working standard into a loo-ml. volumetric flask and dilute to the mark with distilled water. Mix by inversion.
Procedure of the Standard Curve. Pipet 0.0 ml., 1.0 ml., 2.0 ml., 3.0 ml., 4.0 ml., and 5.0 ml. of the working solution into loo-ml. volumetric flasks. To each flask add 10.0 ml. of the sodium chlorate solution followed by 20.0 ml. of the buffered
ANALYSIS
OF
BLOOD
383
IRON
2,2’-bipyridyl or 1 ,lO-phenanthroline solution and 5.0 ml. of the ascorbic acid solution. Dilute to the mark and mix well by inversion. Measure the absorbancy of the resulting red solution in a spectrophotometer at 508 rnp and plot the linear curve shown in Fig. 1, curve A. Determination of Blood Iron. Pipet 0.5 ml. or 1.0 ml. of oxalated blood into a clean 25.0.ml. test tube and to this add 10.0 ml. of the sodium chlorate-perchloric acid solution. Cover tubes with a clean agate and place in a rack in a boiling water bath for 8 min. (or one can use tall-form beakers and several beads and place on a hot plate at low temperature for 5 min. to achieve the same results). The samples are removed from the water bath and placed in an ice bath for cooling. The sides of the tube are washed down with about 1 ml. of the perchloric acid wash water and then filtered through fine filter paper, Whatman No. 42, directly into a lOO-ml. volumetric flask containing 20.0 ml. of sodium acetate buffer and 5.0 ml. of ascorbic reducing agent. The use of a rubber policeman facilitates the removal of protein adhering to the side of the test tube. The proteins on the filter paper are washed several times with small amounts of the acid wash, first from the washed test tubes and then directly from the wash bottle. The solution is diluted to the mark with distilled water, stoppered, and mixed by inversion. The absorbancy is measured at 508 rnp in a spectrophotometer and the concentration of iron obtained from the calibration curve. A conversion factor for iron to hemoglobin makes use of the previously known composition of iron in this molecule (6,7). Cl
4
6
Tim,;in
minutes 16
20
24
0.9 0.8 -
A-Calibration curve E-Time of healing curve 0 Iron in completely ashed
Mwogroms
FIG.
1. Comparative
spectral
curves
blood
per lOOmI.
for standard
iron
and blood
iron.
384
DICKENMAN,
CRAFTS
AND
ZAK
A- Blood sample B- Stondord Fe++
400
500
600
Wavelength
FIG.
2. Calibration
and time of heating curves for blood iron.
TABLE Comparison
of
Hemoglobin Spectrophotometric
Sample
70
In mu
Fe/ml. present” M.
Fe/ml.
found lb-.
I
Content
by Iron
Procedure
to
Hemoglobin Deviation keg.
Hb presenta g.jlOO ml.
Hb found &I100 ml.
Deviation &/loo
ml.
Blood %1 340 350 -1-K) 10.0 10.3 +0.3 15.0 -0.1 Blood %2 513 510 -3 15.1 Blood ~$3 328 320 -8 9.6 9.4 -0.2 326 -10 9.9 9.6 -0.3 Blood #4 336 465 +18 13.3 13.7 +0.5 Blood 85 448 9.7 -0.2 Blood a6 336 330 -6 9.9 Blood W7 350 350 0 10.3 10.3 0.0 Blood ~$8 451 442 -9 13.3 13.0 -0.3 458 +10 13.2 13.5 $0.3 Blood $49 448 320 0 9.4 9.4 0 Blood BlO 320 333 -3 9.9 9.8 -0.1 Blood ~$11 336 a Hemoglobin procedure used here was calibrated by means of manometric Van Slyke and then determined with either Coleman or Beckman spectrophotometers.
ANALYSIS
OF
BLOOD
TABLE Recovery Sample Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood
Xl ~$2 #3 S4 B5 ~$6 X7 B8 %Q 110 ~$11 812
of Iron
MI. used
0.5 1.0 1.0 0.5 1.0 0.5 1.0 0.5 0.5 1.0 0.5 0.5
Added Iron
385
IRON
II to Blood present
before Iron
Ashing added
Iron
found
Pg.
Pg.
Pg.
163 608 350 165 470 160 365 158 163 350 307 160
100 200 200 100 100 100 100 100 100 200 100 100
260 780 545 260 575 258 460 245 255 530 383 268
DISCUSSION
The small amount of chloric acid present in the digestion precipitation agent is ample for the oxidation of iron to the ferric state and its subsequent release from its coordination position in the hemoglobin molecule where it was previously very strongly complexed. This is evidenced by the characteristics of the curve obtained when various aliquots of the same blood are heated from zero time to complete destruction of the proteins present as shown in curve B of Fig. 1. It can be seen that a plateau is reached within 6 min., which means that 8 min. is an ample beating period for this partial digestion. Figure 2 shows the spectral curves obtained for pure iron as well as for the filtrate obtained by the partial digestion of the proteins as described under procedure. In spite of the fact that small degradation products of the proteins may filter through along with the iron, the absorption characteristics of the curve in the visible range are not changed in the region of the absorption maximum. A favorable comparison was made to a spectrophotometric hemoglobin procedure for total hemoglobin as is shown in Table I. The addition of small amounts of iron to the blood before oxidative acid treatment resulted in the excellent recoveries represented in Table II. SUMMARY
A procedure has been presented which makes possible the analysis of blood iron by three short and simple steps which include: (a) partial
386
DICKENMAN,
CRAmS AND ZAK
digestion of the material with a reagent which contains both oxidizing and protein-precipitating reagents, (b) filtering and washing into a buffer containing reducing agent and a color-complexing agent for the reduced form of iron, (c) and finally measuring spectrophotometrically the resulting chromophore which follows Beer’s law over a wide range. REFERENCES 1. TODD, J. B., SANFORD, A. H., AND WELLS, B. B., “Clinical Diagnosis by Laboratory Methods,” pp. 413-14. W. B. S aunders Company, Philadelphia and London, 1948. 2. KENNEDY, R. P., J. Biol. Chem. ?4,385-91 (1927). 3. WONG, S. Y., J. Biol. Chem. 7’7,409-12 (1928); ibid. 66,421 (1923). 4. HANZAL, R. F., Proc. Sot. Ezptl. Biol. Med. 30, 846-S (1933). 5. PONDER, E., J. Biol. Chem. 144, 333-8 (1942). 6. LEMBERG, R., AND LEGGE, J. W., “Hematin Compounds and Bile Pigments,” p. 214. Interscience Publ., New York, 1949. 7. BERNHART, F. W., AND SKEGGS, L., J. Biol. Chem. 147, 19-22 (1943). 8. SUNDERMAN, F. W., AND BOERNER, F., “Normal Values in Clinical Medicine,” pp. 166-7. W. B. Saunders and Company, Philadelphia, 1949. 9. SUNDERMAN, F. W., MACFATE, W. P., MACFADYEN, D. A., STEVENSON, G. F., AND COPELAND, B. E., Am. J. Clin. PathoZ. 23, 519-98 (1953). 10. BELL, G. H., CHAMBERS, J. W., AND WADDELL, M. B. R., Biochcm. J. 39, 60-3 (1945). 11. HALDANE, J., J. Physiol. (London) 26,497-504 (1901). 12. STADIE, W. C., J. Biol. Chem. 41, 23741 (1920). 13. HAWK, P. B., OSER, B. L., AND SUMMERSON, W. H., “Practical Physiological twelfth edition, pp. 566-8. The Blakiston Company, PhilaChemistry,” delphia, 1948.
the Department
of Pathology, Receiving
Wayne Hospital,
Received
B. Crafts and B. Zak University Detroit, April
College Michigan
of Medicine
and Detroit
1, 1954
INTRODUCTION
A number of procedures have appeared in the literature that involve technics used in standardizing the several routine hemoglobin methods now available in many clinical laboratories. These processes include the use of Van Slyke gas analysis equipment as the main tool for calibration purposes. Although nearly all laboratories have photometric instrumentation available, many do not have manometric apparatus. This necessitates the employment of methods which do not involve gas analysis. The determination of total blood iron is a useful means of standardizing the various hemoglobin procedures (1, 2). Since Wong (3) introduced a practical method for this type of analysis and gave impetus to several modifications (4, 5), the procedure has seen much use in the hands of those laboratory personnel who do not have manometric gas analysis devices for the standardization of hemoglobin. The percentage of iron in hemoglobin has been well established (6, 7), while it has been found that the relative percentage of total iron present in the plasma fraction of normal blood is small (8, 9). Therefore, it is convenient to take normal blood, determine its iron content accurately, and then set up a chemical procedure such as the one involving the conversion of hemoglobin present to oxyhemoglobin by means of dilute sodium carbonate, quantitating it finally by spectrophotometric means at the wavelength of maximum absorbance (10). Other spectrophotometric procedures for hemoglobin can be set up in the same manner (11-13). The technic presented in this article involves a simple and rapid pro1 Supported in part by the Receiving Detroit Institute of Cancer Research.
Hospital 381
Research
Corporation
and
the
382
DICKENMAN,
CRAFTS AND ZAK
cedure for the release of iron from its strong complex-binding in hemoglobin, isolation in the supernatant by virtue of the fact that the oxidizing solution is also a protein precipitant, and its final quantitation by filtration into a buffer which contains both reducing agent and color-forming complexing reagent. This enables one to accurately analyze normal blood for iron concentration and to use this latter value as a standard for the calibration of any kind of routine spectrophotometric hemoglobin determination. MATERIALS
AND METHODS
Reagents Buffered 2,2’-Bipyridyl or l,lO-Phenanthroline Solution. Three hundred grams of pure sodium acetate is dissolved in about 700 ml. of iron-free distilled water in a liter volumetric flask. To this is added 1 g. of 2,2’-bipyridyl or 1, lo-phenanthroline which has been dissolved previously in about 200 ml. of distilled water with warming. The solution is diluted to the mark and mixed by inversion. Ascorbic Acid Solution. One gram of ascorbic acid is dissolved in 100 ml. of distilled water and kept refrigerated except when using. This is freshly made for each standardization procedure. Sodium Chlorate-Perchloric Acid Solution. Weigh out 3.2 g. of C.P. sodium chlorate and dissolve in about 500 ml. of distilled water. Cool in an ice bath and then pour in slowly with mixing 85 ml. of 720/, HClO+ which has previously been cooled in an ice bath. Some decomposition of the chlorate may occur which will be evident in the appearance of chlorine. The perchloric acid can be added to the sodium chlorate solution at room temperature without danger, but there may be more decomposition of the sodium chlorate than is desirable. The final mixture contains 0.25% chloric acid and 10% perchloric acid. The former serves as an oxidizing agent while the latter precipitates the protein. Perchloric Acid Wash Solution. Pour 82 ml. of 72yo perchloric acid into a liter volumetric flask containing 700 ml. of distilled water and dilute to the mark with mixing. Transfer to a ground-glass-jointed wash bottle. Iron Stock Standard Solution. Weigh out 1.0 g. of electrolytic iron and transfer to a 11. volumetric flask. Add 10% sulfuric acid and shake until dissolved. Dilute to the mark with mixing. Iron salts may be used in the place of the electrolytic iron, but they must be standardized by some appropriate procedure (13). Standard Iron Working Solution. Pipet 10.0 ml. of working standard into a loo-ml. volumetric flask and dilute to the mark with distilled water. Mix by inversion.
Procedure of the Standard Curve. Pipet 0.0 ml., 1.0 ml., 2.0 ml., 3.0 ml., 4.0 ml., and 5.0 ml. of the working solution into loo-ml. volumetric flasks. To each flask add 10.0 ml. of the sodium chlorate solution followed by 20.0 ml. of the buffered
ANALYSIS
OF
BLOOD
383
IRON
2,2’-bipyridyl or 1 ,lO-phenanthroline solution and 5.0 ml. of the ascorbic acid solution. Dilute to the mark and mix well by inversion. Measure the absorbancy of the resulting red solution in a spectrophotometer at 508 rnp and plot the linear curve shown in Fig. 1, curve A. Determination of Blood Iron. Pipet 0.5 ml. or 1.0 ml. of oxalated blood into a clean 25.0.ml. test tube and to this add 10.0 ml. of the sodium chlorate-perchloric acid solution. Cover tubes with a clean agate and place in a rack in a boiling water bath for 8 min. (or one can use tall-form beakers and several beads and place on a hot plate at low temperature for 5 min. to achieve the same results). The samples are removed from the water bath and placed in an ice bath for cooling. The sides of the tube are washed down with about 1 ml. of the perchloric acid wash water and then filtered through fine filter paper, Whatman No. 42, directly into a lOO-ml. volumetric flask containing 20.0 ml. of sodium acetate buffer and 5.0 ml. of ascorbic reducing agent. The use of a rubber policeman facilitates the removal of protein adhering to the side of the test tube. The proteins on the filter paper are washed several times with small amounts of the acid wash, first from the washed test tubes and then directly from the wash bottle. The solution is diluted to the mark with distilled water, stoppered, and mixed by inversion. The absorbancy is measured at 508 rnp in a spectrophotometer and the concentration of iron obtained from the calibration curve. A conversion factor for iron to hemoglobin makes use of the previously known composition of iron in this molecule (6,7). Cl
4
6
Tim,;in
minutes 16
20
24
0.9 0.8 -
A-Calibration curve E-Time of healing curve 0 Iron in completely ashed
Mwogroms
FIG.
1. Comparative
spectral
curves
blood
per lOOmI.
for standard
iron
and blood
iron.
384
DICKENMAN,
CRAFTS
AND
ZAK
A- Blood sample B- Stondord Fe++
400
500
600
Wavelength
FIG.
2. Calibration
and time of heating curves for blood iron.
TABLE Comparison
of
Hemoglobin Spectrophotometric
Sample
70
In mu
Fe/ml. present” M.
Fe/ml.
found lb-.
I
Content
by Iron
Procedure
to
Hemoglobin Deviation keg.
Hb presenta g.jlOO ml.
Hb found &I100 ml.
Deviation &/loo
ml.
Blood %1 340 350 -1-K) 10.0 10.3 +0.3 15.0 -0.1 Blood %2 513 510 -3 15.1 Blood ~$3 328 320 -8 9.6 9.4 -0.2 326 -10 9.9 9.6 -0.3 Blood #4 336 465 +18 13.3 13.7 +0.5 Blood 85 448 9.7 -0.2 Blood a6 336 330 -6 9.9 Blood W7 350 350 0 10.3 10.3 0.0 Blood ~$8 451 442 -9 13.3 13.0 -0.3 458 +10 13.2 13.5 $0.3 Blood $49 448 320 0 9.4 9.4 0 Blood BlO 320 333 -3 9.9 9.8 -0.1 Blood ~$11 336 a Hemoglobin procedure used here was calibrated by means of manometric Van Slyke and then determined with either Coleman or Beckman spectrophotometers.
ANALYSIS
OF
BLOOD
TABLE Recovery Sample Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood
Xl ~$2 #3 S4 B5 ~$6 X7 B8 %Q 110 ~$11 812
of Iron
MI. used
0.5 1.0 1.0 0.5 1.0 0.5 1.0 0.5 0.5 1.0 0.5 0.5
Added Iron
385
IRON
II to Blood present
before Iron
Ashing added
Iron
found
Pg.
Pg.
Pg.
163 608 350 165 470 160 365 158 163 350 307 160
100 200 200 100 100 100 100 100 100 200 100 100
260 780 545 260 575 258 460 245 255 530 383 268
DISCUSSION
The small amount of chloric acid present in the digestion precipitation agent is ample for the oxidation of iron to the ferric state and its subsequent release from its coordination position in the hemoglobin molecule where it was previously very strongly complexed. This is evidenced by the characteristics of the curve obtained when various aliquots of the same blood are heated from zero time to complete destruction of the proteins present as shown in curve B of Fig. 1. It can be seen that a plateau is reached within 6 min., which means that 8 min. is an ample beating period for this partial digestion. Figure 2 shows the spectral curves obtained for pure iron as well as for the filtrate obtained by the partial digestion of the proteins as described under procedure. In spite of the fact that small degradation products of the proteins may filter through along with the iron, the absorption characteristics of the curve in the visible range are not changed in the region of the absorption maximum. A favorable comparison was made to a spectrophotometric hemoglobin procedure for total hemoglobin as is shown in Table I. The addition of small amounts of iron to the blood before oxidative acid treatment resulted in the excellent recoveries represented in Table II. SUMMARY
A procedure has been presented which makes possible the analysis of blood iron by three short and simple steps which include: (a) partial
386
DICKENMAN,
CRAmS AND ZAK
digestion of the material with a reagent which contains both oxidizing and protein-precipitating reagents, (b) filtering and washing into a buffer containing reducing agent and a color-complexing agent for the reduced form of iron, (c) and finally measuring spectrophotometrically the resulting chromophore which follows Beer’s law over a wide range. REFERENCES 1. TODD, J. B., SANFORD, A. H., AND WELLS, B. B., “Clinical Diagnosis by Laboratory Methods,” pp. 413-14. W. B. S aunders Company, Philadelphia and London, 1948. 2. KENNEDY, R. P., J. Biol. Chem. ?4,385-91 (1927). 3. WONG, S. Y., J. Biol. Chem. 7’7,409-12 (1928); ibid. 66,421 (1923). 4. HANZAL, R. F., Proc. Sot. Ezptl. Biol. Med. 30, 846-S (1933). 5. PONDER, E., J. Biol. Chem. 144, 333-8 (1942). 6. LEMBERG, R., AND LEGGE, J. W., “Hematin Compounds and Bile Pigments,” p. 214. Interscience Publ., New York, 1949. 7. BERNHART, F. W., AND SKEGGS, L., J. Biol. Chem. 147, 19-22 (1943). 8. SUNDERMAN, F. W., AND BOERNER, F., “Normal Values in Clinical Medicine,” pp. 166-7. W. B. Saunders and Company, Philadelphia, 1949. 9. SUNDERMAN, F. W., MACFATE, W. P., MACFADYEN, D. A., STEVENSON, G. F., AND COPELAND, B. E., Am. J. Clin. PathoZ. 23, 519-98 (1953). 10. BELL, G. H., CHAMBERS, J. W., AND WADDELL, M. B. R., Biochcm. J. 39, 60-3 (1945). 11. HALDANE, J., J. Physiol. (London) 26,497-504 (1901). 12. STADIE, W. C., J. Biol. Chem. 41, 23741 (1920). 13. HAWK, P. B., OSER, B. L., AND SUMMERSON, W. H., “Practical Physiological twelfth edition, pp. 566-8. The Blakiston Company, PhilaChemistry,” delphia, 1948.