ANALYTICAL
BIOCHEMISTRY
99,
28-32 (1979)
A Catalytic Method for Determination of Trace Amounts iron in Biological Material A. A. ALEXIEV,* *Department
of Chemistry
V. RACHINA,*
and Biochemistry, Medical Faculty of Chemistry,
of
AND P. R. BoNTcHEvt’l
Faculty, University
Pleven, and TDepartment of Sofia, Bulgaria
of Analytical
Chemistry,
Received October 5, 1978 A rapid, simple, and reproducible method for determination of iron in biological material is suggested using the oxidation of p-phenetidine hydrochloride with hydrogen peroxide in a reaction catalyzed by Fe(lII) and activated by f,lO-phenanthroline. The high sensitivity of the reaction allows a single determination to be carried out with as much as l-5 mg fresh tissue.
In our previous papers (1,2) a rapid method for determination of iron in blood serum was suggested using the oxidation of p-phenetidine with hydrogen peroxide in a reaction catalyzed by Fe(II1) and activated by 1, lophenanthroline. The colored reaction product shows an absorption band at 510 nm, used for photometric measurements. The activator plays an important role in this reaction. It has been shown, that together with p-phenetidine it forms a ternary complex with Fe(II1) (1: 1: 1) and increases sharply its catalytic activity not participating directly as a reagent in the reaction (3). The other metals present in biological samples do not influence the indicator reaction at least at concentrations two times greater than their usual concentration in such samples (1). Even more, in the presence of 1, lo-phenanthroline many of these ions decrease further their catalytic activity. It has been proved also that the solution obtained after mineralization of the biological sample does not contain substances that can affect the catalytic activity. This fact, together with the 1 To whom requests for reprints should be addressed. 0003-2697/79/150028-05$02.00/O Copyright AU rights
0 1979 by Academic Press, Inc. of reproduction in any fomt reserved.
high sensitivity of the reaction (0.001 pg/ ml), its reproducibility, and simplicity of the procedure encouraged us to adopt this method to various biological materials where the iron content is very low. EXPERIMENTAL Reagents
1. 0.2 M and 30% H,O, were used as solutions free of stabilizing agents. The exact concentrations were determined titrimetritally with permanganate. 2. 0.05 M p-phenetidine hydrochloride. 3. 0.00075 M l,lO-phenanthroline. Solutions l-3 should be light protected and stored in a refrigerator. 4. Standard solution of FeCl, in 0.005 M HCl (0.75 pg Fe(III)/ml). Initial solution of FeCl, (-0.1 M) in 0.1 M HCl was used, its exact Fe(II1) concentration being determined by gravimetric and complexometric methods. The standard solution was prepared from it by consecutive dilutions with 0.005 M HCl. 5. Potassium hydrogen phthalate buffer, pH 2.8 (see ref. (2)). 6. Phosphate buffer, pH 5.5. 28
QUANTITATION
OF IRON
7, 0.00068 M solution of disodium bathophenanthroline disulfonate in phosphate buffer, pH 5.5. 8. Standard solution of Fe(II1): 1 pg/ml. 9. Sodium ascorbate. 10. Concentrated HzS04, NH03, and HCl. All reagents used were of analytical grade and solutions were prepared with bidistilled water, Special attention was given to the iron assay of the chemicals. Glassware was kept overnight in HCl (1: 1) and rinsed with bidistilled water in order to be iron free. The optical measurements were performed using single-beam Specol spectrophotometer equipped with a thermostatic cell EK-5. Preliminary
Sample
Treatment
The quantity of iron was determined in samples of cow milk, human skin, and guinea pig kidney, lung, and spleen. After killing the animal the organs were removed, extensively rinsed in bidistilled water and cut into pieces by a plastic knife. This preliminary treatment of the tissue was used as a very simple one, suitable for the aim of the investigation. After mineralization a homogeneous solution is obtained, whose iron content can be determined according to the three methods used. If the exact iron content of the tissue samples is to be determined, they must be perfused before mineralization. Dry ashing. This pretreatment was applied to the samples of skin and milk. A piece of skin (0.5- 1.O g) or IO-20 ml of cow milk were dried in a quartz vessel by heating at 105”C, transferred to a muffle and heated at 450°C for 48 h. Under these conditions the sample of skin was thoroughly ashed, while the milk sample was not completely charred and required an additional treatment consisting in moistening of the material with a few drops of concentrated HN03, evaporation of the latter, and heating the sample for another 1 h at 450°C. This treatment was repeated until the sample was completely
IN BIOLOGICAL
29
MATERIAL
ashed. The charred material thus obtained was ground to powder in an agetate mortar. Aliquots of 5 mg were placed in a quartz tube, 1 ml of hot 1 N HCl was added and the solution brought to 20 ml with distilled water. Digestion with H2S04, HNO, and HzOz. The procedure was applied to samples of animal organs (kidney, lung, and spleen). Digestion was carried out with both fresh and dried material in parallel experiments. In the latter case the sample was dried at 105°C till constant weight, the resulting material powdered in a mortar, and an aliquot weighed for further processing. Concentrated H,SO, and HNO, (1: 1) were added to the sample in a small quartz tube and the mixture was heated gently. As the digestion proceeded, small portions of HNO, (l-2 drops at a time) were added. Digest was cooled just before HN03 to be completely evaporated, l-2 drops of H,O, were added, and the solution again heated to fumes of SO,. Digest was then cooled and brought to the desired volume with bidistilled water. The content of iron in the solutions thus obtained was determined using three different methods: photometrically, by atomic absorption, and by our catalytic method. Because of the high selectivity of the reaction, neither preliminary separation of iron nor use of masking agents were necessary. Analytical of Iron
Methods for Determination
1. Catalytic method. The procedure is illustrated in Table 1. Thirty minutes after the addition ofp-phenetidine the absorbance at 510 nm was measured. The iron content was determined either by using a calibration curve obtained with iron standards or by calculation: pg iron1100 ml solution
=
A, - & Ast - &
x 75,
30
ALEXIEV, TABLE DETERMINATION
1 OF IRON
CATALYTIC
BY THE
METHOD”
Volume Solution
Blank
Standard or sample H,O, (0.2 M) 1, WPhenanthroline (0.00075 M) Buffer (pH 2.8)
2.50
(ml)
Standard
Sample
0.10 2.50
1.00 5.00 (Heating 1.50 (Vigorous
p-Phenetidine (0.05 M)
RACHINA,
0.10 2.50
1.00 1.00 4.90 4.90 10 min at 40°C) 1.50 1.50 shaking; heating 30 min at 40°C)
where A,, Ast, and Ab indicate absorbance of sample, standard, and blank, respectively. 2. Photometric method. The procedure is followed in Table 2. After standing 30-40 min at room temperature the absorption readings were made at 535 nm (4). Calculation: pg iron/100 ml = $-
x 100, st
where A, and Ast indicate absorbance of sample and standard measured against the blank. 3. Atomic absorption method. The analysis were performed in a Perkin-Elmer Model 403 flame atomic absorption spectrophotomTABLE DETERMINATION PHOTOMETRIC
2
AND
eter calibrated with standard solutions of iron (0.5, 1.0, and 1.5 &ml). A lean airacetylene flame was used to avoid interference from HNO, and nickel. The detection wavelength was 248.3 nm (4). The use of graphite furnace is connected with higher sensitivity, but we prefer to compare the results of the catalytic method with the more widely used fiame variant of the atomic absorption method. RESULTS
Volume Solution
Blank
Standard
Buffer (pH 5.5) Bathophenanthroline Bidistilled water Standard or sample Ascorbate (dry substance)
2.00 1.00 1.00 5 mg
2.00 1.00 1.00 5 w
(ml) Sample 2.00 1.00 1.00 5 mg
AND DISCUSSION
Table 3 shows the amounts of iron found in human skin and cow milk after dry ashing of the samples. The content of iron in the tested animal organs is presented in Table 4 (preliminary dried samples) and Table 5 (fresh tissue). In all cases the mean values obtained by the catalytic method were reproducible and in very close agreement with those obtained by the photometric method, independently of the pretreatment procedure used. Additional data for the accuracy of the catalytic method have been obtained using the standard addition technique (Table 6). The standard addition of Fe(II1) was introTABLE IRON
CONTENT
1 2 3 4 5 6 7 8
3
IN ASHED SAMPLES AND HUMAN SKIN”
No. of samples Sample
OF IRON BY THE METHOD
BONTCHEV
Mean value
OF Cow
MILK
Standard deviation
A”
B
A
B
A
B
10 5 4 7 8 2 2 2
5 5 4 7 8 2 2 2
0.59 0.63 0.69 0.44 0.41 0.31 0.37 0.42
0.58 0.62 0.70 0.39 0.38 0.35 0.37 0.45
0.017 0.014 0.014 0.030 0.022 -
0.025 0.026 0.016 0.045 0.020 -
a Iron content is expressed as g/100 g ash. Samples l-5: cow milk; samples 6-8: human skin. b A, Catalytic method. B, photometric method.
QUANTITATION TABLE
OF IRON IN BIOLOGICAL
4
TABLE
Mean values
5 PIG ORGANS TISSUES
IRON CONTENT OF GUINEA DETERMINED OF FRESH
IRON CONTENT OF GUINEA PIG ORGANS DETERMINED AFTER DIGESTION OF DRIED SAMPLW
No. of samples
31
MATERIAL
No. of samples
Standard deviation
Sample
A*
B
A
B
A
B
1 2 3 4 5 6 7 8 9 10 11 12
8 10 12 9 9 6 3 10 6 5 9 8
8 10 12 7 8 4 7 6 5 4 8
7.9 5.7 7.1 12.9 3.2 8.1 5.2 3.8 2.3 6.3 3.2 26.2
8.1 4.9 6.5 11.5 3.6 8.4
1.2 0.69 0.40 1.10 0.83 0.22
0.22 0.37 0.33 0.22 0.68 1.00
4.2 2.4 6.5 4.0 28.7
1.00 0.41 0.53 0.71 2.4
0.94 0.39 0.22 0.35 4.7
n Iron content is expressed as g/100 g dried material x IO-*. Sample weight: 10 mg. Volume of digesting mixture: 0.15 ml. Final volume of digested material: 20 ml. Samples l-8: lung; 9 and 10: kidney; 11 and 12: spleen. b A, Catalytic method; B, photometric method.
duced to the samples of fresh tissue together with the acids used for mineralization. An important advantage of the catalytic method is its higher sensitivity which enables one to perform all analyses with lo20 times less material than that required by the photometric method or the atomic absorption in flame variant. To demonstrate this, 5 mg fresh tissue was digested with H2S04-HNOB-HzOz, the digest was brought to 0.5 ml, and O.l-ml aliquots were used to quantitate the iron by the catalytic method; alternatively, 200 mg fresh tissue was treated in the same way, brought to 20 ml and used to test the iron content by the photometric and atomic methods. The results are shown in Table 7. The high sensitivity of the catalytic reaction allows the application of our method to samples smaller than 5 mg but in this case special attention should be paid to the exact
Mean values
Standard deviation
Sample
A”
B
A
B
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13
13 12 6 7 6 4 4 4 4 4 4 8 6
13 13 6 7 6 4 4 4 4 4 4 8 6
5.4 5.5 5.0 8.8 6.3 6.4 10.5 5.1 6.3 5.2 8.8 15.9 29.1
5.5 5.3 4.8 8.4 6.3 4.8 9.7 4.3 6.8 4.8 8.5 15.1 26.1
3.2 2.3 0.3 1.7 0.3 0.41 0.45 1.2 1.4 0.3 0.22 2.0 7.5
3.00 2.5 0.5 1.5 0.7 1.7 0.53 0.41 0.57 0.3 0.41 1.6 6.7
a Iron content is expressed as g/100 g fresh tissue x 10m3.Sample weight: 200 mg. Volume of digesting mixture: 0.20 ml. Final volume of the digest: 20 ml. Sample 1: lung; 2-11: kidney; 12 and 13: spleen. b A, Catalytic method; B, photometric method.
weight of the starting material. We believe that with small modifications the suggested method could be used for quantitation of TABLE
6
STANDARD ADDITION OF Fe(II1) TO FRESH GUINEA PIG ORGANS~ No. of samples Standard Sample Ab B addition 155 2 3 4 5 6
5 55 55 55 55
5
1.9 3.8 7.6 3.8 3.8 15.2
Found ~ A
B
1.7 2.0 3.9 3.6 7.5 7.8 3.5 3.7 4.0 4.0 15.6 15.0
Standard deviation ~ A B 0.15 0.23 0.19 0.36 0.34 0.96
0.12 0.46 0.15 0.48 0.31 1.05
a Iron content is expressed as g/100 g fresh tissue X 10m3.Sample weight: 200 mg. Volume of digesting mixture: 0.20 ml. Final volume of the digest: 20 ml. Sample 1: lung; 2-5: kidney; 13: spleen. * A, Catalytic method; B, photometric method.
32
ALEXIEV,
RACHINA, TABLE
IRON
CONTENT
OF GUINEA
PIG ORGANS
No. of samples
AND BONTCHEV 7 AFTER DIGESTION
OF FRESH
TISSUE”
Mean values
Standard deviation
Sample
Ab
B
C
A
B
C
A
B
C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
I 4 5 4 6 5 5 6 5 4 4 4 6 6 6 4 4 4 3 4
7 4 6 5 6 6 5 4 4 6 6 4 4 4 3 5
7 6 5 6 5 4 4 6 6 4 4 3 5
1.2 1.7 3.0 1.8 2.6 2.2 1.9 3.1 4.0 5.0 2.5 3.3 5.1 4.1 12.8 2.7 4.9 9.0 17.5 24.0
2.0 1.8 2.6 2.3 1.6 3.3 4.5 3.2 2.1 5.8 4.8 2.5 6.0 8.9 21.3 15.0
2.6 2.3 2.2 3.3 4.3 2.7 3.3 4.8 4.0 4.3 12.6 22.1 30.0
0.33 1.2 0.3 0.8 0.32 0.32 0.41 0.57 0.74 0.26 1.2 1.3 2.4 0.41 1.2 0.20 0.95 1.30 2.80 3.70
1.00 0.66 0.24 0.14 0.17 0.50 0.61 1.3 0.50 1.7 0.77 0.24 0.61 0.50 1.50 7.40
0.20 0.26 0.18 0.46 0.83 0.84 0.32 2.1 1.2 1.5 0.42 1.40 8.50
a Iron content is expressed as g/l00 g fresh tissue X 10m3.Samples 1-8: kidney; 9- 14: lung; 15-20: spleen. b A, Catalytic method. Sample weight: 5 mg; volume of digesting mixture: 0.012 ml; final volume of the digest: 0.5 ml. B, Photometric method; C, atomic absorption method.
iron in biological origin.
material
of different
REFERENCES 1. Alexiev, A. A., Bontchev, P. R., and Raykova, D. (1974) Mikrochim. Ada, 751-758.
2. Bontchev, P. R., Raykova, D., Alexiev, A. A., and Jankova, D. (1974) Clin. Chim. Acta 57, 37-44. 3. Bontchev, P. R. (1975) Complex Formation and Catalytic Activity, p. 120, MIR, Moscow. [In Russian.] 4. Davies, M. I., Bush, K., and Motzik, I. (1972) J. Ass. Of’@. Anal. Chem. 55, 1206-1210.
BIOCHEMISTRY
99,
28-32 (1979)
A Catalytic Method for Determination of Trace Amounts iron in Biological Material A. A. ALEXIEV,* *Department
of Chemistry
V. RACHINA,*
and Biochemistry, Medical Faculty of Chemistry,
of
AND P. R. BoNTcHEvt’l
Faculty, University
Pleven, and TDepartment of Sofia, Bulgaria
of Analytical
Chemistry,
Received October 5, 1978 A rapid, simple, and reproducible method for determination of iron in biological material is suggested using the oxidation of p-phenetidine hydrochloride with hydrogen peroxide in a reaction catalyzed by Fe(lII) and activated by f,lO-phenanthroline. The high sensitivity of the reaction allows a single determination to be carried out with as much as l-5 mg fresh tissue.
In our previous papers (1,2) a rapid method for determination of iron in blood serum was suggested using the oxidation of p-phenetidine with hydrogen peroxide in a reaction catalyzed by Fe(II1) and activated by 1, lophenanthroline. The colored reaction product shows an absorption band at 510 nm, used for photometric measurements. The activator plays an important role in this reaction. It has been shown, that together with p-phenetidine it forms a ternary complex with Fe(II1) (1: 1: 1) and increases sharply its catalytic activity not participating directly as a reagent in the reaction (3). The other metals present in biological samples do not influence the indicator reaction at least at concentrations two times greater than their usual concentration in such samples (1). Even more, in the presence of 1, lo-phenanthroline many of these ions decrease further their catalytic activity. It has been proved also that the solution obtained after mineralization of the biological sample does not contain substances that can affect the catalytic activity. This fact, together with the 1 To whom requests for reprints should be addressed. 0003-2697/79/150028-05$02.00/O Copyright AU rights
0 1979 by Academic Press, Inc. of reproduction in any fomt reserved.
high sensitivity of the reaction (0.001 pg/ ml), its reproducibility, and simplicity of the procedure encouraged us to adopt this method to various biological materials where the iron content is very low. EXPERIMENTAL Reagents
1. 0.2 M and 30% H,O, were used as solutions free of stabilizing agents. The exact concentrations were determined titrimetritally with permanganate. 2. 0.05 M p-phenetidine hydrochloride. 3. 0.00075 M l,lO-phenanthroline. Solutions l-3 should be light protected and stored in a refrigerator. 4. Standard solution of FeCl, in 0.005 M HCl (0.75 pg Fe(III)/ml). Initial solution of FeCl, (-0.1 M) in 0.1 M HCl was used, its exact Fe(II1) concentration being determined by gravimetric and complexometric methods. The standard solution was prepared from it by consecutive dilutions with 0.005 M HCl. 5. Potassium hydrogen phthalate buffer, pH 2.8 (see ref. (2)). 6. Phosphate buffer, pH 5.5. 28
QUANTITATION
OF IRON
7, 0.00068 M solution of disodium bathophenanthroline disulfonate in phosphate buffer, pH 5.5. 8. Standard solution of Fe(II1): 1 pg/ml. 9. Sodium ascorbate. 10. Concentrated HzS04, NH03, and HCl. All reagents used were of analytical grade and solutions were prepared with bidistilled water, Special attention was given to the iron assay of the chemicals. Glassware was kept overnight in HCl (1: 1) and rinsed with bidistilled water in order to be iron free. The optical measurements were performed using single-beam Specol spectrophotometer equipped with a thermostatic cell EK-5. Preliminary
Sample
Treatment
The quantity of iron was determined in samples of cow milk, human skin, and guinea pig kidney, lung, and spleen. After killing the animal the organs were removed, extensively rinsed in bidistilled water and cut into pieces by a plastic knife. This preliminary treatment of the tissue was used as a very simple one, suitable for the aim of the investigation. After mineralization a homogeneous solution is obtained, whose iron content can be determined according to the three methods used. If the exact iron content of the tissue samples is to be determined, they must be perfused before mineralization. Dry ashing. This pretreatment was applied to the samples of skin and milk. A piece of skin (0.5- 1.O g) or IO-20 ml of cow milk were dried in a quartz vessel by heating at 105”C, transferred to a muffle and heated at 450°C for 48 h. Under these conditions the sample of skin was thoroughly ashed, while the milk sample was not completely charred and required an additional treatment consisting in moistening of the material with a few drops of concentrated HN03, evaporation of the latter, and heating the sample for another 1 h at 450°C. This treatment was repeated until the sample was completely
IN BIOLOGICAL
29
MATERIAL
ashed. The charred material thus obtained was ground to powder in an agetate mortar. Aliquots of 5 mg were placed in a quartz tube, 1 ml of hot 1 N HCl was added and the solution brought to 20 ml with distilled water. Digestion with H2S04, HNO, and HzOz. The procedure was applied to samples of animal organs (kidney, lung, and spleen). Digestion was carried out with both fresh and dried material in parallel experiments. In the latter case the sample was dried at 105°C till constant weight, the resulting material powdered in a mortar, and an aliquot weighed for further processing. Concentrated H,SO, and HNO, (1: 1) were added to the sample in a small quartz tube and the mixture was heated gently. As the digestion proceeded, small portions of HNO, (l-2 drops at a time) were added. Digest was cooled just before HN03 to be completely evaporated, l-2 drops of H,O, were added, and the solution again heated to fumes of SO,. Digest was then cooled and brought to the desired volume with bidistilled water. The content of iron in the solutions thus obtained was determined using three different methods: photometrically, by atomic absorption, and by our catalytic method. Because of the high selectivity of the reaction, neither preliminary separation of iron nor use of masking agents were necessary. Analytical of Iron
Methods for Determination
1. Catalytic method. The procedure is illustrated in Table 1. Thirty minutes after the addition ofp-phenetidine the absorbance at 510 nm was measured. The iron content was determined either by using a calibration curve obtained with iron standards or by calculation: pg iron1100 ml solution
=
A, - & Ast - &
x 75,
30
ALEXIEV, TABLE DETERMINATION
1 OF IRON
CATALYTIC
BY THE
METHOD”
Volume Solution
Blank
Standard or sample H,O, (0.2 M) 1, WPhenanthroline (0.00075 M) Buffer (pH 2.8)
2.50
(ml)
Standard
Sample
0.10 2.50
1.00 5.00 (Heating 1.50 (Vigorous
p-Phenetidine (0.05 M)
RACHINA,
0.10 2.50
1.00 1.00 4.90 4.90 10 min at 40°C) 1.50 1.50 shaking; heating 30 min at 40°C)
where A,, Ast, and Ab indicate absorbance of sample, standard, and blank, respectively. 2. Photometric method. The procedure is followed in Table 2. After standing 30-40 min at room temperature the absorption readings were made at 535 nm (4). Calculation: pg iron/100 ml = $-
x 100, st
where A, and Ast indicate absorbance of sample and standard measured against the blank. 3. Atomic absorption method. The analysis were performed in a Perkin-Elmer Model 403 flame atomic absorption spectrophotomTABLE DETERMINATION PHOTOMETRIC
2
AND
eter calibrated with standard solutions of iron (0.5, 1.0, and 1.5 &ml). A lean airacetylene flame was used to avoid interference from HNO, and nickel. The detection wavelength was 248.3 nm (4). The use of graphite furnace is connected with higher sensitivity, but we prefer to compare the results of the catalytic method with the more widely used fiame variant of the atomic absorption method. RESULTS
Volume Solution
Blank
Standard
Buffer (pH 5.5) Bathophenanthroline Bidistilled water Standard or sample Ascorbate (dry substance)
2.00 1.00 1.00 5 mg
2.00 1.00 1.00 5 w
(ml) Sample 2.00 1.00 1.00 5 mg
AND DISCUSSION
Table 3 shows the amounts of iron found in human skin and cow milk after dry ashing of the samples. The content of iron in the tested animal organs is presented in Table 4 (preliminary dried samples) and Table 5 (fresh tissue). In all cases the mean values obtained by the catalytic method were reproducible and in very close agreement with those obtained by the photometric method, independently of the pretreatment procedure used. Additional data for the accuracy of the catalytic method have been obtained using the standard addition technique (Table 6). The standard addition of Fe(II1) was introTABLE IRON
CONTENT
1 2 3 4 5 6 7 8
3
IN ASHED SAMPLES AND HUMAN SKIN”
No. of samples Sample
OF IRON BY THE METHOD
BONTCHEV
Mean value
OF Cow
MILK
Standard deviation
A”
B
A
B
A
B
10 5 4 7 8 2 2 2
5 5 4 7 8 2 2 2
0.59 0.63 0.69 0.44 0.41 0.31 0.37 0.42
0.58 0.62 0.70 0.39 0.38 0.35 0.37 0.45
0.017 0.014 0.014 0.030 0.022 -
0.025 0.026 0.016 0.045 0.020 -
a Iron content is expressed as g/100 g ash. Samples l-5: cow milk; samples 6-8: human skin. b A, Catalytic method. B, photometric method.
QUANTITATION TABLE
OF IRON IN BIOLOGICAL
4
TABLE
Mean values
5 PIG ORGANS TISSUES
IRON CONTENT OF GUINEA DETERMINED OF FRESH
IRON CONTENT OF GUINEA PIG ORGANS DETERMINED AFTER DIGESTION OF DRIED SAMPLW
No. of samples
31
MATERIAL
No. of samples
Standard deviation
Sample
A*
B
A
B
A
B
1 2 3 4 5 6 7 8 9 10 11 12
8 10 12 9 9 6 3 10 6 5 9 8
8 10 12 7 8 4 7 6 5 4 8
7.9 5.7 7.1 12.9 3.2 8.1 5.2 3.8 2.3 6.3 3.2 26.2
8.1 4.9 6.5 11.5 3.6 8.4
1.2 0.69 0.40 1.10 0.83 0.22
0.22 0.37 0.33 0.22 0.68 1.00
4.2 2.4 6.5 4.0 28.7
1.00 0.41 0.53 0.71 2.4
0.94 0.39 0.22 0.35 4.7
n Iron content is expressed as g/100 g dried material x IO-*. Sample weight: 10 mg. Volume of digesting mixture: 0.15 ml. Final volume of digested material: 20 ml. Samples l-8: lung; 9 and 10: kidney; 11 and 12: spleen. b A, Catalytic method; B, photometric method.
duced to the samples of fresh tissue together with the acids used for mineralization. An important advantage of the catalytic method is its higher sensitivity which enables one to perform all analyses with lo20 times less material than that required by the photometric method or the atomic absorption in flame variant. To demonstrate this, 5 mg fresh tissue was digested with H2S04-HNOB-HzOz, the digest was brought to 0.5 ml, and O.l-ml aliquots were used to quantitate the iron by the catalytic method; alternatively, 200 mg fresh tissue was treated in the same way, brought to 20 ml and used to test the iron content by the photometric and atomic methods. The results are shown in Table 7. The high sensitivity of the catalytic reaction allows the application of our method to samples smaller than 5 mg but in this case special attention should be paid to the exact
Mean values
Standard deviation
Sample
A”
B
A
B
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13
13 12 6 7 6 4 4 4 4 4 4 8 6
13 13 6 7 6 4 4 4 4 4 4 8 6
5.4 5.5 5.0 8.8 6.3 6.4 10.5 5.1 6.3 5.2 8.8 15.9 29.1
5.5 5.3 4.8 8.4 6.3 4.8 9.7 4.3 6.8 4.8 8.5 15.1 26.1
3.2 2.3 0.3 1.7 0.3 0.41 0.45 1.2 1.4 0.3 0.22 2.0 7.5
3.00 2.5 0.5 1.5 0.7 1.7 0.53 0.41 0.57 0.3 0.41 1.6 6.7
a Iron content is expressed as g/100 g fresh tissue x 10m3.Sample weight: 200 mg. Volume of digesting mixture: 0.20 ml. Final volume of the digest: 20 ml. Sample 1: lung; 2-11: kidney; 12 and 13: spleen. b A, Catalytic method; B, photometric method.
weight of the starting material. We believe that with small modifications the suggested method could be used for quantitation of TABLE
6
STANDARD ADDITION OF Fe(II1) TO FRESH GUINEA PIG ORGANS~ No. of samples Standard Sample Ab B addition 155 2 3 4 5 6
5 55 55 55 55
5
1.9 3.8 7.6 3.8 3.8 15.2
Found ~ A
B
1.7 2.0 3.9 3.6 7.5 7.8 3.5 3.7 4.0 4.0 15.6 15.0
Standard deviation ~ A B 0.15 0.23 0.19 0.36 0.34 0.96
0.12 0.46 0.15 0.48 0.31 1.05
a Iron content is expressed as g/100 g fresh tissue X 10m3.Sample weight: 200 mg. Volume of digesting mixture: 0.20 ml. Final volume of the digest: 20 ml. Sample 1: lung; 2-5: kidney; 13: spleen. * A, Catalytic method; B, photometric method.
32
ALEXIEV,
RACHINA, TABLE
IRON
CONTENT
OF GUINEA
PIG ORGANS
No. of samples
AND BONTCHEV 7 AFTER DIGESTION
OF FRESH
TISSUE”
Mean values
Standard deviation
Sample
Ab
B
C
A
B
C
A
B
C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
I 4 5 4 6 5 5 6 5 4 4 4 6 6 6 4 4 4 3 4
7 4 6 5 6 6 5 4 4 6 6 4 4 4 3 5
7 6 5 6 5 4 4 6 6 4 4 3 5
1.2 1.7 3.0 1.8 2.6 2.2 1.9 3.1 4.0 5.0 2.5 3.3 5.1 4.1 12.8 2.7 4.9 9.0 17.5 24.0
2.0 1.8 2.6 2.3 1.6 3.3 4.5 3.2 2.1 5.8 4.8 2.5 6.0 8.9 21.3 15.0
2.6 2.3 2.2 3.3 4.3 2.7 3.3 4.8 4.0 4.3 12.6 22.1 30.0
0.33 1.2 0.3 0.8 0.32 0.32 0.41 0.57 0.74 0.26 1.2 1.3 2.4 0.41 1.2 0.20 0.95 1.30 2.80 3.70
1.00 0.66 0.24 0.14 0.17 0.50 0.61 1.3 0.50 1.7 0.77 0.24 0.61 0.50 1.50 7.40
0.20 0.26 0.18 0.46 0.83 0.84 0.32 2.1 1.2 1.5 0.42 1.40 8.50
a Iron content is expressed as g/l00 g fresh tissue X 10m3.Samples 1-8: kidney; 9- 14: lung; 15-20: spleen. b A, Catalytic method. Sample weight: 5 mg; volume of digesting mixture: 0.012 ml; final volume of the digest: 0.5 ml. B, Photometric method; C, atomic absorption method.
iron in biological origin.
material
of different
REFERENCES 1. Alexiev, A. A., Bontchev, P. R., and Raykova, D. (1974) Mikrochim. Ada, 751-758.
2. Bontchev, P. R., Raykova, D., Alexiev, A. A., and Jankova, D. (1974) Clin. Chim. Acta 57, 37-44. 3. Bontchev, P. R. (1975) Complex Formation and Catalytic Activity, p. 120, MIR, Moscow. [In Russian.] 4. Davies, M. I., Bush, K., and Motzik, I. (1972) J. Ass. Of’@. Anal. Chem. 55, 1206-1210.
