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

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.

 

Papers that cite the Iron and Hem Core

J. Chung et al.  Erythropoietin signaling regulates heme biosynthesis. eLIFE. 2017; 6:e24767.

A.Seguin et al.  Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes.  J. Biol. Chem.  2017; 292(39):16284-16299.

Y. Y. Yien et al. Mutation in human CLPX elevates levels of delta-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria. Proc Natl Acad Sci USA.  2017; 114(39):E8045-E8052.

E. R. Rocha et al. C. Bacteroides fragilis requires the ferrous‐iron transporter FeoAB and the CobN‐like proteins BtuS1 and BtuS2 for assimilation of iron released from heme. MicrobiologyOpen. 2018; e669.

Yien YY et al.  FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.  J Biol Chem. 2018; 293(51):19797-19811.

T. M. Bahr et al. Ferritin in serum and urine: A pilot study. Blood Cells, Molecules, and Diseases. Volume 76, 2019; 76: 59-62.

R. Pek et al. Hemazoin produced by mammals confers heme tolerance. eLIFE.  2019; 8:e49503.

B. Frandsen et al. Exploring the underwater silken architectures of caddisworms: comparative silkomics across two caddisfly suborders. Phil. Trans. R. Soc. B.  2019; 374(1784):20190206.

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.

reproduced with permission of themedicalbiochemistrypage, LLC

Porphyrin Analysis (UPLC)

Porphyrins (excluding protporphyrinogen IX (PPIX) which is assayed together with 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 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: the biosynthetic intermediate porphyrins: uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin.  (Standards are available from Frontier Scientific, Logan, UT.)

Porphyrin Isomer Analysis (UPLC)

Uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, and coproporphyrin of both I and III isomers will be assayed with this selection

We may also assay for d-aminolevulinic acid (ALA), bilirubin, biliverdin and nonstandard porphyrins where a standard is available

 

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), 5 million washed mammalian cells, or 100 uL of washed, packed pellet for yeast or bacteria.  Amount of material required will differ between cell types, please contact us for more information. Please remove all buffer prior to sending us cell pellets. Too much PBS or water can dilute digestion. Please keep packed cell volume to less than 100 uL if you wish to digest directly and normalize to cell number. 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 or PBS and bring us > 1 mL of the water/buffer 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.

Per Sample Pricing Information:

 

We have new pricing (as of October 2020)

Heme, Porphyrin and ALA assays now are billed on a per-run-basis, instead of by sample number. Max number of samples per run is listed for each assay type
As before, we have different prices for CIHD members, who are not charged for tech time (paid for by NIDDK grant)
External users will also have a multiplier (1.525) applied to their price to cover F&A (unless they are using a local P&F Award to pay, so not F&A)
If you wish to quantify intermediate porphyrins with their isomers, that is not its own service in order to flag the request
Porphyrin analysis no longer includes Heme & PPIX. Heme and PPIX are requested as a separate assay
Protein assays are included int eh price for all assays except Metal Analysis, where it is billed separately
Sample digestion for Metal Analysis by ICP-MS is not included in the per-sample part of the listed price (see full description below)
All non-academic laboratories, please contact us for commercial pricing
Please let us know if you have any questions

Iron & Heme Core Assays		CIHD Member Pricing		non-CIHD Pricing
	Max number of samples per run	CIHD per-run set-up fee	CHID per-sample price	non-CHID per-run set-up fee	non-CHID per-sample price
Heme & PPIX Analysis (UPLC)	24	340	n/a	890	n/a
Porphyrin Analysis (UPLC)	24	300	n/a	850	n/a
Porphyrin Isomer Analysis (UPLC)	24	300	n/a	990	n/a
ALA Analysis (UPLC)	12	290	n/a	840	n/a
Spectral Analysis of Hemes (pyridine hemochromagen	12	50	11	1150	11
ALAS Enzyme Assay (5-aminolevuline acid synthase)	12	390	n/a	1490	n/a
FECH Enzyme Assay (ferrochelatase)	12	320	n/a	1420	n/a
Reverse FECH (dechelatase) Enzyme Assay	12	360	n/a	1470	n/a
PPOX Enzyme Assay (protoporphyrinogen oxidase)	12	360	n/a	1460	n/a
ALAD/PBGS Enzyme Assay (aminolevuliniate dehydratase / porphobilinogen synthase)	12	290	n/a	1390	n/a
PBGD/HMBS Enzyme Assay (porphobilinogen deaminase / hydroxymethylbilane synthase	12	290	n/a	1390	n/a
U3S Enzyme Assay (uroporphyrinogen III synthase)	12	290	n/a	1390	n/a
UROD Enzyme Assay (uroporphyrinogen decarboxylase)	12	290	n/a	1390	n/a
CPOX Enzyme Assay (coproporphyrinogen III oxidase)	12	360	n/a	1460	n/a
Metal Analysis by ICP-MS (includes digestion)	40	450	2.2	830	2.2
BCA assay (for metal analysis) (includes digestion)	12	15	n/a	110	n/a
Tissue homogenization (each, using bead tubes)	n/a	n/a	6	n/a	8.3

 

Explanation of Metal Analysis pricing (Agilent 7900 ICP-MS):

The pricing includes a one-time set-up fee (per ICP-MS run) with a per sample cost that covers sample digestion (hot block nitric acid/hydrogen peroxide digestion), protein assay & sample homogenization.
Protein Assay is done by BCA Protein Assay and may be used for normalization purposes.
Tissue samples may be homogenized here using bead tubes.
Please provide at least one mL of a sample-appropriate blank (water or buffer used in the sample preparation to determine background) (>1 mL is better) (no charge) (If tissue is homogenized here, we will provide the blank)
Each ICP-MS run can have up to 40 samples (including their replicates and associated blanks)

Shipping Address:

Iron and Heme Core
26 North Medical Dr, Room 621
Maxwell M. Wintrobe Research & Education Building
Salt Lake City, UT 84132

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.

 

For all assays (except for metal analysis by ICP-MS) please send samples as follows:

Sample type	Minimal required amount (per assay)*	preparation
serum, body fluids	1 mL	flash freeze
cells	>100 uL packed cell pellet	wash with PBS, pellet, remove supernatant, freeze
tissue	100 mg	flash freeze
fresh blood	1 standard collection tube	with anticoagulant, in ice bath, covered in foil
feed, complex hard materials	1 g	dried and ground to fine

 

* Each enzyme assay will require this amount of material.

Please send all frozen samples packed in dry ice for overnight shipping and weekday delivery, after receiving an acknowledgement that we are ready to receive it.

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.

 

 

Amount of material required for ICP-MS:

Tissue pieces	100 mg (20 mg ~ OK)
Mammalian cells	10 million cells
S cerevisiae	3 billion yeast
E. Coli bacteria	50 billion bacteria
mouse blood	1 ul

Note: more material will be required for cells and blood if protein assay is requested for normalization purposes. 

 

Core facility can create the homogenates/lysates for users and measure protein (BCA assay).  Please inform core if will be normalizing to cell number or to protein content.

 

The signal (metal levels detected) will depend on amount of material, cell or tissue type, as well as treatment.  Cells must be washed to remove growth media.

 

One way to understand how much material is needed for an adequate ICP-MS signal is through our on-instrument method detection limit (1.3 ppb for Fe).  The limits of detection and quantification will differ run to run.  We advise aiming for a signal above 20 ppb Fe for quantification, and we suggest providing enough material for 50-500 ppb Fe on-instrument.  Rule of thumb is that there will be an on-instrument (diluted) measurement of 3-20 ppb Fe/100 ug protein for most homogenates or lysates.

 

Below we provide examples of the on-instrument (diluted) signals (ppb Fe) we have seen for various cell lines or tissue homogenates (per million cells or per 100 ug protein lysate), using our sample preparation procedure:

cell type	on-instrument ppb Fe / million cells	on-instrument ppb Fe / 100 ug protein
Hek293	2.0
MEL	4.0	2.6
MEL day 6 2% DMSO	7.7	10.8
K562	13.1	3.0
HEPG2	5.7	2.0
RBC	16.7
RCC4 (huge cells)	50.0
ACHN	2.6
786-0	13.0
red blood cell	>17
mitochodria isolated from liver		20
mitochondria isolated from cultured cells (kit)		5
C elegans homogenate		10
cerebellum homogenate		3.5
liver homogenate		15, 62
kidney homogenate		17, 38
soleus homogenate		5
gastronemus homogenate		1.5

The above values are based on cell counts, protein concentrations and material provided by core facility users.

 

Please contact Hector Bergonia for more information (801)581-6870.

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

ALAS Activity Assay

Blood Hemoglobin Assay (Drabkins)

Colorimetric PBG Assay (Ehrlich)

COPOX Activity Assay

FECH Activity Assay

Hemin PPIX ZnPPIX Assay

ICP-MS Method Description

PBGD Activity Assay

Porphyrinogen via Pd-Carbon

Protoporphyrinogen Oxidase

Pyridine Hemochromogen Protocol

Reverse FECH Activity Assay

U3S Activity Assay

UPLC of Intermediate Porphyrins

UPLC of Poryphrins Isomers

 

UROD Activity Assay

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 Internal (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 hector.bergonia@hsc.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.  hector.bergonia@hsc.utah.edu.  It is a good idea to send us an email 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!

Hours of Operation

Monday through Friday   9AM to 5 PM MST

Location

University of Utah

Maxwell M. Wintrobe Research & Education Building

Iron and Heme Core

26 N Medical Dr Room 621

Salt Lake City, UT  84132

STAFF

 

 

Hector Bergonia, MS

Lab Specialist, Tetrapyrrole and Iron Biochemistry

hector.bergonia@hsc.utah.edu

Phone:  801-581-6870