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Serum ferritin, cobalt excretion and body iron status Janet Sorbie, md, John Ludwig, rt
m
sc; Leslie S. Valberg, md, m sc,
Summary: Serum ferritin concentration was measured by immunoradiometric assay in 64 subjects. It was closely related to the size of body iron stores measured by hemosiderin content of bone marrow in all subjects and by the deferoxamine test in 10 patients with iron overload. Urinary cobalt excretion, an indirect measure of iron absorption, was inversely related to hemosiderin content of bone marrow in 34 patients aged 18 to 72 with or
without liver disease, but this relation did not hold in a group of 20 student volunteers aged 17 to 30, indicating that the test is unreliable in young people. A strong inverse correlation was demonstrated between values for cobalt excretion and serum ferritin in the 34 patients and between those for iron absorption and serum ferritin in the 20 students. Serum ferritin concentration appears to reflect accurately the iron status of the healthy individual but high values in liver disease must be interpreted with caution.
Resume: La ferritine serique, l'excretion du cobalt I'dtat des reserves de fer du corps
et
Nous avons mesure par la technique immunoradiometrique la concentration de ferritine serique chez 64 sujets. Cette concentration etait en etroite correlation avec la quantite des reserves organiques de fer, mesuree par la teneur de la moelle osseuse en hemosiderine chez tous les sujets et par I'epreuve a la deferoxamine chez 10 malades hypersideremiques. L'excretion urinaire de cobalt, mesure indirecte de I'absorption du fer, etait inversement proportionnelle a la teneur en hemosiderine de la moelle osseuse chez 34 malades dont I'age variait de 18 a 72 ans et qui avaient ou qui n'avaient pas de maladie hepatique. Cette relation n'etait pas valable chez un group de 20 etudiants volontaires ages de 17 a 30 ans, ce qui indique qu'on ne peut compter sur la valeur de cette epreuve chez de jeunes adultes. Une forte correlation, en sens inverse, existait entre les valeurs de l'excretion de cobalt et la ferritine serique chez les 34 malades et entre celles de I'absorption de fer et la ferritine serique chez les 20 etudiants. La concentration de ferritine serique parait refleter avec precision les reserves organiques de fer From the special investigation unit, Kingston General Hospital, and the departments of medicine and pathology, Queen's University, Kingston, Ont. Supported by a grant from the Medical Research Council of Canada Reprint requests to: Dr. J. Sorbie, Etherington Hall, Queen's
University, Kingston, Ont. K7L 3N6
frcpIc], facp; William E.N. Corbett, md, frcp[c]; chez I'individu sain, mais les valeurs plus elevees qu'on hepatopathies doivent etre interpretees
trouve dans les avec
prudence.
Ferritin was for many years thought to exist merely as an intracellular storage form of iron, although ferritinemia was known to occur in acute hepatocellular disease.1 With the development of a sensitive immunoradiometric assay,2 ferritin has been detected in the circulation of normal sub¬ jects;3'4 its concentration in serum appears to reflect the size of body iron stores over a wide range.2,3,5'7 In addition, an inverse relation was demonstrated between serum ferritin concentration and percentage absorption of iron.4,8 In clin¬ ical studies, patients with anemia due to iron deficiency could be distinguished from those with the anemia of in¬ flammation by the serum ferritin concentration.6 Another test that can differentiate iron deficiency anemia from anemia due to other causes is the cobalt excretion test.910 This provides an indirect measure of iron absorp¬ tion, based on the finding that cobalt absorption responds to mechanisms that enhance iron absorption.11 Cobalt, unlike iron, is excreted rapidly in the urine, in direct propor¬ tion to the amount absorbed from the gastrointestinal tract.11 A close correlation was found between the intestinal absorption of iron given orally and the 6-hour urinary excretion of cobalt given orally in 32 patients with various sizes of iron stores.10 Cobalt excretion, like iron absorption, was inversely related to the amount of hemosiderin in the bone marrow.9 The present study examines the relation between values for serum ferritin, cobalt excretion, iron absorption and bone marrow hemosiderin in a group of student volunteers and in a group of patients with a variety of diseases.
Subjects and methods Subjects studied Serum ferritin concentration, urinary cobalt excretion and other measurements of body iron status were determined in three groups of subjects. The first group comprised 34 patients with a variety of disorders selected from our clin¬ ical practice. Patients with renal failure or malabsorption were excluded. These 34 patients included 16 with and 18 without liver disease. The 14 patients with alcoholic liver disease underwent liver biopsy to establish the diagnosis; the grade of stainable iron ranged from 0 to 3+.11 Two patients with carcinoma of the cclon underwent laparotomy; CMA JOURNAL/MAY 17, 1975/VOL. 112 1173
one had extensive hepatic metastases and was included among those with liver disease, as was a patient with viral
hepatitis.
The second
was a
group of 10
patients
with
body
iron
overload established by finding an increased amount (4-b) of stainable iron in the liver biopsy specimen.11 Of the seven studied before treatment five had iron overload sec¬ ondary to alcoholic cirrhosis, one had taken oral iron with large amounts of brandy for 30 years, and one had primary sideroblastic anemia. Three patients with idiopathic hemochromatosis were studied after venesection. The third group consisted of 20 healthy students, 18 men and 2 women (ages, 17 to 30 years). All but 4 students had donated blood previously, i2 of them within 3 to 12 months, and 10 had donated between 5 and 10 pints of blood in total. Informed consent was obtained for the investigations from all of the students and the patients. Serum ferritin Serum ferritin was measured by a two-site immunoradio¬ metric assay.12 Iron absorption A whole-body counting technique was used to measure the absorption of an oral test dose cf 10 fimol of iron ascorbate labelled with 4 /xCi of 59Fe.10 When iron ab¬ sorption and cobalt excretion were measured in the same subject, the iron solution was given first, and the next day the cobalt test was done. Radioactive iron in the body was distinguished from residual cobalt activity by differential counting. The normal range of values for the technique is 6 to 52%.13 Cobalt excretion A test dose of 20 /xmol of cobaltous chloride labelled with 0.5 /xCi of 57Co was given orally and the percentage excreted in the urine in 6 hours was measured. In an earlier study the range of values in control subjects aged 31 to 75 years with plentiful iron stores was 4 to 11%.10 Patients with iron deficiency anemia excreted more than 12% of the dose in 6 hours, whereas patients in this age group with anemia from other causes excreted less than 11%. A broader range, 4 to 14%, was found in subjects aged 15 to 30 years with plentiful iron stores. Assessment of body iron stores Bone marrow iron stores: The amount of stainable iron in smears of a bone marrow aspirate was estimated by one of us (W.E.N.C.), as described previously,9 without knowledge of the clinical status of the subjects. The iron in reticuloendothelial cells was graded from 0 (no stainable iron) to 4+ (stainable iron visible in 100% of high power
fields).
Liver iron stores: Liver biopsy sections stained with Prussian blue were graded on a scale of 0 to 4+ for hemo¬ siderin;11 grades 0 to 34- were considered normal. A grade of 4+ represented iron in 100% of the cells in mas¬ sive amounts; subjects with this grade were considered to have iron overload. Deferoxamine test: This was carried out as described by Balcerzak et al.1* Urine was collected for 24 hours before and for 24 hours after the intramuscular injection of 1000 mg of deferoxamine mesylate. Urinary iron concentration was determined by spectrophotometry after acid digestion and colour development with dipyridyl. A value of more than 4 mg/24 h is highly suggestive of iron overload. Other methods The hemoglobin concentration was measured with the Model S Coulter Counter (Coulter Electronics Inc, Hialeah, Florida). The technique for measurement of serum iron 1174 CMA
JOURNAL/MAY 17, 1975/VOL. 112
and unsaturated iron-binding capacity has been described previously.15 The normal range of values for serum iron by this method is 55 to 185 /xg/dl and that for total ironbinding capacity is 250 to 430 /xg/dl; transferrin saturation in normal subjects ranges from 15 to 47%.15
Statistical methods The statistical difference between any two mean values was assessed by the /-test. The least squares method of linear regression was used to analyse the relation between serum ferritin values and other measurements.16 Because serum ferritin values are normally distributed on a logarithmic scale,4 they were converted to logarithms for re¬
gression analysis.
Results The serum ferritin values for all 64 subjects are shown in Fig. 1. The mean was lowest in the group of healthy students, 59 ng/ml (range, 7 to 170 ng/ml); a little higher in the patients with no liver disease, 73 ng/ml (range, 5 to 282 ng/ml); and considerably higher in the patients with
10,000 6,000-
4,000 2,0001,000 600 H
oo
400 c
200-
t 100£
60
©
4.
40 o
20
o© o o
10H o
FIG. 1.Serum ferritin values, on logarithmic scale, in four groups of subjects. Arithmetic mean of each group indicated by horizontal bar. Bone marrow iron graded as absent (open circles), intermediate (dotted circles) or plentiful (solid circles). Three patients with iron overload treated by venesection denoted by arrows.
liver disease, 393 ng/ml (range, 11 to 1063 ng/ml). When the students and patients without liver disease were con¬
Patients with low
CO
normal iron stores The patients with 0 to 3+ marrow iron, including those with liver disease, were subdivided into three groups on the basis of the stainable iron content of the bone marrow (Table I). In the patients with absent marrow hemosiderin the mean serum ferritin value was significantly lower than in those with 1+ or 2+ to 3+ stainable iron, and there was no overlap in the range of values. The mean value for cobalt excretion in the group with absent marrow iron was significantly greater than that in the group with intermediate marrow iron, which in turn was higher than the mean value in the group with plentiful marrow iron. No overlap was present between the ranges of values for the cobalt excretion test in subjects with 0 and 2+ to 3-f- marrow iron. The results in the 1+ group overlapped the ranges of both of these groups. The relation between serum ferritin values and the results of the other tests is shown in Table II. The inverse correla¬ tion between serum ferritin concentration and percentage excretion of cobalt is shown in Fig. 2. Patients with iron overload The serum ferritin value was increased in all seven un¬ treated patients but the range was very broad (Table I). It was also above normal in two of the treated patients and was in the range of iron deficiency in a patient whose marrow iron stores had been completely depleted by vene¬ section (Fig. 1). There was no relation between serum fer¬ ritin value and the results of any of the other tests except the deferoxamine test when all 10 patients were considered (Table II). A strong positive correlation was evident be¬ tween values for iron absorption and cobalt excretion (Fig. 3 a), similar to the correlation in patients with 0 to 4+ marrow iron reported in a previous study.10
FIG. 2.Relation between serum ferritin concentration and cobalt excretion in patients with absent (open circles), intermediate (dotted circles) or plentiful (solid circles) bone marrow iron stores (r = .0.764, P < 0.001).
Students None of the students was anemic and all 20 had a normal serum iron value, iron-binding capacity and transferrin saturation (Table III). When they were divided into three groups according to stainable iron in the bone mar¬ row, no significant difference was found among the 0, 1-f and 2-f to 3+ groups in the mean hemoglobin value, serum iron value, total iron-binding capacity or transferrin saturation. The mean serum ferritin value in the students with absent marrow hemosiderin was significantly less than the value in those with 2+ to 3-f marrow iron. The differ¬ ences between the average value of the four students with 1+ marrow iron and those of the other two groups did not achieve statistical significance. The mean cobalt excretion
sidered together, the ranges were 5 to 38 ng/ml in those with absent marrow hemosiderin and 59 to 282 ng/ml in those with plentiful marrow iron. The average serum fer¬ ritin value in the group with iron overload was 2854 ng/ml and values ranged from 406 to 12 204 ng/ml in untreated patients and from 12 to 494 ng/ml in patients treated by venesection. The relation between serum ferritin concentration and the results of various tests for the assessment of iron status were analysed for each of three groups of subjects.
O)
c
.
P^p :M v"4- &* '3§H»J« ::$j0*%?i ffeltf11 '¦^S^lSf#^^33^';s#^'? pW
Serum ferritin, cobalt excretion and body iron status Janet Sorbie, md, John Ludwig, rt
m
sc; Leslie S. Valberg, md, m sc,
Summary: Serum ferritin concentration was measured by immunoradiometric assay in 64 subjects. It was closely related to the size of body iron stores measured by hemosiderin content of bone marrow in all subjects and by the deferoxamine test in 10 patients with iron overload. Urinary cobalt excretion, an indirect measure of iron absorption, was inversely related to hemosiderin content of bone marrow in 34 patients aged 18 to 72 with or
without liver disease, but this relation did not hold in a group of 20 student volunteers aged 17 to 30, indicating that the test is unreliable in young people. A strong inverse correlation was demonstrated between values for cobalt excretion and serum ferritin in the 34 patients and between those for iron absorption and serum ferritin in the 20 students. Serum ferritin concentration appears to reflect accurately the iron status of the healthy individual but high values in liver disease must be interpreted with caution.
Resume: La ferritine serique, l'excretion du cobalt I'dtat des reserves de fer du corps
et
Nous avons mesure par la technique immunoradiometrique la concentration de ferritine serique chez 64 sujets. Cette concentration etait en etroite correlation avec la quantite des reserves organiques de fer, mesuree par la teneur de la moelle osseuse en hemosiderine chez tous les sujets et par I'epreuve a la deferoxamine chez 10 malades hypersideremiques. L'excretion urinaire de cobalt, mesure indirecte de I'absorption du fer, etait inversement proportionnelle a la teneur en hemosiderine de la moelle osseuse chez 34 malades dont I'age variait de 18 a 72 ans et qui avaient ou qui n'avaient pas de maladie hepatique. Cette relation n'etait pas valable chez un group de 20 etudiants volontaires ages de 17 a 30 ans, ce qui indique qu'on ne peut compter sur la valeur de cette epreuve chez de jeunes adultes. Une forte correlation, en sens inverse, existait entre les valeurs de l'excretion de cobalt et la ferritine serique chez les 34 malades et entre celles de I'absorption de fer et la ferritine serique chez les 20 etudiants. La concentration de ferritine serique parait refleter avec precision les reserves organiques de fer From the special investigation unit, Kingston General Hospital, and the departments of medicine and pathology, Queen's University, Kingston, Ont. Supported by a grant from the Medical Research Council of Canada Reprint requests to: Dr. J. Sorbie, Etherington Hall, Queen's
University, Kingston, Ont. K7L 3N6
frcpIc], facp; William E.N. Corbett, md, frcp[c]; chez I'individu sain, mais les valeurs plus elevees qu'on hepatopathies doivent etre interpretees
trouve dans les avec
prudence.
Ferritin was for many years thought to exist merely as an intracellular storage form of iron, although ferritinemia was known to occur in acute hepatocellular disease.1 With the development of a sensitive immunoradiometric assay,2 ferritin has been detected in the circulation of normal sub¬ jects;3'4 its concentration in serum appears to reflect the size of body iron stores over a wide range.2,3,5'7 In addition, an inverse relation was demonstrated between serum ferritin concentration and percentage absorption of iron.4,8 In clin¬ ical studies, patients with anemia due to iron deficiency could be distinguished from those with the anemia of in¬ flammation by the serum ferritin concentration.6 Another test that can differentiate iron deficiency anemia from anemia due to other causes is the cobalt excretion test.910 This provides an indirect measure of iron absorp¬ tion, based on the finding that cobalt absorption responds to mechanisms that enhance iron absorption.11 Cobalt, unlike iron, is excreted rapidly in the urine, in direct propor¬ tion to the amount absorbed from the gastrointestinal tract.11 A close correlation was found between the intestinal absorption of iron given orally and the 6-hour urinary excretion of cobalt given orally in 32 patients with various sizes of iron stores.10 Cobalt excretion, like iron absorption, was inversely related to the amount of hemosiderin in the bone marrow.9 The present study examines the relation between values for serum ferritin, cobalt excretion, iron absorption and bone marrow hemosiderin in a group of student volunteers and in a group of patients with a variety of diseases.
Subjects and methods Subjects studied Serum ferritin concentration, urinary cobalt excretion and other measurements of body iron status were determined in three groups of subjects. The first group comprised 34 patients with a variety of disorders selected from our clin¬ ical practice. Patients with renal failure or malabsorption were excluded. These 34 patients included 16 with and 18 without liver disease. The 14 patients with alcoholic liver disease underwent liver biopsy to establish the diagnosis; the grade of stainable iron ranged from 0 to 3+.11 Two patients with carcinoma of the cclon underwent laparotomy; CMA JOURNAL/MAY 17, 1975/VOL. 112 1173
one had extensive hepatic metastases and was included among those with liver disease, as was a patient with viral
hepatitis.
The second
was a
group of 10
patients
with
body
iron
overload established by finding an increased amount (4-b) of stainable iron in the liver biopsy specimen.11 Of the seven studied before treatment five had iron overload sec¬ ondary to alcoholic cirrhosis, one had taken oral iron with large amounts of brandy for 30 years, and one had primary sideroblastic anemia. Three patients with idiopathic hemochromatosis were studied after venesection. The third group consisted of 20 healthy students, 18 men and 2 women (ages, 17 to 30 years). All but 4 students had donated blood previously, i2 of them within 3 to 12 months, and 10 had donated between 5 and 10 pints of blood in total. Informed consent was obtained for the investigations from all of the students and the patients. Serum ferritin Serum ferritin was measured by a two-site immunoradio¬ metric assay.12 Iron absorption A whole-body counting technique was used to measure the absorption of an oral test dose cf 10 fimol of iron ascorbate labelled with 4 /xCi of 59Fe.10 When iron ab¬ sorption and cobalt excretion were measured in the same subject, the iron solution was given first, and the next day the cobalt test was done. Radioactive iron in the body was distinguished from residual cobalt activity by differential counting. The normal range of values for the technique is 6 to 52%.13 Cobalt excretion A test dose of 20 /xmol of cobaltous chloride labelled with 0.5 /xCi of 57Co was given orally and the percentage excreted in the urine in 6 hours was measured. In an earlier study the range of values in control subjects aged 31 to 75 years with plentiful iron stores was 4 to 11%.10 Patients with iron deficiency anemia excreted more than 12% of the dose in 6 hours, whereas patients in this age group with anemia from other causes excreted less than 11%. A broader range, 4 to 14%, was found in subjects aged 15 to 30 years with plentiful iron stores. Assessment of body iron stores Bone marrow iron stores: The amount of stainable iron in smears of a bone marrow aspirate was estimated by one of us (W.E.N.C.), as described previously,9 without knowledge of the clinical status of the subjects. The iron in reticuloendothelial cells was graded from 0 (no stainable iron) to 4+ (stainable iron visible in 100% of high power
fields).
Liver iron stores: Liver biopsy sections stained with Prussian blue were graded on a scale of 0 to 4+ for hemo¬ siderin;11 grades 0 to 34- were considered normal. A grade of 4+ represented iron in 100% of the cells in mas¬ sive amounts; subjects with this grade were considered to have iron overload. Deferoxamine test: This was carried out as described by Balcerzak et al.1* Urine was collected for 24 hours before and for 24 hours after the intramuscular injection of 1000 mg of deferoxamine mesylate. Urinary iron concentration was determined by spectrophotometry after acid digestion and colour development with dipyridyl. A value of more than 4 mg/24 h is highly suggestive of iron overload. Other methods The hemoglobin concentration was measured with the Model S Coulter Counter (Coulter Electronics Inc, Hialeah, Florida). The technique for measurement of serum iron 1174 CMA
JOURNAL/MAY 17, 1975/VOL. 112
and unsaturated iron-binding capacity has been described previously.15 The normal range of values for serum iron by this method is 55 to 185 /xg/dl and that for total ironbinding capacity is 250 to 430 /xg/dl; transferrin saturation in normal subjects ranges from 15 to 47%.15
Statistical methods The statistical difference between any two mean values was assessed by the /-test. The least squares method of linear regression was used to analyse the relation between serum ferritin values and other measurements.16 Because serum ferritin values are normally distributed on a logarithmic scale,4 they were converted to logarithms for re¬
gression analysis.
Results The serum ferritin values for all 64 subjects are shown in Fig. 1. The mean was lowest in the group of healthy students, 59 ng/ml (range, 7 to 170 ng/ml); a little higher in the patients with no liver disease, 73 ng/ml (range, 5 to 282 ng/ml); and considerably higher in the patients with
10,000 6,000-
4,000 2,0001,000 600 H
oo
400 c
200-
t 100£
60
©
4.
40 o
20
o© o o
10H o
FIG. 1.Serum ferritin values, on logarithmic scale, in four groups of subjects. Arithmetic mean of each group indicated by horizontal bar. Bone marrow iron graded as absent (open circles), intermediate (dotted circles) or plentiful (solid circles). Three patients with iron overload treated by venesection denoted by arrows.
liver disease, 393 ng/ml (range, 11 to 1063 ng/ml). When the students and patients without liver disease were con¬
Patients with low
CO
normal iron stores The patients with 0 to 3+ marrow iron, including those with liver disease, were subdivided into three groups on the basis of the stainable iron content of the bone marrow (Table I). In the patients with absent marrow hemosiderin the mean serum ferritin value was significantly lower than in those with 1+ or 2+ to 3+ stainable iron, and there was no overlap in the range of values. The mean value for cobalt excretion in the group with absent marrow iron was significantly greater than that in the group with intermediate marrow iron, which in turn was higher than the mean value in the group with plentiful marrow iron. No overlap was present between the ranges of values for the cobalt excretion test in subjects with 0 and 2+ to 3-f- marrow iron. The results in the 1+ group overlapped the ranges of both of these groups. The relation between serum ferritin values and the results of the other tests is shown in Table II. The inverse correla¬ tion between serum ferritin concentration and percentage excretion of cobalt is shown in Fig. 2. Patients with iron overload The serum ferritin value was increased in all seven un¬ treated patients but the range was very broad (Table I). It was also above normal in two of the treated patients and was in the range of iron deficiency in a patient whose marrow iron stores had been completely depleted by vene¬ section (Fig. 1). There was no relation between serum fer¬ ritin value and the results of any of the other tests except the deferoxamine test when all 10 patients were considered (Table II). A strong positive correlation was evident be¬ tween values for iron absorption and cobalt excretion (Fig. 3 a), similar to the correlation in patients with 0 to 4+ marrow iron reported in a previous study.10
FIG. 2.Relation between serum ferritin concentration and cobalt excretion in patients with absent (open circles), intermediate (dotted circles) or plentiful (solid circles) bone marrow iron stores (r = .0.764, P < 0.001).
Students None of the students was anemic and all 20 had a normal serum iron value, iron-binding capacity and transferrin saturation (Table III). When they were divided into three groups according to stainable iron in the bone mar¬ row, no significant difference was found among the 0, 1-f and 2-f to 3+ groups in the mean hemoglobin value, serum iron value, total iron-binding capacity or transferrin saturation. The mean serum ferritin value in the students with absent marrow hemosiderin was significantly less than the value in those with 2+ to 3-f marrow iron. The differ¬ ences between the average value of the four students with 1+ marrow iron and those of the other two groups did not achieve statistical significance. The mean cobalt excretion
sidered together, the ranges were 5 to 38 ng/ml in those with absent marrow hemosiderin and 59 to 282 ng/ml in those with plentiful marrow iron. The average serum fer¬ ritin value in the group with iron overload was 2854 ng/ml and values ranged from 406 to 12 204 ng/ml in untreated patients and from 12 to 494 ng/ml in patients treated by venesection. The relation between serum ferritin concentration and the results of various tests for the assessment of iron status were analysed for each of three groups of subjects.
O)
c
.
