Relationship between hemoglobin concentration and transferrin saturation in iron-sufficient 2

Relationship between hemoglobin concentration and transferrin saturation in iron-sufficient 2 Martti A. Siimes,3 M. D., Ulla M. Saarinen,4 M. D., and Peter R. Dallman,5 M.D. ABSTRACT Ten groups of healthy infants and children from 2 months to 15 years of age were studied, each consisting of 98 to 238 subjects. In young infants whose serum ferritin values indicated ample storage iron, the concentration of hemoglobin was found to bear a significant relationship to the degree of iron saturation of transferrin. This phenomenon was evident throughout the range oftransferrin saturation until 1 year ofage but became undetectable or less significant from 2 to 15 years of age. We postulate that the production of hemoglobin could be influenced through a broader range of transferrin saturation in rapidly growing infants than in the older child or adult. Am. J. Clin. Nutr. 32: 000-000, 1979. Developmental changes in concentration of hemoglobin have been recognized since the early years of this century (1). Between the 1920 s and 1950 s, efforts were made to interpret these changes by distinguishing a physiological anemia present in healthy, well-nourished infants and children from the frequently superimposed consequences of nutritional iron deficiency (2, 3). More recently it has become possible to minimize the influence of iron deficiency and other common causes of anemia as sources of error in the estimation of normal developmental changes in the concentration of hemoglobin by controlled studies of iron supplemented and unsupplemented infants and by the use of newer laboratory tests (4, 5). As a result, there are now normative data for infants and children that should represent a closer approximation of the physiological changes in hemoglobin concentration in a healthy, adequately nourished population (6, 7). These changes can be summarized as follows: in the newborn infant, the concentration of hemoglobin averages about 17 g/dl, a value substantially higher than in the adult. There is a progressive postnatal decrease in concentration until a low value of about 1 1 g/dl is reached at 2 months of age. Between 2 and 6 months of age there is a relatively rapid rise in hemoglobin values followed by a more gradual increase during later infancy and childhood. At about 1 1 years of age the mean values begin to diverge according to sex, those in male rising more rapidly to approach adult values during puberty (5, 6). Several explanations have been proposed for the developmental alterations in concentration of hemoglobin. The rapid decrease in rate of production of hemoglobin after birth is believed to be due primarily to a decreased release of erythropoietin as a consequence of greater oxygen availability (8). The maintenance ofa lower concentration of hemoglobin in children than in adults has been attributed to their high plasma concentrations of inorganic phosphate that result in a shift of the oxygen dissociation curve to the right and facilitate oxygen delivery (9). Lastly, the divergence of hemoglobin values by sex at puberty is attributed to the influence of androgens ( 10). In this paper, we present evidence that the concentration of hemoglobin in presumably iron-sufficient infants is a dependent variable of transferrin saturation. This suggests the possibility that transferrin-bound From the Children s Hospital. University of Helsinki. Finland, and the Department of Pediatrics, University of California, San Francisco, California. 2Supported by funds from Sigrid Juselius Foundation, Finland and the National Institutes of Health Grant AM HD 13897..1 Assistant Professor of Pediatrics. Fellow in Pcdiatric Hematology. Professor of Pediatrics. The American JournalofClinical Nutrition 32: NOVEMBER 1979, pp. 2295-2300. Printed in U.S.A. 2295

2296 SIIMES ET AL. iron may play a role in influencing the concentration of hemoglobin during infancy even when iron stores are adequate. Subjects and methods Subjects Laboratory data were analyzed from two groups of healthy Finnish infants and children in each of whom iron deficiency anemia was unusually rare. The first group consisted of 238 full-term infants who were followed longitudinally through their first year of life with laboratory studies at the ages of2, 4, 6, 9, and 12 months (6). Only six of the infants developed iron deficiency anemia by the criteria of hemoglobin less than I I g/dl. mean corpuscular volume (MCV) less than 70 ft. and/or transferrin saturation less than 10%. These patients were started on iron medication. The second group of subjects consisted of 803 healthy children that represented a large sampling of the total population from a suburban and a rural community. The suburban children included about 50% of the total in the community and the rural group included 90%. The ages and numbers of subjects at each age were 2 (n = 101), 4 (n = 98), 7 (n = 190), 10 (n = 220), and IS (n = 194) years of age. All blood samples were drawn between 8 AM and noon. Only 10 children fulfilled the criteria of iron deficiency anemia by anemia plus low MCV or transferrin saturation <16%. Anemia and MCV were defined as a hemoglobin below the 3rd percentile for age according to recently developed normative data. The sexes were equally represented and no subjects were excluded. The numbers of subjects listed in Figures 1 and 2 and Table 1 do not correspond exactly to the total numbers listed for the groups above due to incomplete data in a small number of subjects. Methods Hemoglobin concentration and red blood cell indices were determined by a Model S Coulter Counter, serum iron and total iron-binding capacity were measured spectrophotometrically using ferrozine as the color reagent, and serum ferritin was estimated from venous blood by a radioimmunological method as described earlier (6). Statistical analysis SE s of the means were calculated and comparisons between groups were made by Student s t test. Since serum ferritin values approximate a logarithmic distribution, mean values were calculated after logarithmic transformation. Regression coefficients (slopes) and their significances were calculated by linear regression analysis. Results The developmental rise in concentration of hemoglobin is shown by the increase in the overall height of the columns with increasing age in Figure 1 and the mean values in Table 1. The lowest mean concentration of hemoglobin was at 2 months of age after which there was a gradual rise to 15 years of age when the values for boys approach those of adult males. This increase in concentration of hemoglobin also occurred within any given range of transferrin saturation (Fig. 1). Between 2 and 12 months of age (top of Fig. 1) hemoglobin concentration was a dependent variable of transferrin saturation. If this were evident primarily in the lower ranges of transferrin saturation, it would be most readily attributable to iron deficiency. However, the tendency for a higher transferrim saturation to be associated with a higher hemoglobin concentration was present at all ages studied between 2 and 12 months of age and throughout the range of transferrin saturation. This is shown to varying degrees at each age group by the increase in mean hemoglobin concentration with progressively higher categories of transferrin saturation, from left to right. The magnitude of this relationship is shown by the regression coefficient of hemoglobin concentration on transferrin saturation (Table 1). In each age, category between 2 and 12 months, the correlation of transferrin saturation with hemoglobin values was highly significant (P < 0.01 at 4 months and < 0.001 at all other ages). Between 2 and 15 years of age there is far less evidence for a similar relationship between transferrin saturation and hemoglobin concentration. The lower portion of Figure 1 shows that the concentration of hemoglobin remained relatively constant through the middle and upper categories of transferrin saturation that accounted for most of the values at each age. Slightly lower concentrations of hemoglobin at the lowest transferrin saturations occurred only at 2, 4, and 15 years of age. These are likely to represent cases of mild iron deficiency. The linear regression for the total group in Table 1 show that there was no significant relationship between transferrin saturation and concentration of hemoglobin at 2, 10, and I 5 years of age. At 4 and 7 years of age, there was evidence of a relationship but at a lower level of significance than in infants (P < 0.05). After the decrease in neonatal iron stores between 4 and 6 months age, much of infancy and childhood is characterized by low iron

IRON SATURATION OF TRAFERRIN IN INFANTS 2297 2mo 4mo 6mo 9mo l2mo 14 13 7 * /, // / / / I fl/ / I#{149} 4 - -. 12 11 $7 / r / /1/ lu 14 2yr 4yr lyr loyr 15yr1! 13 ci 7 I // /// / I ::,/ I // /, z 12 -j III / / / // // // // I/ / I/I / / I I TRAFERRIN SATURATION, PER CENT FIG. 1. Concentration of hemoglobin in various ranges of transferrin saturation from 2 months through 15 years ofage. At each age. the ranges ofthe categories represented by bars from left to right were: less than 10%, 10 to 16%, 16 to 22%, 22 to 30%, 30 to 50%, and above 50%. Mean values ± SEM of hemoglobin concentrations and number of subjects in each category are shown. TABLE I Hemoglobin concentration as a function of transferrin saturation Age No. Total group Mean,,. Slope hemoglobin P After exclusion of transfemn saturation < l6 or serum ng/ml Excluded No. Mean hemoglobin Slope. ferritin <10 P 2 mo 235 11.1 0.0134 <0.001 I 232 11.1 0.0124 k czo.oos 4mo 216 I1.9 O.0I28 <0.01 24 164 12.0 0.0110 h 6 mo 231 12.3 0.0212 <0.001 33 154 12.4 0.0146 <0.01 9 mo 230 12.4 0.0335 <0.001 30 161 12.6 0.0210 <0.01 12 mo 224 12.5 0.0241 <0.001 30 156 12.6 0.0188 <0.005 2yr 4yr 7yr loyr I5yr 15 yr a Regress 99 90 186 219 102 92 12.5 0.0081 12.8 0.0158 13.1 0.0082 13.6 0.0006 13.7-0.0106 14.8 0.0140 <0.05 <0.05 21 78 14 77 13 162 21 173 16 86 2 90 12.6 0.0011 12.6 0.0132 13.1 0.0104 13.6-0.0052 13.9 0.0053 14.8 0.0094 ion coe fficient; hemoglobin, g/d 1, per unit of transferrin saturation, %. bnot significant. <0.05

2298 SIIMES ET AL. stores and a high dietary iron requirement for growth. Although the data described in Subjects and methods indicated a remarkably low prevalence of iron deficiency, additional analyses were done to determine whether mild iron deficiency in our populations was likely to explain the findings. Since serum ferritin tends to reflect the abundance of storage iron or iron reserves, we calculated the mean serum ferritin at various levels of hemoglobin concentration in each of the age groups between 2 and 12 months of age. There was little evidence of iron deficiency on the basis of serum ferritin values even in the lowest hemoglobin categories (Fig. 2). Laboratory findings suggestive of iron deficiency include a serum ferritin value <10 ng/ml and a transferrin saturation of <16%. The right side of Table 1 shows the regression coefficients (slopes) and their significance after excluding subjects who were most likely to have iron deficiency anemia by having met either one or both of these conditions. At 2 months ofage, only one subject was excluded. The largest percentage ofsubjects, 24 to 33%, was excluded between 4 and 12 months of age. However, even at these ages, the mildness and/or rarity ofany iron-deficiency anemia was indicated by the small effect of the exclusion on the mean concentration of hemoglobin in the remaining group, no more than 0.2 g/dl. The percentage ofsubjects excluded should not be considered indicative of relative prey- w LIalence of mild iron deficiency because the transferrin saturation is normally lower in infancy and early childhood than in older children and adults ( I 1 ). The lower limit of the 95% range oftransferrin saturation is 10% in infants between 4 and 12 months of age who receive ample iron supplementation and who have no other laboratory evidence of iron deficiency. This is in accord with our finding that all exclusions at 2 and 4 months of age were on the basis of transferrin saturation and none on the basis ofserum ferritin. From 2 to 12 months of age, 4 to 10 times as many infants were excluded for transferrin saturation alone than on the basis of serum ferritin. Presumably most of these exclusions were iron-sufficient subjects. At 2 years of age and subsequently, serum ferntin alone or with transferrin saturation became the basis for excluding more subjects than transferrin saturation alone at all ages except 7 years where the number was nearly equal. Discussion Our data indicate a relationship between the concentration of hemoglobin and transferrin saturation that occurs in infancy in the absence of any evidence of iron deficiency by the usual clinical criteria. Even though the instance of such a relationship would not demonstrate causality, it does suggest the possibility that transferrin saturation in the range that is considered normal may be a rate-lim- E 44 z I.- HEMOGLOBIN, G/DL FIG. 2. Concentration of serum ferritin in various ranges of hemoglobin concentration from 2 to 12 months of age. Mean values ± SEM of serum ferritin and the number of subjects in each category are shown.

IRON SATURATION OF TRAFERRIN IN INFANTS 2299 iting factor in the production of hemoglobin between 2 and 12 months of age. One possible basis for an association between transferrin saturation and hemoglobin concentration in iron-sufficient infants is related to the unusually high rate of erythropoiesis that is required to keep pace with rapid growth and to raise the concentration of hemoglobin from its postnatal low point at 2 months of age. Between 2 and 4 months of age, for example, the body weight in this group of healthy term infants increased from 5.4 to 7.2 kg, an increment of 33%. Concurrently the concentration of hemoglobin increased from a mean of 1 1.2 to a value of 12.2 (6). It seems reasonable to postulate that a transferrin saturation that is adequate for maintaining hemoglobin production at a steady state in the adult could be rate-limiting under these conditions in the infant. Perhaps the strongest evidence for a relationship between transferrin saturation in the range that is considered normal and hemoglobin concentration are the data in the 2- month-old infants. At this age, a high serum ferritin concentration reflects abundant iron stores. The 95% range for serum ferritin concentration was 80 to 400 ng/ml (12). Similarly, the 95% range for transferrin saturation was 2 1 to 63%, above the range associated with iron deficiency anemia in children and adults ( 13, 14). Accordingly, there is no rcason to suspect a lack of iron either in individual infants or in 2-month-old infants as a group. At 4 months of age, the same considerations still apply. Serum ferritin and transferrin saturation values have declined, but there are virtually no subjects with serum ferritin concentrations <10 ng/ml or transferrin saturation < 10%, the lower limit of the 95% range for iron-supplemented infants. Several studies are in accord with our hypothesis that hemoglobin production in the infant may be rate limited within the range of transferrin saturation that is considered normal, in contrast to the older child or adult. One example is the finding that maximal rates of erythropoiesis in adults who were subjected to phlebotomy could not be maintamed unless the concentration of iron in plasma was artificially raised to levels above 200 jg/dl (15). It was calculated that normal men could mobilize no more than 40 to 60 mg of storage iron per day for erythropoiesis or enough to support approximately twice the normal base-line rate of erythropoiesis. This amount, on the basis of body weight, would correspond to about 3.5 mg in a 2-month-old infant weighing 5 kg, only slightly more than the average daily need of about 2.5 mg that we calculate to be necessary for growth (assuming a proportional increase in blood volume) and for the observed developmental increase in concentration of hemoglobin (6, 12). Increased rates of erythropoiesis in small infants might be considered analogous to phlebotomy in adults, particularly at 2 months of age when the 95% range of the reticulocyte counts is from 0.5 to 4.9% (12) in comparison to 0.5 to 1.5% in children and adults. Studies in the rabbit also support the view that the concentration of hemoglobin may be iron responsive even in the presence of substantial iron stores during early development or after phlebotomy (16, 17). An alternative explanation for our findings is that the probability of mild iron-deficiency anemia is greater with decreasing transferrin saturation. The biological variability of serum iron, and consequently transferrin saturation, is far greater than for hemoglobin or serum ferritin (18, 19) and a single blood sample is a poor approximation of the average value for an individual. It can be argued that transferrin saturation is so highly variable that increasing values within the normal range merely indicate a gradually diminishing but never disappearing probability of iron deficiency. Cook et al. (20) have pointed out an analogous situation regarding the distribution of hemoglobin values of normal individuals which overlap substantially with individuals who have mild iron deficiency in populations where iron deficiency is common. Following this line of reasoning, we might expect to fmd a relationship between transferrin saturation and hemoglobin concentration but only if there were large proportion of subjects with mild iron deficiency. These considerations cannot be applied to the 2-month-old infant in whom there was virtually no iron deficiency by any criteria. Also against this argument in the older infants is the rarity of significant iron-deficiency anemia, even in those subjects with the lowest values for transferrin saturation or serum ferritin, and the persistence of the correlation after exclusion of such individuals.

2300 SIIMES ET AL. Regardless of the interpretation of our findings, it seems worth emphasizing that a relationship between transferrin saturation and hemoglobin concentration in apparently iron-sufficient individuals is unlikely to be of sufficient magnitude to complicate the diagnosis of iron deficiency. It would only be likely to become evident by comparing mean values oflarge populations and not in clinical situations where data from individual patients are examined. For example, with a rise in transferrin saturation from 20% to 40%, one would calculate from the regression coefficients in Table 1 an increase in mean concentration ofhemoglobin between 0.3 and 0.7 g/dl in infants. Despite the significance of this difference, it is small in relation to the 95% limits for normal hemoglobin that encompass a range of 2.6 to 4.0 g/dl for any given age or sex (5, 6). Consequently, the relationship between transferrin saturation and hemoglobin concentration is unlikely to be of sufficient magnitude to substantially influence the interpretation of the values we currently consider to be reference standards in clinical practice (6, 1 1, 14). However, the implications of the phenomenon could be interesting from other points of view. The rate of production of hemoglobin could be to some extent limited by the supply of serum iron in rapidly growing infants whose dietary iron intake is considered to be within the physiological range. If so, this may be a contributing factor to the developmental changes in concentration of hemoglobin. References 1. WILLIAMSON, C. S. Influence of age and sex on hemoglobin. A spectrophotometric analysis of nine hundred and nineteen cases. Arch. Internal Med. 18: 505, 1916. 2. MERRITT, K. K., AND L. T. DAVIDSON. The blood during the first year of life I. Normal values for erythrocytes, hemoglobin, reticulocytes and platelets, and their relationship to neonatal bleeding and coagulation time. Am. J. Dis. Child. 46: 990, 1933. 3. GUEST, G. M., AND E. W. BROWN. Erythrocytes and hemoglobin of the blood in infancy and childhood. Am. J. Diseases Children 93: 486, 1957. 4. O BRIEN, R. T., AND H. A. PEARSON. Physiologic anemia of the newborn infant. J. Pediat. 79: 132, 1971. 5. DALLMAN, P. R. New approaches to screening for iron deficiency. J. Pediat. 90: 678, 1977. 6. SAARINEN, U. M., AND M. A. SItMES. Developmental changes in red blood cell counts and indices of infants after exclusion of iron deficiency by laboratory criteria and continuous iron supplementation. J. Pediat. 92: 412, 1978. 7. DALLMAN, P. R., AND M. A. SuMES. Percentile curves for hemoglobin and red cell volume in infancy and childhood. J. Pediat. 94: 26, 1979. 8. HALVORSEN, S. Plasma erythropoietin levels in cord blood and in blood during the first weeks of life. Acta Paediat. 52: 425, 1963. 9. CARD, R. T., AND M. C. BRAIN. The anemia of childhood. Evidence for a physiologic response to hyperphosphatemia. New EngI. J. Med. 288: 388, 1973. 10. DANIEL. W. A. Hematocrit: maturity relationship in adolescence. Pediatrics 52: 388, 1973. I I. SAARINEN, U. M., AND M. A. SIIMES. Developmental changes in serum iron, total iron-binding capacity, and transferrin saturation in infancy. J. Pediat. 9 1: 875, 1977. 12. SAARINEN, U. M., AND M. A. SIIMES. Serum ferritin in assessment of iron nutrition in healthy infants. Acta Paediat. Scand. 67: 745, 1978. 13. BAINTON, D. F., AND C. A. FINCH. The diagnosis of iron deficiency anemia. Am. J. Med. 37: 62, 1964. 14. KOERPF.R, M. A., AND P. R. DALLMAN. Serum iron concentration and transferrin saturation in the diagnosis of iron deficiency in children: Normal developmental changes. J. Pediat. 91: 870, 1977. 15. HILLMAN, R. S., AND P. A. HENDERSON. Control of marrow production by the level of iron supply. J. Clin. Invest. 48: 454, 1969. 16. HALVORSEN, K., AND S. HALVORSEN. The early anemia its relation to postnatal growth rate, milk feeding, and iron availability. Experimental study in rabbits. Arch. Diseases Childhood 48: 842, 1973. 17. JACOBS, P., AND C. A. FINCH. Iron for erythropoiesis. Blood 37: 220, 1971. 18. CooK, J. D., D. A. LIPSCHITZ, L. E. M. MILES AND C. A. FINCH. Serum ferritin as a measure of iron stores in normal subjects. Am. J. Clin. Nutr. 27: 681, 1974. 19. STATLAND, B. E., AND P. WINKEL. Relationship of day-to-day variation of serum iron concentrations to iron-binding capacity in healthy young women. Am. J. Clin. Pathol. 67: 84, 1977. 20. CooK, J. D., J. ALVARADO, A. GUTNISKY, Fr AL. Nutritional deficiency and anemia in Latin America: a collaborative study. Blood 38: 591, 1971.