Determination of Iron Metabolism-related ... - Clinical Chemistry

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between anemia from iron deficiency and anemia from chronic disease (4). Because the soluble transferrin recep- tor (sTfR) concentration is not influenced by ...
Clinical Chemistry 47, No. 8, 2001

Determination of Iron Metabolism-related Reference Values in a Healthy Adult Population, Glenn Van den Bosch,1 Jan Van den Bossche,2 Carola Wagner,3 Pieter De Schouwer,2 Martine Van De Vyvere,2 and Hugo Neels1* (Laboratories of 1 Clinical Biochemistry and 2 Hematology, Algemeen Centrum Ziekenhuis Antwerpen, Campus Stuivenberg, Lange Beeldekensstraat 267, B-2060 Antwerp, Belgium; 3Dade-Behring Marburg GMBH, PO Box 11 49, 35001 Marburg, Germany; * author for correspondence: fax 323-2177358, e-mail hugo.neels@ocmw. antwerpen.be) Although iron evaluation on bone marrow aspirates remains the gold standard for assessing iron status, several other methods have been implemented that are less invasive and more practical. Serum iron, percentage of saturation, and total iron-binding capacity, however, lack sensitivity and are too labile to be of value as single determiners (1 ). Indirect measures of the functional iron compartment, such as mean cell volume and red cell distribution width, have the disadvantage of becoming indicators relatively late in the development of iron deficiency (2 ). Serum ferritin can be used as a marker of the iron storage compartment because it is the earliest marker to decrease with iron depletion. However, because ferritin is an acute-phase reactant in serum, its concentration may rise disproportionately to the iron storage status during inflammation, infection, or neoplasia (3 ), an occurrence that limits utility of ferritin in the differential diagnosis between anemia from iron deficiency and anemia from chronic disease (4 ). Because the soluble transferrin receptor (sTfR) concentration is not influenced by acute-phase reactions, it remains within reference values in patients with anemia of chronic disease. sTfR, therefore, can be used as a more reliable index of iron deficiency anemia (5, 6 ). The correlation between sTfR and bone marrow erythropoetic activity allows the use of sTfR for monitoring erythropoetin therapy, with sTfR increasing 4 weeks before the first increase of hemoglobin (7 ). However, conditions associated with erythroid hyperplasia can also lead to an increase of sTfR in the absence of iron deficiency (8 ). The sTfR/log ferritin ratio (sTfR/ferritin ratio) is reported to be even more sensitive in the presence of borderline normal ferritin and/or sTfR concentrations. This index may also be useful in distinguishing iron deficiency from conditions with hyperplastic erythropoiesis (2 ). Because of the lack of international standardization, different reference values for sTfR can be found in the literature (9, 10 ). It also has been shown that the 95% ranges of the sTfR distribution in healthy and irondeficient individuals may overlap (11 ), emphasizing the need for well-established reference values. Therefore, we measured iron status characteristics in 456 volunteers and determined reference values for male and female adults. Subsequently, the influence of blood donation among volunteers was evaluated, as well as estrogen therapy and menopausal status in female volunteers. Blood was collected from 527 healthy volunteers in the

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morning with the Sarstedt blood collection system in accordance with NCCLS guidelines (C28-A, H3-A3, and H18-A) (12–14 ) and after a 15-min recumbent rest. All individuals were living in Antwerp at 7.5 m above sea level and were asked to refrain from participation if they were aware of any preexisting illnesses or were taking medication. The institutional committee for medical ethics approved the study protocol and all volunteers gave informed consent. From the total number of 527 Caucasian adults enrolled, 71 subjects were excluded because of a C-reactive protein concentration ⬎7 mg/L, a body mass index ⬎28 kg/m2 [weight/length2 (kg/m2)], and/or a positive drug screening result (abuse of alcohol or drugs). We measured iron-related analytes in 456 apparently healthy individuals (216 men and 240 women) with a mean age of 39 years (range, 19 – 60 years) within the 6 weeks of the study period. Seventy-four were regular blood donors (38 men and 36 women). In the female subgroup of 193 premenopausal and 47 postmenopausal women, 87 and 26 women received hormone therapy, respectively. According to WHO guidelines (15 ), 79 subjects in this survey were identified as anemic (hemoglobin limit of 130 g/L for males and 120 g/L for females). Hemoglobin concentration, hematocrit, mean cellular volume, mean cellular hemoglobin, mean cellular hemoglobin concentration, and erythrocyte counts were measured on a Coulter STKS hematology analyzer (Coulter Electronics) within 4 h after sampling. Iron measurement was performed on the Ektachem Vitros 950IRC analyzer (Ortho-Clinical Diagnostics). Transferrin measurement was done on the BM/Hitachi 911 (Boehringer-Manheim Diagnostics). Soluble transferrin receptor and ferritin concentrations were nephelometrically determined by N Latex reagent sets (Dade Behring) according to the manufacturer’s instructions after storage of the centrifuged (2600g; 10 min) serum at ⫺80 °C. These latex tests are based on microagglutination of latex particles coated with a monoclonal antibody. The intraassay CVs for the ferritin and sTfR assays were 1.0 – 4.6% and 1.4 –2.1%, respectively. Interassay CVs for ferritin and sTfR were 1.2–3.1% and 0.8 –1.2%, respectively, as determined according to NCCLS guidelines (16 ). For sTfR and ferritin, means and SDs were calculated. Frequency distribution histograms were plotted, and 2.5 and 97.5 percentiles were determined (Fig. 1). To assess the effects of gender, pre- or postmenopausal status in women, blood donation status, and hormone therapy, the Wilcoxon 2-sample test was applied. For the correlation analysis, a nonparametric approach was chosen using Spearman rank correlation coefficients. Statistical parameters for estimating the regression line were calculated according to the procedure described by Passing and Bablok (17 ). All statistical evaluations were performed with the statistical analysis software package SAS (SAS Institute Inc.). Median and 2.5–97.5 percentiles of all iron-related analytes that we obtained from our study population are presented in Table 1. Statistical analysis of our data showed significantly higher ferritin concentrations in men

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Technical Briefs

Fig. 1. Distribution of sTfR and ferritin in the study population.

than in premenopausal women (␹2⫽ 157.35; P ⫽ 0.0001). Premenopausal women had ⬃38% lower values for ferritin than males, whereas no significant difference was found between males and postmenopausal women. Moreover, 14.65% of the 193 premenopausal women included in the study had hemoglobin concentrations ⬍120 g/L, a finding that was far different from the 2.5 percentile

Table 1. Results of laboratory tests reflecting the iron status of the reference population. Median (2.5–97.5 percentiles) Analyte

Males (n ⴝ 216)

Females (n ⴝ 240)

Hemoglobin, g/L Hematocrit Erythrocyte count, ⫻1012/L Serum iron, ␮mol/L Serum ferritin, ␮g/L Serum transferrin, mg/L Serum transferrin saturation sTfR, mg/L sTfR-ferritin index

146 (131–165) 0.424 (0.384–0.477) 4.74 (4.14–5.51)

128 (111–146) 0.376 (0.332–0.423) 4.18 (3.70–4.74)

21.5 (10.7–35.6) 91.5 (20.0–291.0) 251 (186–342)

20.6 (8.8–36.2) 36.5 (4.5–182.0) 267 (196–393)

0.33 (0.15–0.62)

0.29 (0.11–0.64)

1.14 (0.76–1.74) 0.58 (0.38–1.07)

1.09 (0.76–1.82) 0.70 (0.39–2.27)

usually excluded at each end of the reference distribution for calculating reference intervals. These findings demonstrate that the prevalence of iron deficiency among premenopausal women may be higher than generally assumed. All reference values were calculated without the exclusion of these anemic subjects because no significantly different reference values were found when these individuals were excluded (data not shown). Moreover, exclusion of these subjects would lead to “ideal” reference values, instead of reference values that would be obtained from the general population. The mean cell volume of 88 fL (SD, 4.15) in the anemic population lies within the reference interval, which confirms the low diagnostic value of the mean cell volume in iron deficiency, as was found in earlier studies (18 ). From our data, ferritin reference intervals of 20 –291 ␮g/L for males and 5–182 ␮g/L for females could be determined. Statistical analysis showed a significant correlation between ferritin and age (r ⫽ 0.2449; P ⬍0.0001), hemoglobin concentration (r ⫽ 0.5522; P ⬍0.0001), and transferrin saturation (r ⫽ 0.3653; P ⬍0.0001). These correlations, however, are too weak to justify the need for age-specific ferritin reference intervals in a clinical setting. Individual sTfR measurements were 0.58 –2.50 mg/L with a mean (⫾ SD) of 1.15 (⫾0.26), although sTfR concentrations did not significantly differ between sexes, which is in agreement with earlier findings (18, 19 ). Calculation of the 2.5–97.5 percentiles led to a sTfR reference interval for adults of 0.83–1.76 mg/L. This finding relates closely with the 0.78 –1.38 mg/L reference interval found by Vernet and Doyen (18 ), although that study was performed on a small population of 61 subjects and used 5–95 percentiles. Other studies, however, show different reference intervals because of different methods: 3– 8.2 mg/L for the TfRTM ELISA assay (Ramco Laboratories; Inc.) (20 ); 0.85–3.05 mg/L for the QuantikineTTM Soluble Transferrin Receptor ELISA (R&D SysMIVD tems Inc.) and the ClinigenTM assay (Amgen Diagnostics) (11 ); 1.1–3.3 mg/L for the IDeATM Soluble Transferrin Receptor IEMA (Orion Diagnostics) (21 ); and 2.16 – 4.54 mg/L (males) and 1.79 – 4.63 mg/L (females) for the TinaquantTM sTfR assay (Roche Diagnostics, GmBH) (22 ). For sTfR, a correlation with transferrin saturation could be found (r ⫽ ⫺0.3043; P ⬍0.001); correlations with other iron metabolism variables were weak but statistically significant for hemoglobin (r ⫽ 0.1036; P ⫽ 0.0274) and ferritin (r ⫽ ⫺0.0995; P ⫽ 0.0337). Unlike ferritin, no correlation was found between sTfR concentrations and age, as previously demonstrated by Allen et al. (11 ), who found that in 225 healthy adults, the status of sTfR was an independent valuable. The sTfR-ferritin index correlated well with the hemoglobin measured (r ⫽ ⫺0.2968; P ⬍0.0001). Postmenopausal women had statistically higher ferritin concentrations than premenopausal women (␹2 ⫽ 6.5076; P ⫽ 0.0107), indicating the influence of menstrual bleeding on the iron depletion in premenopausal women. However, estrogen intake in the form of birth control pills increased the ferritin concentrations significantly in pre-

Clinical Chemistry 47, No. 8, 2001

menopausal women (␹2 ⫽ 10.988; P ⫽ 0.0009), whereas hormone replacement therapy in postmenopausal women did not significantly alter ferritin concentrations. Whether this is because of different dosage schedules of the estrogens used or because of less blood loss in postmenopausal women has to be further evaluated. When subjects donated blood on a regular basis, a significantly lower ferritin concentration could be observed for men (␹2 ⫽ 23.242; P ⫽ 0.0001) as well as for premenopausal women (not shown). This correlates well with the results of Punnonen and Rajama¨ki (23 ), who showed that 17% of Finnish women who frequently donated blood had completely lost their iron stores. Unlike the findings of Vernet and Doyen (18 ), who found increased sTfR concentrations in males who regularly donated blood, we found that blood donation did not affect sTfR concentrations. This may be caused by compensation of the chronic blood loss by mobilizing iron from storage pools. These findings suggest that serum sTfR concentrations will only be increased when erythropoiesis becomes deprived of iron, whereas a decrease in serum ferritin will reflect changes over a broad range of body iron stores. In premenopausal women, we found significantly higher sTfR concentrations compared with postmenopausal women (␹2 ⫽ 6.5076; P ⫽ 0.0107), which is in contrast with the earlier findings of Allen et al. (11 ). In our opinion, this difference may be a result of the more rigid exclusion criteria or the low number of postmenopausal women involved in their study.

We would like to acknowledge Annick Wauters and Ermine Van Boeckel for their valuable advice in performing this study. References 1. Baynes RD. Assessment of iron status. Clin Biochem 1996;29:209 –15. 2. Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor: a quantitative measure of tissue iron deficiency. Blood 1990;75:1870 – 6. 3. Cook JD, Skikne BS. Iron deficiency: definition and diagnosis. J Intern Med 1989;226:349 –55. 4. Mast AE, Blinder MA, Gronowski AM, Chumley C, Scott MG. Clinical utility of the soluble transferrin receptor and comparison with serum ferritin in several populations. Clin Chem 1998;44:145–51. 5. Huebers HA, Beguin Y, Pootrakul P, Einspahr D, Finch CA. Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood 1990;75:102–7. 6. Ferguson BJ, Skikne BS, Simpson KM, Baynes RD, Cook JD. Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency. J Lab Clin Med 1992;119:385–90. 7. Dore F, Bonflgli S, Gaviano E, Pardini S, Longinotti M. Serum transferrin receptor levels in patients with thalassemia intermedia during rHuEPO administration. Haematologica 1996;81:37–9. 8. Feelders RA, Kuiper-Kramer EPA, van Eijk HG. Structure, function and clinical significance of transferrin receptors. Clin Chem Lab Med 1999;37:1–10. 9. Kohgo Y, Niitsu Y, Kondo H, Kato J, Tsushima N, Sasaki K, et al. Serum transferrin receptor as a new index of erythropoiesis. Blood 1987;70: 1955– 8. 10. Flowers CH, Skikne BS, Covell AM, Cook JD. The clinical measurement of serum transferrin receptor. J Lab Clin Med 1989;114:368 –77. 11. Allen J, Backstrom KR, Cooper JA, Cooper M, Detwiler TC, Essex DW, et al. Measurement of soluble transferrin receptor in serum of healthy adults. Clin Chem 1998;44:35–9. 12. National Committee for Clinical Laboratory Standards. How to define, determine, and utilize reference intervals in the clinical laboratory; approved guideline C28-A. Wayne, PA: NCCLS, 1995. 13. National Committee for Clinical Laboratory Standards. Procedures for col-

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Determination of Blood Total, Reduced, and Oxidized Glutathione in Pediatric Subjects, Anna Pastore,1* Fiorella Piemonte,2 Mattia Locatelli,3 Anna Lo Russo,1 Laura Maria Gaeta,2 Giulia Tozzi,2 and Giorgio Federici1 (1 Laboratory of Biochemistry, 2 Molecular Medicine Unit, and 3 Scientific Directorate, Children’s Hospital and Research Institute “Bambino Gesu`”, Piazza S. Onofrio, 4, 00165 Rome, Italy; * author for correspondence: fax 39-0620902270, e-mail [email protected]) Glutathione (l-␥-glutamyl-l-cysteinylglycine), which is present in virtually all mammalian tissues, provides reducing capacity for several reactions and plays an important role in detoxification of hydrogen peroxide, other peroxides, and free radicals (1 ). The synthesis and degradation of glutathione are controlled by reactions of the ␥-glutamyl cycle; a decrease in blood reduced glutathione (GSH) has been reported in patients affected by deficiencies of the enzymes involved in the synthesis of glutathione (1 ). In cells, total glutathione can be free or bound to proteins; measurement of free glutathione in blood samples is essential for evaluation of the redox and detoxification status of cells in relation to its protective role against oxidative and free radical-mediated cell injury; moreover, GSH measurement is important for the diagnosis of ␥-glutamyl cycle disorders. Recently, several methods to measure glutathione in blood have been described, but little is known about the concentrations of various forms of blood glutathione in pediatric subjects (2– 6 ). We report a rapid and fully automated HPLC method for determining total (tGSH), reduced (GSH), and oxidized glutathione (GSSG) in