Iron&Heme

Overview

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The Iron and Heme Core provides analysis of metals, precursor porphyrins and heme. The core will measure activity of the enzymes responsible for heme biosynthesis. Analysis and quantification of heme and its precursors can be obtained for cell pellets, tissue, whole blood, urine, feces and other complex biological materials. Analysis of enzyme activity can be provided for cell pellets, tissue and blood.

The Iron and Heme Core is also prepared to assay for Total Iron Binding Capacity (TIBC) in serum, and to assay for human serum ferritin and mouse serum ferritin. Hepcidin will be assayed in the Metabolomics core (our sister CIHD core) by mass spectrometry.

The Iron and Heme Core will collect and distribute reagents from the iron and heme research community, including plasmids for the expression of proteins involved in heme biosynthesis. We expect that these reagents will begin to be available in August. Please watch this website for updates on availability.

Protocols for many of our assays are downloadable from this website in PDF form. Core staff will provide additional assay protocols and consultation upon request.

Referencing Us

Heme/porphyrin/metal analysis was performed at the Iron and Heme Core facility at the University of Utah, supported in part by a grant from the NIH National Institute of Diabetes and Digestive and Kidney Diseases, Grant number U54DK110858.

Services

The Iron and Heme Core’s primary mission is to facilitate research into the role of heme, heme precursors and transition metals in both normal and disease states. The iron and heme core lab has extensive experience with the separation and identification of tetrapyrroles and with running and developing heme biosynthesis pathway enzyme assays. We are offering the following services:

UPLC Analysis of Total Heme and PPIX

A Waters Acquity ultra performance liquid chromatography (UPLC) system equipped with a reverse phase C18 column, a photodiode array detector and a fluorescence detector is used to determine total heme and protoporphyrin IX (PPIX). Samples are subjected to solvent extraction and all noncovalently associated heme and PPIX are transferred into the organic phase and quantified. Hemin chloride (the Fe⁺³ equivalent of heme formed during sample processing) and PPIX are used as standards for quantitation.

While PPIX may be at extremely low to undetectable levels in normal unperturbed biological samples (due to conversion to heme), PPIX and ZnPPIX are detectable (in our standard analysis) in perturbed systems such as in MEL cells induced to differentiate, and in liver samples of mice with ALA in the drinking water. Ferrochelatase is known to catalyze the insertion of other divalent ions, mainly zinc, into protoporphyrin IX, hence the observation of elevated ZnPPIX accompanying PPIX concentration that is raised under certain experimental conditions.

Spectral Analysis of Heme

An Agilent 8453 diode array spectrophotometer can also be used for the spectrophotometric analysis of hemes. Typically, samples are solubilized in 200 mM NaOH in the presence of pyridine to obtain the pyridine-ferrohemochrome derivative spectra. Simultaneous determination of hemes a, b, and o, is possible from the pyridine hemochrome spectra. Each heme species is quantified using the well-established alpha band extinction coefficients at characteristic absorbance maxima obtained from the reduced-minus-oxidized spectra.

3hemes

reproduced with permission of themedicalbiochemistrypage, LLC

Porphyrin Analysis (with heme and other compounds involved in heme metabolism)

Porphyrins and heme from urine, feces, cell lysates, serum, blood, media concentrates from mammalian cells and yeast and other biologic samples are analyzed using a Waters UPLC system. Porphyrins and heme are separated on a reverse-phase C18 column, 2.1x 100 mm BEH, and visualized with the tandem high sensitivity photodiode array and fluorescence detectors of the UPLC.

Molecules analyzed include: heme (as hemin chloride), the precursor porphyrins (uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin) of both I and III isomers, protoporphyrin IX and zinc protoporphyrin IX. We may also assay for d-aminolevulinic acid, bilirubin and biliverdin. (Standards are available from Frontier Scientific, Logan, UT.)

Our typical analysis here will give you heme and PPIX (see UPLC Analysis of Total Heme and PPIX procedure for details) as well as total porphyrins (uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin). We can break this analysis down into individual porphyrins at your request. Analysis for nonstandard porphyrins, bilirubin and biliverdin can also be done if requested at the time of sample submission.

There are instances where additional absorptive or fluorescent peaks are observed with “tetrapyrrole signatures” that do not correspond to the standard molecules listed above. When standards are not available and there is enough sample, fractions can be collected, pooled and analyzed by our sister CIHD core, the Metabolomics Core, at the University of Utah.

Assays of Heme Biosynthetic Enzymes

Activity may be measured for the following enzymes:

ALAS 5-aminolevulinic acid synthase

ALAD/PBGS aminolevulinate dehydratase/porphobilinogen synthase

PBGD porphobilinogen deaminase

U3S uroporphyrinogen III synthase

UROD uroporphyrinogen decarboxylase

COPOX coproporphyrinogen III oxidase

PPOX protoporphyrinogen IX oxidase

FECH ferrochelatase

Protocols for these enzyme assays are available for download through the Shared Resources – Protocols tab in this website [link]

reproduced with permission of themedicalbiochemistrypage, LLC

Analytical Service – Metals analysis ICP-MS

ICP-MS analaysis will be used to determine concentrations for metals of interest such as Fe, Mn, Mg, Zn and Cu in biological samples. If you are interested in assaying for additional metals, please contact us. We expect that our Agilent 7900-ICP mass spectrometer will be installed by late August.

Hepcidin, Transferrin Saturation and Serum Ferritin

The following assays may be conducted using core personnel for academic labs or academic investigators who are not set up to perform these assays themselves. These assays are offered in order to permit investigators to identify the biochemical basis of normal and malregulated iron homeostasis.

Assay – Transferrin Saturation

The levels of iron and total iron-binding capacity in human serum may be measured via a ferene-based colorimetric method using the TECO Diagnostics IRON/TIBC REAGENT SET

Total iron binding capacity (TIBC) will be assayed for serum samples. We will measure iron in serum and unsaturated iron binding capacity (UIBC). TIBC: iron_level + UIBC

Assay – ELISA for Human Serum Ferritin

Ferritin levels in human serum may be quantified using the MP Biomedicals Ferritin Enzyme Immunoassay Test Kit.

Assay – ELISA for Mouse Serum Ferritin

Ferritin levels in mouse serum may be quantified using the Immunology Consultants Laboratory, Inc. Immunoperoxidase Assay for Determination of Ferritin in Mouse Samples.

Measurement for Hepcidin levels may be obtained through our sister CIHD core, the Metabolomics Core [link].

Clone and Antibody distribution

We will be collecting and distributing reagents for investigators researching Iron and Heme disorders, including:

– plasmids for the expression of proteins in heme biosynthesis

– antibodies reactive to heme biosynthesis proteins

– unique strains of bacteria that can be used to purify porphyrin pathway intermediates for use in other experimental systems

For more information please refer to our Plasmids and Reagents for Distribution tab [link]

Additional Information:

Please allow at least two weeks for service completion.

Heme and porphyrin analysis will yield data in picomoles per mg sample protein.

Heme biosynthesis assays will yield data in nanomoles per mg sample protein per hour.

Pricing

Per Sample Pricing Information:

*Some assays include a setup fee in addition to a per sample price

All prices (with the exception of ICP-MS) include triplicate assays/reads.

	CIHD Internal Member Rates (No Tech Time)	CIHD External Member Rates (No Tech Time)	CIHD Internal Non-Member Rates (Includes Tech Time)	CIHD External Non-Member Rates (Includes Tech Time)	Commercial Rates
UPLC analysis of total Heme and PPIX	$8.03	$12.13	$22.71	$34.29	$45.42
Spectral Analysis of Hemes	$12.30	$18.57	$30.65	$46.27	$61.29
Porphyrin Analysis	$13.11	$19.80	$24.85	$37.53	$49.71

Heme Biosynthetic Enzyme Assays:

	CIHD Internal Member Rates (No Tech Time)	CIHD External Member Rates (No Tech Time)	CIHD Internal Non-Member Rates (Includes Tech Time)	CIHD External Non-Member Rates (Includes Tech Time)	Commercial Rates
ALAS (5-aminolevulinic acid synthase)	Set Up Fee: $42.41, Price Per Sample: $17.78	Set Up Fee: $64.04, Price Per Sample: $26.85	Set Up Fee: $42.41, Price Per Sample: $25.12	Set Up Fee: $64.04, Price Per Sample: $37.93	Set Up Fee: $84.82, Price Per Sample: $50.25
ALAD/PBGS (aminolevulinate dehydratase/porphobilinogen synthase)	Set Up Fee: $12.37, Price Per Sample: $11.32	Set Up Fee: $18.68, Price Per Sample: $17.09	Set Up Fee: $12.37, Price Per Sample: $23.42	Set Up Fee: $18.68, Price Per Sample: $35.37	Set Up Fee: $27.74, Price Per Sample: $46.85
PBGD (porphobilinogen deaminase)	Set Up Fee: $12.91, Price Per Sample: $11.15	Set Up Fee: $19.49, Price Per Sample: $16.83	Set Up Fee: $12.91, Price Per Sample: $23.25	Set Up Fee: $19.49, Price Per Sample: $35.11	Set Up Fee: $25.82, Price Per Sample: $46.51
U3S (uroporphyrinogen III synthase)	Set Up Fee: $15.12, Price Per Sample: $12.10	Set Up Fee: $22.84, Price Per Sample: $18.27	Set Up Fee: $15.12, Price Per Sample: $24.20	Set Up Fee: $22.84, Price Per Sample: $36.55	Set Up Fee: $30.25, Price Per Sample: $48.51
UROD (uroporphyrinogen III decarboxylase)	Set Up Fee: $12.57, Price Per Sample: $12.08	Set Up Fee: $18.98, Price Per Sample: $18.24	Set Up Fee: $12.57, Price Per Sample: $24.18	Set Up Fee: $18.98, Price Per Sample: $36.52	Set Up Fee: $25.14, Price Per Sample: $48.37
COPOX (coproporphyrinogen III oxidase)	Set Up Fee: $18.21, Price Per Sample: $14.77	Set Up Fee: $27.50, Price Per Sample: $22.30	Set Up Fee: $18.21, Price Per Sample: $26.87	Set Up Fee: $27.50, Price Per Sample: $40.58	Set Up Fee: $36.43, Price Per Sample: $53.75
FECH (ferrochelatase)	Set Up Fee: $14.61, Price Per Sample: $10.56	Set Up Fee: $22.07, Price Per Sample: $15.94	Set Up Fee: $14.61, Price Per Sample: $25.23	Set Up Fee: $22.07, Price Per Sample: $38.10	Set Up Fee: $29.23, Price Per Sample: $50.46

Other Assays:

The pricing for the ELISAs below reflects the amount of samples that can be run on a plate. If multiple investigators submit samples at the same time, we may be able to reduce the first sample price.

	CIHD Internal Member Rates (No Tech Time)	CIHD External Member Rates (No Tech Time)	CIHD Internal Non-Member Rates (Includes Tech Time)	CIHD External Non-Member Rates (Includes Tech Time)	Commercial Rates
Metal Analysis: ICP-MS	$9.01	$13.61	$19.81	$29.91	$39.62
Transferrin Saturation (Total Iron Binding Capacity of serum)	Set Up Fee: $4.22, Price Per Sample: $7.70	Set Up Fee: $6.37, Price Per Sample: $11.63	Set Up Fee: $4.22, Price Per Sample: $27.43	Set Up Fee: $6.37, Price Per Sample: $41.42	Set Up Fee: $8.44, Price Per Sample: $54.86
ELISA human Ferritin (run in quadruplicate)	Set Up Fee: $112.95, Price Per Sample: $0.63	Set Up Fee: $170.55, Price Per Sample: $0.95	Set Up Fee: $112.95, Price Per Sample: $22.66	Set Up Fee: $170.55, Price Per Sample: $34.22	Set Up Fee: $225.90, Price Per Sample: $45.32
ELISA mouse Ferritin (run in quadruplicate)	Set Up Fee: $33.87, Per Sample Price: $16.94	Set Up Fee: $51.14, Per Sample Price: $25.57	Set Up Fee: $33.87, Per Sample Price: $27.95	Set Up Fee: $51.14, Per Sample Price: $42.20	Set Up Fee: $67.74, Per Sample Price: $55.90
Clone distribution	$3.00	$4.53	$19.44	$29.35	$38.88
Clone distribution (Single sequence confirmation of clone)	$12.00	$18.12	$28.44	$42.94	$56.88

Sample Preparation & Shipping

For most assays please send the samples as follows:

Sample type Form/condition

Serum, body fluids quickly frozen

Cells washed with PBS, pelleted with supernatant removed, and frozen

Tissue flash frozen

Fresh blood with anticoagulant, in ice bath, and covered with foil

Feed and other complex hard materials dried and ground to fine powder

Sample type Minimum amount

Serum*, body fluids 1 mL

Cells >100 µL packed cell pellet

Tissue ~100 mg

Fresh blood 1 standard collection tube

Feed and other complex hard materials 1 g

*For Mouse Ferritin ELISA assays please send us at least 20 uL of serum. For TIBC/Transferrin saturation assay please send 1.5 – 2 ml to allow for replicates.

Please send all frozen samples packed in dry ice for overnight shipping and weekday delivery.

Please inquire for the amount needed for multiple assays per sample.

Results of analyses heme and its precursor compounds shall be reported in nano- or picomoles per mg protein in the sample. Enzyme activity shall be reported as nano- or picomoles product per mg of total sample protein per hour.

Please let us know if you wish to receive leftover sample, after processing and after the assay has been completed. We will require a FedEx number if this is an external user. All samples will be discarded 30 days after results have been sent to the investigator’s lab, unless we receive a return request.

Shared Resources - Protocols

You may find protocols for download in PDF format from this page.

SOP for ICP-MS Sample Preparation

Pyridine Hemochromogen Protocol

Hemin PPIX ZnPPIX Assay

FECH Activity Assay

Porphyrinogen via Pd-Carbon

Colorimetric PBG Assay (Ehrlich)

Iron Assay (ICP-OES)

Blood Hemoglobin Assay (Drabkins)

UPLC of Porphyrins

ALAS Activity Assay

Account Setup

Detailed information for setting up an account and placing an order:

To set up a user account: click on the red “Account Setup” button at the top of the overview page http://cihd.cores.utah.edu/ironheme/ . This will take you to links with a form to fill out. There will be different forms if you are an on-campus user or an off-campus user. When you fill out the form completely, the last line on the page that says “(complete form to submit)” will turn from red to green and then you will be able to submit it (This includes filling out the Acknowledge Legal Responsibility part near the bottom of the form).

Once you submit a work authorization form, your PI will get an Email message with a hyperlink, used to approve or deny the work authorization. There are 72 hours to use that link. If your PI does not approve or deny within 72 hours we will need to set up a new link. After approval, people in the core administration office at the U will enter the authorization into the system and will send you an Email with a username and with password information that will allow you to place your orders.

To place an order: click on the red “Place an Order” button at the top of the overview page http://cihd.cores.utah.edu/ironheme/ . This will take you to a login page. Once you have logged in, you will see a page that will allow you to select for various U of Utah Services. The CIHD specific services are: Iron and Heme (this core), Metabolomics, and Mutation Generation. Click on the appropriate selection. Once you have reached the Services for Iron & Heme page you will be able to select the desired service. There are five different types of buttons for each service that we provide (colored differently). Different types of users pay different rates. The five types are: CIHD Member Internal (U of Utah) academic, CIHD Member External academic, CIHD-NonMember Internal (U of Utah) academic, CIHD-NonMember External academic, and Commercial. Members of the CIHD do not pay tech time. To apply to become a CIHD member, please contact us at iron-and-heme@cores.utah.edu.

Some of our services have a one-time setup fee. For those services, please leave the field in the “one time setup fee” line set to 1, and then in the “number of samples” line, enter the number of samples you are submitting for service. Thank you!

There are tabs on this page with shipping and sample preparation instructions. Please contact us with any questions. L.jackson@cores.utah.edu. It is a good idea to send us a mail when you are about to ship a package to us so we can keep an eye out for it. Also, we are working to update this site so that it can accept sample names. In the meantime, please send an Email to: iron-and-heme@cores.utah.edu with a list of your sample names, the service requested and material type. This will ensure that we can correctly record the name of the sample that you will be sending to our core, to properly name your results. Thank you!