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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 2  |  Page : 105-110

Urinary hepcidin concentration in assessment of iron homeostasis in pediatrics


1 Department of Pediatrics, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
3 Department of Pediatrics, Mansoura Insurance Hospital, Dakahlia, Egypt

Date of Submission04-Mar-2019
Date of Acceptance10-Apr-2019
Date of Web Publication15-Nov-2019

Correspondence Address:
Amany M El-Kelany
Pediatrics Department, Faculty of Medicine, Suez Canal University, 4.5 Km the Ring Road, Ismailia 41522
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_9_19

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  Abstract 


Background Iron deficiency is a worldwide health problem that can cause long term consequences. Hepatic hormone hepcidin regulates systemic iron homeostasis. Urinary hepcidin level could be an effective tool in assessment of iron status.
Aim The present study aims to evaluate the diagnostic role of urinary hepcidin to predict different stages of iron deficiency (ID) in children.
Methods We studied 75 children with iron deficiency and 25 healthy control children. The diagnostic performance of urinary hepcidin was estimated by analyzing the receiver operating characteristic curve. Diagnostic cut-off point with a high predictive value for iron deficiency were selected.
Results Urinary hepcidin levels were significantly lower in all stages of iron deficiency than in the control group. Significant positive correlations between urinary hepcidin level with hemoglobin, mean corpuscular volume, serum iron, ferritin and Tsat had been confirmed. Hepcidin cutoff values of ≤ 369 ng/ml in ID stage -1 , 315 ng/ml ≤ in ID stage-2 and 293 ng/ml in ID stage-3 were associated with a high diagnostic likelihood for iron deficiency.
Conclusion We found that in all stages of ID, hepcidin levels were significantly lower than the control group. Urinary hepcidin assay provides a reliable non-invasive screening mean of diagnosing ID state in children.

Keywords: Hepcidin, iron, pediatrics


How to cite this article:
Al Sharkawy SG, El-Kelany AM, Anani MM, El-shahat El Sayed H. Urinary hepcidin concentration in assessment of iron homeostasis in pediatrics. Egypt J Haematol 2019;44:105-10

How to cite this URL:
Al Sharkawy SG, El-Kelany AM, Anani MM, El-shahat El Sayed H. Urinary hepcidin concentration in assessment of iron homeostasis in pediatrics. Egypt J Haematol [serial online] 2019 [cited 2019 Dec 5];44:105-10. Available from: http://www.ehj.eg.net/text.asp?2019/44/2/105/271084




  Introduction Top


Iron is an essential micronutrient involved in energy metabolism, oxygen transport, and immune response and plays an essential role in brain development [1],[2]. Iron deficiency (ID) in infancy affects different neurodevelopmental processes with increasing risk of psychomotor impairment and/or mental development that persist forever in spite of iron supplementation [3],[4].

ID is a public health problem and considered the commonest nutritional deficiency worldwide, especially in developing countries. Important risk factors for ID state include low birth weight, prematurity, lead exposure, prolonged exclusive breastfeeding, and weaning to foods without iron-fortification [5].

ID is defined as a decrease in total body iron to a degree that iron stores are fully exhausted with some degree of tissue ID. ID passes through three stages: first stage is iron depletion, where body iron stores are reduced; the second stage is iron-deficient erythropoiesis, defined as laboratory evidence of an impaired supply of iron to the erythroid bone marrow required for hemoglobin synthesis; and the third stage is iron-deficiency anemia (IDA), which refers to the combination of ID and anemia [3].

Screening children for ID is recommended to prevent its long-term consequences [5]. There is no single investigation currently approved to characterize the iron status of a child. Development of a simple, easy, noninvasive, and sensitive tool to diagnose ID is mandatory.

Hepcidin (hepatic bactericidal protein discovered by Krause et al. [6] and Park et al. [7]) is a hepatic-derived peptide hormone that is known as an iron regulatory hormone. Hepcidin decreases serum iron by decreasing both intestinal iron absorption and macrophage iron release [8]. Hepcidin mediates its functions by a single biochemical mechanism: the hepcidin–ferroportin interaction [9]. Dysregulation of hepcidin synthesis is linked to a variety of iron disorders [10].

Three hepcidin isoforms (hepcidin-20, −22, and −25) are present and excreted in urine [7]. Hepcidin-25 is the only isoform that plays a key role in iron regulation [11]. Hepcidin assay can be a helpful tool in distinguishing anemia of chronic illness from IDA, as it is well known that hepcidin production is increased by inflammation and reduced in IDA [12],[13].

Urinary hepcidin provides an indirect assay of the circulating hormone level that is less affected by diurnal variation, which may allow the development of noninvasive mean of diagnosing IDA [14]. This could become particularly useful for children, as a screening test.

The aims of the current study were to assess urinary hepcidin levels and their significance as an indicator of ID and to determine correlations between urinary hepcidin levels and other iron parameters in children.


  Patients and methods Top


This is a case–control study that was conducted on children recruited from Outpatient Pediatric Clinic in Ismailia University Hospital, Ismailia, Egypt, from October 2015 to January 2017.

The study investigated 75 children with ID and 25 healthy children of matched age and sex as a control group with an age range from 3 to 10 years. Children were assorted as follows:
  1. ID stage-1 group: 25 children with normal hematological and iron parameters except for low serum ferritin level (≤20 ng/ml).
  2. ID stage-2 group: 25 children with normal hemoglobin level, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) for age, but had low serum ferritin (<12 ng/ml) and low serum transferrin saturation (Tsat) less than 16%.
  3. ID stage-3 group: 25 children with low serum ferritin levels (<12 ng/ml), low Tsat (<16%), and microcytic hypochromic anemia (low hemoglobin level, low MCV, and low MCHC). The cut-off levels of hemoglobin used to define anemia is 11.5 g%.
  4. Control group: 25 healthy children matched for age and sex with normal hematological iron parameters and serum ferritin level more than 20 ng/ml.


Exclusion criteria

Children were excluded if they had liver or renal function abnormalities or signs of infection or inflammation (ferritin is known as an acute phase reactant). Children who received iron therapy in the previous three months were also excluded.

All children were subjected to careful clinical examination, including general examination with a focus search for pallor, brittle nails or spooning of nails, or a red glazed tongue. Anthropometric measurements were taken, including weight and height measurements, and BMI was calculated. Abdominal examination was done, with a special attention to detect any organomegaly.

Blood samples were collected to measure complete blood count, serum ferritin, and serum iron parameters. Serum iron and total iron-binding capacity (TIBC) concentrations were assessed by Ferrozine/MgCO2 colorimetric method (Tulip Diagnostics India, Bambolimcomplex, India). Tsat is the value of serum iron divided by TIBC. Serum ferritin was assessed by enzyme-linked fluorescent assay (Mini Vidas).

Midstream fresh urine samples were collected in the morning between 9 and 11 am to avoid diurnal variation. Then samples were centrifuged and were stored in aliquots at −20°C. We avoid freeze–thaw cycles to prevent hepcidin loss. Urinary hepcidin levels were measured by ELISA kits from Uscn Life Science Inc.

Ethical consideration

This study was approved by the Medical Ethical Committee of Faculty of Medicine, Suez Canal University. Written parental informed consent consistent with the ethical principles of International Conference of Harmonization guidelines and Good Clinical Practice (ICH-GCP) was obtained for all participants.

Statistical analysis

Data processing and statistical analysis of results were studied using SPSS software, version 21. Continuous variables were expressed as mean and SD and were studied using analysis of variance. Categorical variables were presented as absolute numbers. The degree of relationship between different variables was calculated using Pearson’s correlation analysis, and the results were demonstrated in the form of correlation coefficient (r) and P values. We used receiver operating characteristic (ROC) curve analysis to determine distinctive characteristic of different cutoff levels of urinary hepcidin. We considered P value as statistically significant if less than 0.05.


  Results Top


There were no significant differences between patients and control group regarding age, sex, and BMI (P>0.05). Urinary hepcidin levels in control children were 443.92±91.84 ng/ml, with range from 344 to 650 ng/ml. Children with different stages of ID had significantly lower urinary hepcidin level than in the control group with more decrement with advanced severity of ID stage (P<0.00) ([Table 1]).
Table 1 Demographic and laboratory characteristics of the study population

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Urinary hepcidin level had a significant positive correlation with hemoglobin, MCV, MCHC, serum iron, serum ferritin level ([Figure 1]), and Tsat (P<0.01). There were significant negative correlation between urinary hepcidin level and TIBC ([Table 2]).
Figure 1 Correlation of urinary hepcidin with serum ferritin in ID. ID, iron deficiency.

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Table 2 Pearson’s correlation between urinary hepcidin and clinicolaboratory parameters

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ROC curve was used to assess the cutoff points for urinary hepcidin levels for different stages of ID from control children. Cutoff points for ID stage-1, stage-2, and stage-3, respectively, from normal children were less than or equal to 369 ng/ml, less than or equal to 315 ng/ml, and less than or equal to 293 ng/ml, with 95% confidence intervals of 0.797–0.977, 0.988–1.005, and 0.866–1.02, respectively ([Table 3]). The three cutoff points’ positive predictive values were 80, 96, and 92%, respectively, and the negative predictive values for cutoff points were 85, 95, and 95%, respectively ([Table 3]).
Table 3 Predictive values of urinary hepcidin level in detection of iron deficiency

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These three cutoff points’ sensitivity (84, 96, and 96%) and specificity (80, 96, and 92%) were high and are illustrated in [Figure 2],[Figure 3],[Figure 4].
Figure 2 ROC curve for urinary hepcidin in diagnosis of ID stage-1. ID, iron deficiency; ROC, receiver operating characteristic.

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Figure 3 ROC curve for urinary hepcidin in diagnosis of ID stage-2. ID, iron deficiency; ROC, receiver operating characteristic.

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Figure 4 ROC curve for urinary hepcidin in diagnosis of ID stage-3. ID, iron deficiency; ROC, receiver operating characteristic.

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  Discussion Top


ID and IDA remain a worldwide health problem, especially in pediatrics as young infant and children are more vulnerable to the effects of ID because of rapid growth and development. Iron metabolism was comprehended by the discovery of hepcidin, which also promoted the inquiry about the use of hepcidin as a diagnostic and therapeutic tool in many diseases. Hepcidin is a key iron controller hormone that regulates systemic iron homeostasis [15]. Hepcidin synthesis is affected by iron body level, inflammation, hypoxia, and anemia [16],[17],[18]. Hepcidin is decreased in ID to allow increment of dietary iron absorption and iron stores [19].

Hepcidin sounds to be a sensitive and reliable marker of ID. Decrease in hepcidin and Tsat is an early indicator of ID than decrease in hematocrit or hemoglobin [20]. Use of urinary hepcidin level as a sensitive, noninvasive investigation that can evaluate iron state in children, even in early stages before reaching stage-3 (IDA), deserves study. Urine tests were used as an alternative for serum assays because it is less affected by diurnal variation and the noninvasive nature of sampling [11].

In the present study, we found a significant reduction in urinary hepcidin levels in all stages of ID than the control group, which decrease more with the severity of ID. These data broadly consistent with Sanad and Gharib [20], who found hepcidin levels were markedly lower in all phases of ID. In addition, a highly significant decrease was reported in its level with the progress in severity of ID. Cherian et al. [11] also reported that levels of urinary hepcidin were significantly lower in ID and IDA. In 2018, Culafic et al. [21] concluded that anemic patients had significantly lower hepcidin levels than control group.

This study shows that urinary hepcidin levels were positively correlated with hemoglobin level, MCV, MCHC, serum iron, serum ferritin level, and Tsat. These results are in agreement with results of Al-Mazahi et al. [22] who reported significant correlation between hemoglobin, MCV, MCH, serum iron, and ferritin levels and urinary hepcidin levels. Moreover, Bregman et al. [23] stated that hepcidin levels were positively correlated with ferritin levels.

Our results are in agreement with Choi et al. [24] who demonstrated that hepcidin level was significantly positively associated with hemoglobin levels, serum ferritin, and Tsat and significantly negatively correlated with TIBC. Culafic et al. [21] confirmed a positive correlation between serum hepcidin level and serum iron, ferritin, MCV, and Tsat.

Urinary hepcidin cutoff points that distinguish different iron-deficiency stages (stage-1, stage-2, and stage-3) from healthy children in the current study had marked significant confidence intervals with valuable predictive potentials. These results are comparable with data clarified by Sanad and Gharib, who stated that urinary hepcidin levels at cutoff point could diagnose ID stage-1 with sensitivity of 88% and specificity of 88%, could expect ID stage-2 with a sensitivity of 96% and specificity of 92% and could expect ID stage-3 with a sensitivity of 96% and specificity of 100% [21].

Al-Mazahi et al. [22] used the ROC curve to determine the cutoff point of urinary hepcidin levels that could diagnose ID state from normal children. The sensitivity and specificity at their cutoff point were 91 and 51%, respectively. The positive predictive value was calculated at 65% and negative predictive value at 85%.

The effectiveness of using urinary hepcidin level in detection of IDA can be more clear when we comparing the ROC curve results of urinary hepcidin in our study with those for serum ferritin level in other studies [25],[26], as ferritin had been considered the best single assay for the diagnosis of IDA. Guyatt et al. [27] calculated serum ferritin predictive value and the area under the ROC curve in the diagnosis of IDA. Area under the ROC curve was 0.95 (P<0.001), compared with 0.94 for urinary hepcidin in our study.


  Conclusion Top


Urinary hepcidin assay provides a reliable screening means of diagnosing iron-deficiency state especially for children, as it is simple, sensitive, and noninvasive and reflects serum iron and iron store stage.

Acknowledgements

The authors acknowledge the parents of children included in this study. We thank all Suez Canal University Hospital staff at pediatrics and clinical pathology departments for their assistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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