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.

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.

Our Agilent 7900-ICP mass spectrometer is now available for metal analysis. Please check our “Services” tab for more information.

Facility Description

The Iron and Heme Core is part of the NIDDK sponsored, Center for Iron and Heme Disorders. It is also one of the University of Utah shared-resource Health Science Cores facilities, which are established service/recharge centers offering services to research facilities across the country. The Iron and Heme Core provides analysis of metals, precursor porphyrins and heme; all from a variety of biological samples. The core specializes in the UPLC quantification of heme and porphyrin levels, and the measurement of activity of enzymes responsible for heme biosynthesis. This facility is equipped with an Agilent 7900-ICP mass spectrometer for metal analysis. For porphyrin analysis, this facility is equipped with a Waters Acquity ultra-high pressure liquid chromatography (UPLC) system equipped with a reverse-phase C18 column and tandem high sensitivity photodiode array and fluorescence detectors, an HPLC Waters Alliance HT HPLC system, a spectrophotometer, as well as centrifuges, cell disruptors and a variety of miscellaneous small equipment needed to prepare and process samples.

Major Core Equipment:
Agilent 7900-ICP mass spectrometer
Waters Acquity ultra-high pressure liquid chromatography (UPLC) system
HPLC Waters Alliance HT HPLC system
Agilent 8453 diode array spectrophotometer
Dark room
Fume hood
Deoxygenation system

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.

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

An Agilent 7900-ICP mass spectrometer is available for the determination of metals, such as Fe, in biological samples.

For this service, we would like 30-100 mg of sample, sent frozen, and will prepare the sample for metal analysis here. For cells, please wash the pellet, and remove liquid prior to freezing. The media or wash buffer can be a possible source of metal contamination. Please also send us a sample of your wash buffer to test. If you are able to use metal-free or trace metal materials/reagents that is preferable. If you choose to send us homogenates, please prepare the homogenate in pure, metal-free (trace) water, and send a matching control (containing homogenization water).

Please let us know your preferred method of sample normalization. We typically normalize samples to protein concentration after running a BCA protein assay on the homogenate. For tissue, we can also weigh the sample. If you wish the sample to be normalized to cell number, please count the cells prior to sending and let us know how many cells in the pellet.

If you wish to test a metal other than Fe, Mn, Zn, & Cu, please contact us first to discuss.

ICP -MS Useful Information:

Instrument: Agilent 7900 ICP mass spectrometer

Sample digestion: Our core digests biological samples using Nitric Acid, Hydrogen Peroxide and heat (hot block method). We digest small samples in polypropylene tubes, heat them until dry and resuspend in 2% Nitric Acid for ICP-MS analysis. We utilize Agilent certified multi-element calibration standards to quantify your element of interest.

The metals that we most commonly measure are: Fe, Cu, Zn, Mn

Our users also request: Ca, Mg, Ni, K, Rb, P, Co, Cd

We have the following multi-element ICP-MS calibration standard solutions in stock:

Agilent Standard2A: Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn

Agilent Environmental Standard: Ca, Fe, K, Mg, Na, Ag, Al, As, Ba, Be, Se, Th, Tl, U, V, Zn

Agilent Multi-Element Calibration Standard-4: B, Ge, Mo, Nb, P, Re, S, Si, Ta, Ti, W, Zr

We request a minimum of 20 mg of tissue (we prefer at least 30 mg), 4 million washed mammalian cells, or 100 uL of washed, packed pellet for yeast or bacteria. Please send us a sample of your wash buffer to allow us to subtract background. For any type of experiment please send us multiple control samples (buffer) so that we can subtract background. These are free.

We can measure several elements at once (no increase in price if the elements are in the same calibration standard). We do need for you to let us know each time (by Email), at the time that you submit your samples, which elements that you are interested in. The elements of interest must be entered into the software prior to the ICP-MS run for a quantitative measurement.

The instrument also does a qualitative element scan (all elements) in each run. Feel free to ask us for this qualitative data if you have an interest.

We are able to homogenize tissue here using Ceramic Bead Tubes.

We recommend biological replicates for each sample type. Please let us know each time you submit your samples if you want us to quantify protein for you, for normalization purposes. We use a Pierce BCA Protein Assay for protein quantification. We typically lyse the sample for the protein assay as follows (bead tubes and water for tissue, add water and sonicate for cells, add water and freeze-thaw for mitochondria). You can also do this in your own lab (including protein quantification) and bring us the remaining lysed sample. Please only use ultra-pure water and bring us 500 uL of the water that you used in your sample preparation. Iron can be found in just about everything so washes, and choice of buffer can impact signal to noise. Since each experiment is different, we are happy to speak with you to help you to prepare your samples.

If you are shipping the sample, please ship the sample frozen. If you deliver the sample and want protein to be quantified here for you, please bring the sample to us cold or preferably frozen.

Please see pricing tab for information on charge breakdown. We typically use the Optima Nitric Acid in our digests and runs.

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.

For those with an interest, we may be able to run a human Hepcididin assay for you, utilizing an excellent kit from DRG Diagnostics (Hepcidin 25 bioactive HS ELISA). Please contact us for availability.

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: $64.68, Price Per Sample: $27.11	Set Up Fee: $42.41, Price Per Sample: $25.12	Set Up Fee: $64.68, Price Per Sample: $38.31	Set Up Fee: $84.82, Price Per Sample: $50.24
ALAD/PBGS (aminolevulinate dehydratase/porphobilinogen synthase)	Set Up Fee: $12.37, Price Per Sample: $11.32	Set Up Fee: $18.86, Price Per Sample: $17.26	Set Up Fee: $12.37, Price Per Sample: $23.42	Set Up Fee: $18.86, Price Per Sample: $35.72	Set Up Fee: $24.74, Price Per Sample: $46.84
PBGD (porphobilinogen deaminase)	Set Up Fee: $12.91, Price Per Sample: $11.15	Set Up Fee: $19.69, Price Per Sample: $17.00	Set Up Fee: $12.91, Price Per Sample: $23.25	Set Up Fee: $19.69, Price Per Sample: $35.46	Set Up Fee: $25.82, Price Per Sample: $46.50
U3S (uroporphyrinogen III synthase)	Set Up Fee: $15.12, Price Per Sample: $12.10	Set Up Fee: $23.06, Price Per Sample: $18.45	Set Up Fee: $15.12, Price Per Sample: $24.20	Set Up Fee: $23.06, Price Per Sample: $36.91	Set Up Fee: $30.24, Price Per Sample: $48.40
UROD (uroporphyrinogen III decarboxylase)	Set Up Fee: $12.57, Price Per Sample: $12.08	Set Up Fee: $19.17, Price Per Sample: $18.42	Set Up Fee: $12.57, Price Per Sample: $24.18	Set Up Fee: $19.17, Price Per Sample: $36.87	Set Up Fee: $25.14, Price Per Sample: $48.36
COPOX (coproporphyrinogen III oxidase)	Set Up Fee: $18.21, Price Per Sample: $14.77	Set Up Fee: $27.77, Price Per Sample: $22.52	Set Up Fee: $18.21, Price Per Sample: $26.87	Set Up Fee: $27.77, Price Per Sample: $40.98	Set Up Fee: $36.42, Price Per Sample: $53.74
FECH (ferrochelatase)	Set Up Fee: $14.61, Price Per Sample: $10.56	Set Up Fee: $22.28, Price Per Sample: $16.10	Set Up Fee: $14.61, Price Per Sample: $25.23	Set Up Fee: $22.28, Price Per Sample: $38.48	Set Up Fee: $29.22, Price Per Sample: $50.46

Pricing for Metal Analysis (Agilent 7900 ICP-MS):

The pricing includes a one-time set-up fee (per ICP-MS run) and can include a per-sample cost of sample digestion (hot block nitric acid/hydrogen peroxide digestion), protein assay & sample homogenization.
The set up fee is different if Trace Metal Grade (TMG) or OPTIMA nitric acid is used in the run (very small samples should use OPTIMA).
A BCA Protein Assay may be used to assay protein content in lysed samples, to use for normalization purposes.
Tissue samples may be homogenized here using bead tubes.
Each ICP-MS run can have up to 40 samples (including their replicates and associated blanks), and an ICP-MS run can be divided between users in the same run.
These sample fees include up to three digestion replicates per sample, and three blank control replicates.
Please send us a good control for background if your sample is in buffer, or your water (unless it is in a tissue sample that we homogenize).

	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
Set-up fee (TMG)	$166.33	$251.99	$220.33	$333.80	$440.66
Set-up fee (OPTIMA)	$184.47	$279.47	$238.47	$361.28	$476.94
Sample digestion	$3.81	$5.77	$5.81	$8.80	$11.62
Protein Assay	$3.01	$4.56	$4.01	$6.08	$8.02
Sample Homogenization	$1.18	$1.79	$2.68	$4.06	$5.36

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.

Different types of users pay different rates. Members of the CIHD get a discount and do not pay tech time. There is also different pricing for Interna (University of Utah labs), External (non U of Utah academic labs), and Commercial. Please see the Pricing tab for your pricing information. Some of our services have a one-time setup fee. This setup fee will be added to the total per-sample charge. Please feel free to contact us with questions.

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!