Document#:

FOR RESEARCH USE ONLY

Catalog # CFE-005

MC Reagent Biochemical Assay Series

Iron Assay Kit
Ferrozine Chromogenic method

Biochemical Significance and Test Summary
Iron is one of the most important elements, which function as enzyme cofactor. Iron in the blood is bounded withtransferrin and transported throughout the body to synthesize globin proteins such as myoglobin and hemoglobin.
Iron is crucial for synthesis of oxygen-transport protein. Its deficiency causes iron deficiency anemia, chronic
blood loss anemia and infectious anemia.
This product is a colorimetric assay kit without sample deproteinization. Iron bounded with transferrin is
dissociated from proteins in weakly acidic region. Fe(III) is reduced to Fe(II), and blue colored complex withFrrozine [Fe(II)–ferrozine complex] is formed. The concentration of iron in sample is determined by theabsorbance of Fe(II)–ferrozine complex at 560 nm.

1. Kit contents
R-1
R-2
STD

BufferChelate color
200 µg/dL Fe Standard

1 x 20 mL
1 x 0.8 mL
1 x 4.0 mL

Ready to use
Ready to use
Ready to use*Storage conditions: Store at 2-8°C. Don’t freeze.
*Expiration: 1 year. After the vials are opened, the kit should be used in one month.
*Measuring range: 5-1000 µg/dL

2. Materials required but not provided(1) Distilled water(2) Micropipettors and pipette tips
(3) Clear flat-bottom 96-well plate(4) Microplate reader with 560 nm capability

3. Assay preparation(1) Bring all reagents to room temperature before use.
(2) Wash test tube and glassware by 1M HNO3 or 1M HCl, and rinse with distilled water.

4. Sample preparation
Serum/Plasma: Insoluble substances in serum/plasma samples should be removed by filtration orcentrifugation. EDTA-plasma cannot be used.
Urine (24 hour pooled urine)/Tissue homogenate/Cell lysate/Saliva: Add 6M HCl to the sample and adjust
pH 1.5-3.0 (e.g. 5-10 µL of 6M HCl/1 mL of sample). Centrifuge at 6,000 rpm for 15 minutes. Collect thesupernatant and use it for assay.
Tissue: Add 5% TCA solution, voltex for 1 minute and incubate for 30 minutes at 4-8°C. Centrifuge at 6,000 rpm
for 15 minutes. Collect the supernatant and use it for assay.

5. Assay protocol(1) Add 40 µL of distilled water (Blank)/STD (Standards)/samples to each well.
(2) Add 200 µL of R-1 to each well and incubate for 5 minutes at room temperature.
(3) Read the absorbance at 560 nm (540-580 nm). --> OD1(4) Add 8 µL of R-2 to each well and incubate for 5 minutes at room temperature.
(5) Read the absorbance at 560 nm (540-580 nm). --> OD2

6. Calculation
OD = OD2 - OD1
ΔODStandard = ODStandard - ODBlank, ΔODSample = ODSample - ODBlank
Iron (µg/dL) = ΔODSample / ΔODStandard x 200
Iron (µM) = ΔODSample / ΔODStandard x 35.8(Assay example)
DW (Blank)
StandardSample

OD20.030
0.1330.099

OD10.030
0.0290.044

ΔOD (OD2 - OD1)
0.104
0.055

Iron (µg/L)
106

ΔODStandard = (OD2Standard - OD1Standard) - (OD2Blank - OD1Blank) = (0.133 - 0.029) - (0.030 - 0.030) = 0.104
ΔODSample = (OD2Sample - OD1Sample) - (OD2Blank - OD1Blank) = (0.099 - 0.044) - (0.030 - 0.030) = 0.055
IronSample = ΔODSample / ΔODStandard x 200 = 0.055 / 0.104 x 200 = 106 (µg/dL)
Iron (µM) = ΔODSample / ΔODStandard x 35.8 = 0.055 / 0.104 x 35.8 = 19 (µM)

7. Interferences
EDTA inhibits iron to chromogenic system. The test is not affected by presence of bilirubin-F and bilirubin-C up to
40 mg/dL, hemoglobin up to 0.2 g/dL and chyle up to 1,000 FTU.

8. Quality Control
Use of control sera is recommended to monitor the assay quality.

9. References(1) Yamada M, Tanioka K, Kishi K, Hatanaka U, Ohnishi M. An automated method for measurement of serumiron and unsatur- ated iron binding capacity using nitroso-PSAP. Jpn J Lab Auto,13, p659-63 (1988).
(2) Ikeda. Y, Tajima. S, Izawa-Ishizawa. Y, Kihira. Y, Ishizawa. K, Tomita. S, Tsuchiya. K, Tamaki. T: Estrogen
regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes, PLoS One, 7(7)(2012).
(3) Tsugawa. H, Suzuki. H, Matsuzaki. J, Hirata. K, Hibi. T: FecA1, a bacterial iron transporter, determines thesurvival of Helicobacter pylori in the stomach, Free Radic Biol Med. 15, 52(6), p1003-10 (2012).
(4) Nakaya. M, Tajima. M, Kosako. H, Nakaya. T, Hashimoto. A, Watari. K, Nishihara. H, Ohba. M, Komiya. S,Tani. N, Nishida. M, Taniguchi. H, Sato. Y, Matsumoto. M, Tsuda. M, Kuroda. M, Inoue. K, Kurose. H: GRK6
deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance, Nature Commun, 4,p1532 (2013).
(5) Hayashi. K, Nakamura. M, Sakamoto. W, Yogo. T, Miki. H, Ozaki. S, Abe. M, Matsumoto. T, Ishimura. K:
Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging
and hyperthermia treatment, Theranostics. 23, 3(6), p366-76 (2013).

10. Technical support & troubleshooting
Please visit our web site http://www.jaica.com/e/or FAX: +81-538-49-1267(Supported languages: Japanese and English)

Japan Institute for the Control of Aging (JaICA),
Nikken SEIL Co., Ltd.
Haruoka 710-1, Fukuroi, Shizuoka 437-0122, Japan
TEL: +81-538-49-0125 FAX: +81-538-49-1267
www.jaica.com/e/ E-mail: biotech@jaica.com