Brief Communication: Association of Serum Insulin-Like Growth Factor-I and Erythropoiesis in Relation to Body Iron Status

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1 324 Annals of Clinical & Laboratory Science, vol. 34, no. 3, 2004 Brief Communication: Association of Serum Insulin-Like Growth Factor-I and Erythropoiesis in Relation to Body Iron Status Jong Weon Choi 1 and Soon Ki Kim 2 1 Department of Laboratory Medicine, and 2 Department of Pediatrics, College of Medicine, Inha University, Inchon, South Korea Abstract. This study investigated the associations between serum insulin-like growth factor-i (IGF-I) concentrations and erythropoietic activities in relation to body iron status. Serum IGF-1 concentrations, free erythrocyte protoporphyrin (FEP), hemograms, and serum iron markers were measured in 71 female adolescents, age 14 to 17 yr. No significant differences were observed in hemograms, iron parameters, or FEP between the subjects with IGF-I <681.2 ng/ml and IGF-I ng/ml. H owever, blood hemoglobin and serum iron concentrations averaged 13.4 ± 0.8 g/dl and 93.7 ± 41.2 µg/dl in the subjects with IGF-I >809 ng/ml, which were above the values in those with IGF-I <523 ng/ml (12.3 ± 0.9 g/dl and 50.5 ± 30.8 µg/dl, p < 0.05, respectively). On the other hand, FEP was significantly lower in the adolescents with IGF- I >809 ng/ml than in those with IGF-I <523 ng/ml (38.9 ± 16.2 µg/dl vs 63.4 ± 23.1 µg/dl, p <0.05). Prevalences of iron deficiency or iron deficiency anemia were 3- or 5-fold higher in the subjects with IGF- I <523 ng/ml, compared to those with IGF-I >809 ng/ml. Serum IGF-I correlated significantly with FEP (r = -0.45, p <0.05) and serum iron concentrations (r = 0.40, p <0.05) in iron deficient subjects. In summary, IGF-I seems to have an important relationship to iron metabolism and protoporphyrin synthesis in adolescents. (received 1 April 2004; accepted 1 May 2004) Keywords: insulin-like growth factor-i, iron deficiency anemia, erythrocyte protoporphyrin Introduction Insulin-like growth factor-i (IGF-I) is a family of cytokines produced by the liver, osteoblasts, and many other cells. IGF-I mediates chondrogenic effects of growth hormone in promoting long bone growth, and regulates the proliferation and differentiation of various cell types, including bone marrow cells. IGF-I has partial amino acid homology with the pro-insulin peptide, and also has insulinlike bioactivities [1,2]. Address correspondence to Jong Weon Choi, MD, PhD, Department of Laboratory Medicine, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon, , South Korea; tel ; fax ; jwchoi@inha.ac.kr. Iron is an essential component of hemoglobin, and two-thirds or more of the body s total iron content resides in the normoblasts and erythrocytes. The development of iron deficiency progresses in an orderly sequence of events, ie, iron depletion, iron-deficient erythropoiesis, and iron deficiency anemia, and each stage correlates with clinical laboratory abnormalities [3]. The formation of protoporphyrin is the last step in the synthesis of heme. Normally, iron is inserted rapidly into protoporphyrin by ferrochelates. In an iron deficiency state, a larger proportion of protoporphyrin is not converted to heme and continues to exist in the erythrocyte [4]. Recent studies demonstrated that IGF-I directly stimulates the proliferation of primitive erythroid /04/ $ by the Association of Clinical Scientists, Inc.

2 Serum insulin-like growth factor-i and erythropoiesis 325 progenitor cells [5,6]. To date, few studies have examined the relationships between serum IGF-I concentrations and protoporphyrin synthesis during the late phases of erythropoiesis. In this study, we tested whether serum IGF-I level is correlated with free erythrocyte protoporphyrin (FEP) in mature red blood cells and with several parameters of iron metabolism during 3 stages of iron deficiency in adolescents. Materials and Methods Serum IGF-I concentrations, FEP, hemograms, and serum iron markers were measured in 71 female adolescents, age 14 to 17 yr (mean 15.4 yr). The subjects were all Korean volunteers. This survey was explained to and approved by their parents and the directors at each participating school. This study was approved by the Ethical Committee of Inha University Hospital, and informed consent was obtained from all subjects. Approximately 9 ml of venous blood was collected into 2 tubes: 3 ml of blood in an EDTAanticoagulated tube for the measurement of complete blood cell count (CBC) and FEP; 6 ml of blood in an iron-free evacuated tube for measurement of serum IGF-I and serum iron markers. Serum IGF-I concentrations were measured by radioimmunoassay using Biosource IGF-I-D-RIA- CT kit (BioSource Europe, Nivelles, Belgium). Erythrocyte protoporphyrin levels were measured as free erythrocyte protoporphyrin IX by using a fluorometric procedure [7]. In brief, protoporphyrin was extracted into ethyl acetate and acetic acid, and re-extracted in hydrochloric acid. Protoporphyrin concentrations were measured fluorometrically and the results expressed as µg of free protoporphyrin IX /dl of whole blood. CBC was determined with an electronic counter (SE 9000; Sysmex, Kobe, Japan). Serum iron and total iron-binding capacity (TIBC) were assayed with a chemical analyzer (Hitachi 747; Hitachi, Tokyo, Japan) and serum ferritin was measured by a chemiluminescent method (ACS 180, Chiron, MA). The subjects were assigned to 2 groups based on serum IGF-I concentrations: lower 20% group (IGF-I <523 ng/ml, n = 14) and upper 20% group (IGF-I >809 ng/ml, n = 14). The subjects were also divided into other groups based on their body iron status: iron-depletion phase (stage I, n = 17), irondeficient erythropoiesis (stage II, n = 13), iron deficiency anemia (stage III, n = 16), and healthy controls (n = 25). Non-anemic subjects with normal serum iron level (>50 µg/dl), but with decreased serum ferritin concentration (<12 µg/l), were considered to be in the iron-depletion phase (stage I). Iron-deficient erythropoiesis (stage II) was defined as serum ferritin concentration <12 µg/l and serum iron level <50 µg/dl without overt anemia. Subjects showing decreased serum ferritin concentration, decreased serum iron level, and decreased blood hemoglobin level (<12 g/dl) were considered to have iron deficiency anemia (stage III). To investigate the prevalence of iron deficiency, we assigned the subjects in the iron-depletion phase (stage I) or the iron-deficient erythropoiesis (stage II) to the iron deficiency group. Data analyses were performed with the SAS software package (SAS Institute, Cary, NC). The Mann-Whitney U test was used to evaluate the differences of means between 2 groups. Correlation coefficients were analyzed by Spearman s method. All p values <0.05 were considered statistically significant. Results and Discussion Mean values of hematologic parameters, iron markers, and FEP in relation to IGF-I concentrations are summarized in Table 1. There were no significant differences in hemograms and iron parameters between the subjects with IGF-I <681.2 ng/ml and IGF-I ng/ml, which was the provisional cutoff point based on mean IGF-I concentrations of the subjects. However, the mean values of white blood cells (WBCs), hemoglobin, serum iron, and FEP concentrations showed large differences when we compared the subjects with IGF-I >809 ng/ml (upper 20%) to those with IGF- I <523 ng/ml (lower 20%). WBCs, hemoglobin, and serum iron averaged 6.8 ± 1.2 (x 10 3 /µl), 13.4 ± 0.8 g/dl, and 93.7 ± 41.2 µg/dl in the subjects with IGF- I >809 ng/ml, which were above the values in those with IGF-I <523 ng/ml [5.2 ± 0.7 (x 10 3 /µl), 12.3

3 326 Annals of Clinical & Laboratory Science Table 1. Mean values (±SD; [median]) of hematologic parameters, FEP, and iron markers, and prevalence of iron deficiency in relation to serum IGF-I concentrations. Parameters Total Serum IGF-I concentrations Lower 20% group (IGF-1 <523 ng/ml) Upper 20% group (IGF-1 >809 ng/ml) Subjects (n) Age (yr) 15.4 ± 1.9 [15] 15.7 ± 1.6 [15] 15.1 ± 1.8 [15] Serum IGF-I (ng/ml) ± [679] ± 41.2 [478] 882.4± 68.6 [870] a Blood FEP (µg/dl) 49.4 ± 20.7 [45.5] 63.4 ± 23.1 [60.1] 38.9 ± 16.2 [36.5] a Hematologic parameters White blood cells (10 3 /µl) 6.1 ± 1.4 [5.9] 5.2 ± 0.7 [5.3] 6.8 ± 1.2 [6.7] a Red blood cells (10 6 /µl) 4.39 ± 0.31 [4.41] 4.34 ± 0.35 [4.32] 4.49 ± 0.19 [4.53] Hemoglobin (g/dl) 12.9 ± 1.1 [13.1] 12.3 ± 0.9 [12.3] 13.4 ± 0.8 [13.5] a Hematocrit (%) 37.9 ± 2.9 [38.2] 37.2 ± 3.3 [37.8] 38.6 ± 2.8 [38.9] Platelets (10 3 /µl) ± 52.5 [259] ± 35.1 [227] ± 51.6 [276] Iron markers Serum iron (µg/dl) 72.1 ± 46.2 [76] 50.5 ± 30.8 [67] 93.7 ± 41.2 [115] a Total iron-binding capacity (µg/dl) ± 31.5 [334] ± 37.2 [342] ± 36.5 [331] Serum ferritin (µg/l) 19.2 ± 10.7 [16.5] 14.8 ± 9.5 [11.2] 21.6 ± 11.4 [20.4] Prevalences of iron deficiency Iron deficiency anemia (n, %) 16 (22.5%) 5 (35.7%) 1 (7.1%) a Iron deficiency b (n, %) 30 (42.3%) 9 (64.2%) 3 (21.4%) a FEP >50 µg/dl (n, %) 25 (35.2%) 11 (78.6%) 2 (14.3%) a IGF-I, insulin-like growth factor-i; FEP, free erythrocyte protoporphyrin. a Statistically significant (p < 0.05) vs lower 20% group, computed by Mann-Whitney U test. b Iron deficiency group includes subjects with iron depletion (stage I) or iron-deficient erythropoiesis (stage II) Table 2. Serum IGF-I and blood FEP concentrations during 3 stages of iron deficiency in adolescents. Parameters Mean values (mean ± SD; [median]) Iron-depletion Iron-deficient Iron-deficiency Healthy phase erythropoiesis anemia controls (Stage I, n = 17) (Stage II, n = 13) (Stage III, n = 16) (n = 25) Age (yr) 15.3 ± 2.1 [15] 14.8 ± 1.6 [15] 15.9 ± 1.4 [16] 15.1 ± 1.7 [15] IGF-I (ng/ml) ± [697] ± [681] ± 94.6 [550] a ± [760] FEP (µg/dl) 42.9 ± 25.6 [41.5] 49.5 ± 21.6 [48.4] 67.0 ± 23.1 [74.2] a 41.6 ± 10.9 [42.1] Hemoglobin (g/dl) 12.8 ± 0.6 [12.7] 12.7 ± 0.9 [12.6] 11.0 ± 0.7 [11.0] a 13.5 ± 0.8 [13.6] Serum iron (µg/dl) 93.8 ± 21.6 [91] 41.5 ± 18.1 [39] a 35.4 ± 14.3 [32] a ± 37.5 [104] Serum ferritin (µg/l) 10.4 ± 2.1 [9.6] a 8.9 ± 2.4 [9.1] a 8.3 ± 2.7 [8.5] a 37.4 ± 13.9 [34.9] a Statistically significant (p < 0.05) vs healthy controls, computed by Mann-Whitney U test.

4 Serum insulin-like growth factor-i and erythropoiesis 327 Table 3. Correlation coefficients of hematologic parameters, iron markers, and blood FEP vs serum IGF-I concentrations according to body iron status. Parameters Correlation coefficients (r) with serum IGF-I Iron depleted group a (n = 46) Healthy controls (n = 25) Hematologic parameters White blood cells (10 3 /µl) 0.27 b 0.15 b Red blood cells (10 6 /µl) 0.16 b 0.13 b Hemoglobin (g/dl) 0.24 b 0.19 b Platelets (10 3 /µl) Iron markers Serum iron (µg/dl) 0.40 b 0.09 Total iron-binding capacity (µg/dl) 0.38 b 0.13 b Serum ferritin (µg/l) 0.23 b 0.02 Blood FEP (µg/dl) b b a Iron depleted group includes the subjects with iron depletion (stage I), iron-deficient erythropoiesis (stage II), or iron deficiency anemia (stage III). b Statistically significant (p < 0.05). ± 0.9 g/dl and 50.5 ± 30.8 µg/dl, p <0.05, respectively] (Table 1). On the other hand, FEP levels were significantly lower in the subjects with IGF-I >809 ng/ml than in those with IGF-I <523 ng/ml (38.9 ± 16.2 µg/dl vs 63.4 ± 23.1 µg/dl, p <0.05). Our data for hemoglobin, FEP, and WBC levels suggest that IGF-I is not only related to heme synthesis of mature erythrocytes, but also associated with leukocytopoiesis; however, the influence of IGF-I on hematopoiesis is evident only when serum IGF-I concentrations are moderately increased. In the present study, IGF-I concentrations showed significant correlations with blood hemoglobin (r = 0.24, p <0.05) and WBCs (r = 0.27, p <0.05) in iron deficient subjects. These results corroborate partly the report of Anttila et al [8], who found that serum IGF-I was positively correlated with blood hemoglobin concentrations in healthy prepubertal or early pubertal boys. Prevalences of iron deficiency or iron deficiency anemia were significantly higher in the subjects with IGF-I <523 ng/ml, compared to those with IGF-I >809 ng/ml (64.2 and 35.7% vs 21.4 and 7.1%). In particular, the incidences of subjects showing an increase in FEP concentration (>50 µg/dl) were 5.4- fold higher in adolescents with IGF-I <523 ng/ml than in those with IGF-I >809 ng/ml. As shown in Table 2, serum IGF-I levels decrease gradually as body iron status falls, reaching minimum values (582.6 ± 94.6 ng/ml) when the subjects attain unmistakable iron deficiency anemia; their serum IGF-I levels were significantly lower than those of non-anemic healthy controls (741.2 ± ng/ml, p <0.05). IGF-I concentrations were more strongly correlated with FEP in the iron-deficient adolescents (r = , p <0.05) than in the healthy controls (r = , p <0.05) (Table 3). Considering that heme is formed by insertion of iron into protoporphyrin IX, our results suggest that IGF-I plays roles in iron metabolism and protoporphyrin synthesis, especially in adolescents with negative iron balance. Soliman et al [9] reported that serum ferritin concentration was negatively correlated with circulating IGF-I levels in children with betathalassemia. In contrast, in the present study, serum IGF-I concentrations correlated positively with serum ferritin levels in iron-deficient subjects. These discrepancies may be explained by the differences in iron metabolism between thalassemias and iron deficiency anemia. In thalassemias, serum ferritin

5 328 Annals of Clinical & Laboratory Science and bone marrow iron levels are normal or slightly increased, which is the opposite of what is found in iron deficiency anemia. In addition, blood FEP is usually elevated in iron deficiency anemia, but not in thalassemia. In the present study, we studied female adolescents with iron deficiency or iron deficiency anemia who were otherwise healthy. In our study, serum IGF-I concentrations of iron-depleted subjects showed high correlation coefficients with hematologic parameters, compared to those of healthy controls, implying that the effects of IGF-I on erythropoiesis may differ according to body iron status. In conclusion, female adolescents with iron deficiency anemia exhibited a significant decrease in serum IGF-I concentrations compared to healthy controls. The subjects with decreased IGF-I concentrations showed high incidences of iron deficiency anemia vs those with increased IGF-I concentrations, suggesting that IGF-I is closely associated with erythropoietic activities and may influence erythropoiesis through iron metabolism. Acknowledgement This work was supported by Inha University Research Grant (INHA-31640). References 1. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 1989;10: Froesch ER, Hussain MA, Schmid C, Zapf J. Insulin-like growth factor I: physiology, metabolic effects and clinical uses. Diabetes Metab Rev 1996;12: Choi JW, Pai SH. Associations between serum transferrin receptor concentrations and erythropoietic activities according to body iron status. Ann Clin Lab Sci 2003;33: Serdar MA, Sarici SU, Kurt I, Alpay F, Okutan V, Kurnaz L, Kutluay T. The role of erythrocyte protoporphyrin in the diagnosis of iron deficiency anemia of children. J Trop Pediatr 2000;46: Miyagawa S, Kobayashi M, Konishi N, Sato T, Ueda K. Insulin and insulin-like growth factor I support the proliferation of erythroid progenitor cells in bone marrow through the sharing of receptors. Br J Haematol 2000; 109: Claustres M, Chatelain P, Sultan C. Insulin-like growth factor I stimulates human erythroid colony formation in vitro. J Clin Endocrinol Metab 1987;65: Gunter EW, Turner WE, Huff DL. Investigation of protoporphyrin IX standard materials used in acidextraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX. Clin Chem 1989;35: Anttila R, Koistinen R, Seppala M, Koistinen H, Siimes MA. Insulin-like growth factor I and insulin-like growth factor binding protein 3 as determinants of blood hemoglobin concentration in healthy subjects. Pediatr Res 1994;36: Soliman AT, El Banna N, Ansari BM. GH response to provocation and circulating IGF-I and IGF-binding protein-3 concentrations, the IGF-I generation test and clinical response to GH therapy in children with betathalassaemia. Eur J Endocrinol 1998;138: