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Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

Staroń R, Van Swelm RP, Lipiński P, Gajowiak A, Lenartowicz M, Bednarz A, Gajewska M, Pieszka M, Laarakkers CM, Swinkels DW, Starzyński RR - PLoS ONE (2015)

Bottom Line: Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder.Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets.We also found a high correlation between urine hepcidin level and hepatic non-heme iron content.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Animal Breeding PAS, Department of Molecular Biology, Jastrzębiec, Poland.

ABSTRACT
Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

No MeSH data available.


Related in: MedlinePlus

WCX-TOF MS analysis of urine porcine hepcidin.(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [15]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.
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pone.0136695.g001: WCX-TOF MS analysis of urine porcine hepcidin.(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [15]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.

Mentions: Due to its small size (2.7 kDa), hepcidin molecule is in part eliminated from blood by glomerular filtration, but then it is taken up and degraded in the proximal tubule. A small fraction of the filtered hepcidin passes intact into the urine where it is readily detectable. To verify the usefulness of urine hepcidin as a marker of iron status in piglets, we quantified hepcidin-25 in urine samples collected from 28-day old iron-deficient and iron-replete piglets using the previously described method for pig plasma hepcidin measurement (Fig 1). Fig 1A shows the over-time increase of a mass peak of m/z 2749 (which correlates to the m/z of human hepcidin-25), in the urine profiles of iron dextran-treated pigs, but not in that of control animals. Using immunocapture with an anti-hepcidin antibody, we were able to remove this particular mass peak from the urine profile, confirming its identity as pig hepcidin-25 (Fig 1B). The concentration of urinary pig hepcidin-25, corrected for urine creatinine concentration, reflected plasma pig hepcidin-25 concentrations as determined by Spearman rank correlation (Fig 1C).


Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

Staroń R, Van Swelm RP, Lipiński P, Gajowiak A, Lenartowicz M, Bednarz A, Gajewska M, Pieszka M, Laarakkers CM, Swinkels DW, Starzyński RR - PLoS ONE (2015)

WCX-TOF MS analysis of urine porcine hepcidin.(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [15]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556373&req=5

pone.0136695.g001: WCX-TOF MS analysis of urine porcine hepcidin.(A) Urine and plasma pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS) [15]. The spectra (1–4) illustrate the appearance of hepcidin-25 in plasma on day 3, 14, 21 and 28 upon iron dextran injection to piglets on day 3 after birth. Spectra 5 and 6 show hepcidin-25 in the urine of piglets on day 28. Spectrum 5 corresponds to piglet injected with iron dextran on day 3 and spectrum 6 corresponds to non-supplemented, control piglet. IS—internal standard. (B) Immune capture of pig hepcidin-25 and isoform hepcidin-20 in piglet urine. Pig urine was incubated with anti-hepcidin overnight at 4°C, and subsequently hepcidin was measured by WCX-TOF MS. Hepcidin was almost not detectable in the urine sample incubated with anti-hepcidin antibody (upper spectrum); in the spectrum below (pig urine without anti-hepcidin antibody) both a high hepcidin-25 peak and a hepcidin-20 peak (mass 2152 Da) are present. (C) Correlation of pig hepcidin-25 in plasma with pig hepcidin-25 in urine measured with WCX-TOF-MS in piglets injected with iron dextran. Data from 4 measurements of urine and 4 measurements of plasma samples from day 28 were used for the statistical calculations. Spearman correlation r 0.9063, p = 0.0067.
Mentions: Due to its small size (2.7 kDa), hepcidin molecule is in part eliminated from blood by glomerular filtration, but then it is taken up and degraded in the proximal tubule. A small fraction of the filtered hepcidin passes intact into the urine where it is readily detectable. To verify the usefulness of urine hepcidin as a marker of iron status in piglets, we quantified hepcidin-25 in urine samples collected from 28-day old iron-deficient and iron-replete piglets using the previously described method for pig plasma hepcidin measurement (Fig 1). Fig 1A shows the over-time increase of a mass peak of m/z 2749 (which correlates to the m/z of human hepcidin-25), in the urine profiles of iron dextran-treated pigs, but not in that of control animals. Using immunocapture with an anti-hepcidin antibody, we were able to remove this particular mass peak from the urine profile, confirming its identity as pig hepcidin-25 (Fig 1B). The concentration of urinary pig hepcidin-25, corrected for urine creatinine concentration, reflected plasma pig hepcidin-25 concentrations as determined by Spearman rank correlation (Fig 1C).

Bottom Line: Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder.Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets.We also found a high correlation between urine hepcidin level and hepatic non-heme iron content.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Animal Breeding PAS, Department of Molecular Biology, Jastrzębiec, Poland.

ABSTRACT
Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

No MeSH data available.


Related in: MedlinePlus