Iron deficiency and hepcidin by laboratory diagnosis

Hepcidin in general

The first of the twenty-first century was the finding of hepcidin in human urine and serum, which was initially documented in 2000. The functional hormone, hepcidin-25, is the result of the HAMP gene’s cleavage of prohormone (60 amino acids [AAs]), which is a split product of phytohormone (84-AAs). In the blood, hepcidin-25 binds to albumin and α-2-macroglobulin.^[2] Three distinct peptide forms are often produced from the 84-AA prepropeptide hepcidin: Hepcidin-20, hepcidin-22, and hepcidin-25. Controlling functional iron deficiency requires the active form of hepcidin-25.^[8] Hepcidin-25 is a 25-AA polypeptide that has bioactivity.^[4,9] However, after N-terminal degradation, smaller isoforms (hepcidin-20, -22, and -24) are produced. Although the amounts of degraded isoforms vary greatly throughout investigations, large levels have been seen in conditions like sepsis and renal failure that have high hepcidin concentrations. However, the biological significance of these smaller isoforms in iron control has not been shown.^[9] However, hepcidin was first identified as a liver-expressed antimicrobial peptide because of its antibacterial and antifungal characteristics. Both its hepatic origin and antibacterial activity are reflected in the present name. Nonetheless, the primary source is the liver; additional hepatic hepcidin production in the kidney,^[10] heart, and hepcidin-25 is a bioactive 25-AA polypeptide.^[4,9] Nevertheless, smaller isoforms (hepcidin-20, -22, and -24) are generated following N-terminal degradation. Large quantities of degraded isoforms have been seen in circumstances with high hepcidin concentrations, such as sepsis and renal failure, despite the fact that the amounts vary widely between research. Nevertheless, it has not been demonstrated that these smaller isoforms have any biological function in iron regulation.^[9] However, due to its antibacterial and antifungal properties, hepcidin was initially discovered to be an antimicrobial peptide expressed in the liver. The current name reflects both its hepatic origin and antibacterial action. However, the liver is the main source; further hepatic hepcidin synthesis occurs in the kidney,^[10] heart, and additionally, the stomach,^[11] retina, and adipocytes^[12] have been described. And pancreatic β cells,^[13-15] alveolar cells, and monocytes and macrophages.^[16] However, as these organs have been demonstrated to produce the hepcidin gene at a much lower level than the liver, their significance is still uncertain.^[14,17] The significant association between serum hepcidin levels and specific hepatic hepcidin expression levels indicates that hepatic hepcidin is the main regulator of systemic iron balance. The data were reported by Kulaksiz et al.,^[13] Hepcidin binds, internalizes, and degrades FPN, the gatekeeper of transcellular iron transport that is confined to the cell membrane. FPN is found in hepatocytes, enterocytes, reticuloendothelial macrophages, and adipocytes. Iron availability hence opposes rising and falling hepcidin levels. Hepcidin is both upregulated and downregulated by the factors listed in Table 1. Hepcidin overexpression causes iron-restricted anemia, whereas complete lack of hepcidin causes juvenile monochromatism.^[2] Conversely, excessive hepcidin can result in anemia that is resistant to iron therapy, whereas low serum hepcidin may be more likely to iron overload with widespread iron deposition in the tissue.^[14,18] Numerous clinical situations, such as sepsis, inflammation, and neoplastic diseases, have been shown to increase serum hepcidin levels.^[14] Hepatocytes are the main source of circulating hepcidin, which regulates systemic iron homeostasis. However, cardiomyocytes, dendritic cells, and keratinocytes also produce hepcidin mRNA, and these hepcidin sources have both autocrine and paracrine effects on the local distribution of iron. While hepcidin from cardiomyocytes appears to maintain the heart’s baseline iron homeostasis, hepcidin from keratinocytes and dendritic cells enhances the local response to infection and inflammation.^[4]

Diagnostic assays for hepcidin in different conditions with different methods

There are now just a few locations that can afford and easily access the assays that have been developed for the determination of serum and urine hepcidin levels. Separating the physiologically active hepcidin 25 from other hepcidin isoforms is also crucial. The majority of commercially available tests are used to detect hepcidin in urine and serum. Assays based on MS^[6,19] include Weak Cation Exchange Time of Flight Mass Spectrometry (WCX TOF MS).^[14,20] and surface-enhanced laser desorption/ionization TOF MS (SELDI TOF MS), which are better than antibody-based assays.^[21] Because they can distinguish between different isoforms.^[14] There are other mass spectrometric techniques that employ a synthetic hepcidin reference standard and immunoassays based on antihepcidin antibodies.^[19] It determines the mass of the active 25-amino-acid hepcidin species and measures the peak’s strength in relation to an internal standard that has been spiked.^[22] Hepcidin levels showed diurnal fluctuations in healthy conditions, with lower morning and greater afternoon amounts.^[23] as well as gender disparities, with women having lower levels of hepcidin than males.^[24] Iron deficiency, β-thalassemia intermedia, and juvenile forms of hereditary hemochromatosis were shown to have very low levels of hepcidin in pathological stages, while adult forms of hereditary hemochromatosis had slightly greater levels [Table 2].^[25] Increased overall mortality has also been associated with elevated hepcidin levels.^[8] Unless they have excessive inefficient erythropoiesis, as some patients with β-thalassemia major have, individuals with secondary iron overload from transfusions have high hepcidin readings.^[26] Despite frequently severe iron shortage, individuals with iron-refractory iron-deficiency anemia (IRIDA) have high-normal hepcidin levels.^[27] Serum hepcidin levels are also elevated in multiple myeloma, inflammatory diseases, Hodgkin’s disease, and several malignancies.^[28] Hepcidin is markedly increased in infections, where it may contribute to host defense by lowering blood iron levels and preventing microorganisms from accessing this vital nutrient.^[29] Chronic kidney disease (CKD) patients had elevated hepcidin levels^[30] and correspond to how severe renal impairment is. Since high-normal or elevated blood hepcidin in the context of iron deficiency is a characteristic of IRIDA, clinical hepcidin tests may be helpful in the identification of the condition.^[27] Hepcidin levels are predictive of acute kidney injury (AKI) following heart surgery or in critical illness, deaths in patients with severe AKI, and deaths in septic patients in intensive care.^[31,32] In addition, hepcidin levels are greater in postmenopausal women than in premenopausal women, and they have a strong correlation with ferritin serum levels.^[33]

Moreover, neither cardiovascular disease nor atherosclerosis was associated with hepcidin or hepcidin to ferritin ratios.^[34] Hepcidin levels were higher during prolonged fasting; however, findings from smaller studies indicated that there was some within-subject variability^[35] and showed a significant daily variation (27–50%) as well as a circadian rhythm. Hepcidin-25 levels drop in 1–2 days at room temperature but remain constant for 1 week, 4 weeks, and 2 years at 4°C, −20°C, and −80°C, respectively.^[19] Hepcidin assay standardization will be necessary for the widespread use of hepcidin measurements as well as for testing of other indications that may benefit from hepcidin measurement.^[4,36] In addition, it is difficult to produce antibodies for laboratory testing because hepcidin is a short, evolutionarily conserved peptide that prefers to cluster and adhere to lab plastics.^[37] Hepcidin isoforms can be distinguished from one another, despite the higher cost of testing based on MS. Immunoassays frequently lack the specificity of hepcidin-25 and the ability to detect total hepcidin levels.

As a result, it is debatable whether quantifying hepcidin-25 in particular or total hepcidin is more crucial for clinical decision-making. Without a portable calibrator, a reference method, and a primary source of reference material, absolute hepcidin levels can differ significantly (up to ten times) across tests.^[19] Even though harmonization research is still ongoing, these differences presently make it difficult to compare the data and establish a uniform reference range.^[38] Rather, each method/lab should provide tight reference ranges for hepcidin to serum ferritin (SF), hepcidin to transferrin saturation ratios, and age and gender in addition to hepcidin.^[39] Hepcidin levels in the general population have been found to fluctuate, and it is clear that as the number of metabolic syndrome features grew, hepcidin levels rose noticeably.^[40] Moreover, as a study of some articles induced that hepcidin has a strong positive correlation with iron deficiency. And after treatment of iron deficiency, the level of hepcidin has become increasing.^[41-43]

Hepcidin and diseases

Hepcidin overproduction is linked to iron-restricted anemia, which is seen in people with congenital IRIDA, chronic renal illness, chronic inflammatory disorders, and some types of cancer.^[44]

In pregnant women with and without anemia conditions

The progressive depletion of iron stores results in iron deficiency. Among the reasons include inadequate food intake, malabsorption issues, and bleeding. Sufficient quantities of iron in the mother are necessary during pregnancy to fulfill the induced requirement of the growing feto-placental unit. How it impacts its functioning depends on the severity of the iron deficiency. Severe iron deficiency disrupts normal erythropoietin-mediated erythropoiesis and causes anemia or a drop in hemoglobin concentration and RBC count.^[2] Therefore, iron deficiency may or may not be accompanied with anemia. Although folic acid and vitamin B12 deficiencies can also cause anemia, iron deficiency is by far the most common cause of anemia during pregnancy, Organization for World Health.^[45] The paper states that approximately 38% (32 million) of pregnant women globally suffer from anemia and that 75% of all anemia diagnoses are due to iron deficiency.^[46] Women in the third trimester of pregnancy were more likely to develop anemia than those in the first and second trimesters.^[47] Iron-deficiency anemia in pregnant women is linked to low-birth-weight newborns, preterm delivery, and decreased iron storage for the fetus, which may lead to poor development.^[21] The WHO criteria for anemia in the second trimester were a reduction in hemoglobin RBC <3.8 million/µL, level^[48] <110 g/L, and hematocrit <33%. Laboratory examinations of pregnant women in the second trimester of pregnancy revealed that similar hematological parameter tests were performed for all observed patients to determine the specificities of iron metabolism. Each subject’s SF, Fe, total Fe-binding capacity, and latent Fe-binding capacity were measured. Hepcidin levels were determined using the direct ELISA.^[6] Table 3 displays the indicators that were obtained. Mean corpuscular hemoglobin (25 pg), mean corpuscular volume (82 fl), and red cell distribution width (14.6%) were lower and higher, respectively, in the control group than in the receiving laboratory indications (28.7 pg, 88.3 fl, and 13.3%) (22).

In inflammation

The prolonged duration of hepcidin production in infections and systemic inflammatory diseases provided evidence of hepcidin’s role as an innate immune stimulant. Hepatocyte hepcidin synthesis is transcriptionally regulated by interleukin (IL)-6 through the signal transducer and activator of transcription-3 signaling pathway; bone morphogenetic proteins (BMP) signaling enhances hepcidin in conjunction with IL-6 [Figure 1].^[4,49] Elevated hepcidin during infections or inflammatory diseases results in early-stage hypoferremia [Figure 2].^[4] The inflammatory hepcidin response is probably developed to minimize the production of non-transferrin-bound iron (NTBI) during infection, when the risk of transferrin oversaturation is highest, because erythropoiesis is suppressed (reducing plasma iron consumption) and macrophage recycling of iron from injured cells is increased.^[4] To prevent the rapid growth of extracellular bacteria that rely on NTBI, hepcidin’s role in host defense is to block NTBI production. However, iron sequestration and hypoferremia caused by inflammation-induced hepcidin also limit the availability of iron for erythropoiesis, resulting in anemia of inflammation (also known as anemia of chronic disease).^[4,49]

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Figure 1:

Hepcidin regulation molecular pathways. Center: Hepcidin is controlled by iron in two different ways. The two transferrin receptors (TFR1 and TFR2) detect extracellular iron in the form of holotransferrin (Fe-Tf). Hemochromatosis protein (HFE) interacts with TFR2 rather than TFR1 when Fe-Tf binds to its receptors. The HFE/TFR2 complex subsequently makes the bone morphogenetic protein (BMP) receptor more sensitive to its ligands, BMP2 and -6 or the BMP2/6 heterodimer. Additionally, by blocking its ubiquitination, HFE may directly stabilize the BMP receptor. The activation of the BMP receptor is enhanced by hemojuvelin (HJV), a membrane-linked BMP coreceptor. BMP receptors trigger SMAD signaling, which increases the transcription of hepcidin, once they are active. Hepatocytes’ BMP receptor is activated as a result of increased intracellular iron in the liver, which also increases the synthesis of BMP6 and -2 by liver sinusoidal endothelial cells. Hepcidin mRNA expression and BMP pathway activation is both reduced in low-iron environments due to low Fe-Tf concentrations and low intracellular iron. The SMAD signaling is further reduced by matriptase-2 (MT-2) protease, which is stable in low-iron environments and inhibits and cleaves HJV and other BMP pathway components. Left: By raising hepcidin transcription via the interleukin (IL)-6JAK-STAT pathway, inflammation promotes the synthesis of hepcidin. Right: When erythropoiesis is activated, erythroblasts produce more erythroferrone. By attaching to BMP2/6 and preventing it from interacting with the BMP receptor, ERFE subsequently suppresses BMP signaling, reducing hepcidin and increasing the amount of iron available for erythropoiesis. By attaching to BMP2/6 and preventing it from interacting with the BMP receptor, ERFE subsequently suppresses BMP signaling, reducing hepcidin and increasing the amount of iron available for erythropoiesis.

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Figure 2:

Hepcidin has a homeostatic role in conditions associated with (a) physiological hepcidin stimulation or (b) suppression and (c) a pathological role in iron-restrictive disorders and (d) iron-overload disorders. Iron flows are shown in shades of blue indicative of intensity. Hepcidin and its effects are shown in red; normal and pathological hepcidin modulators are shown in gold. Fe-Tf: Iron-transferrin, MT-2: Matriptase-2, RBC: Red blood cell.

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Inflammatory in bowel diseases

Iron-deficiency anemia in inflammatory bowel illness has been studied and proposed.^[50] It is believed that iron-deficiency anemia-related chronic fatigue has a major impact, much like diarrhea and stomach pain^[51] and having a major impact on patient-reported outcomes, such as mental health, fatigue, quality of life, cognitive function, and occupational competence.^[52-55] Despite the negative impact iron-deficiency anemia has on the patient’s life and the course of the illness, it appears that half of the cases go untreated.^[50,56,57] Hepcidin levels are elevated in inflammatory bowel disease.^[25]

Inflammation in kidney

Hepcidin concentrations change according to the conditions of blood circulation. In particular, situations with higher hepcidin concentrations, such as sepsis and renal failure, have been seen to exhibit high levels of hepcidin-25.^[9] Hepcidin levels in iron deficiency and iron-deficiency anemia have been shown to be very low, often dropping below the assays’ lower limit of detection. Although both illnesses have high blood hepcidin levels, AKI has lower urinary hepcidin levels, which may assist in differentiating the disorder from chronic renal disease.^[58] Hepcidin inhibitors can be used therapeutically when hepcidin overexpression arises in a disease. The hemojuvelin and BMP6 pathway regulates hepcidin synthesis during inflammatory responses. One potential treatment for anemia linked to CKD is LY3113593, a monoclonal antibody that targets BMP6.^[59] In addition, previous studies have demonstrated that the sTfR/hepcidin ratio is a sensitive mechanism of response to IV iron supply in patients with pre-dialysis CKD.^[60] It is still challenging to make clinical observations on individual hepcidin-25 levels. Hepcidin is produced for a variety of often conflicting reasons.^[36] Serum hepcidin levels roughly correspond to the daily excretion of the normal kidneys.^[61] Significant inter-individual differences in hepcidin circulation levels and, hence, wide reference ranges have been reported in healthy controls.^[33,61-63] Hepcidin levels in the blood can be affected by iron status, anemia, hypoxia, renal insufficiency, and inflammation in a medical condition.^[61,64] Multifactorial control needs additional biochemical and clinical context for accurate hepcidin-25 level determination.^[65] Iron deficiency can manifest as a subtle symptom or illness throughout a spectrum of severity.^[61] Elevated hepcidin levels have been associated with anemia in chronic renal disease, which may be a contributing factor to the illness.^[66,67] Moreover, CKD patients may have inadequate renal clearance of plasma hepcidin as kidney function declines, potentially leading to increased hepcidin levels.^[61] An important part of the pathogenesis of CKD anemia is inflammation, as demonstrated by the positive effects of anti-IL-6 therapy on anemia in hemodialysis-dependent patients, some of whom were able to totally cease taking erythropoiesis-stimulating drugs.^[4] An increase in hepcidin is the source of overall renal inflammation.

Statistical analysis

As a qualitative statistical paper written by a researcher without the use of automated technology, I discovered two key issues about the use of laboratory analysis in various illnesses. The first concern is the expense of analyzing different approaches to identify hepcidin isoform differences, as MS-based testing is more costly. It is possible to distinguish between distinct hepcidin isoforms. Immunoassays frequently lack the specificity of hepcidin-25 and the ability to detect total hepcidin levels. And the second one is about the serum determining levels that have been determined and displayed the variations in the general population and age.