The diagnosis of iron deficiency

iron determination assays
Iron is essential for a list of biological processes in almost every living organism. Enzymes, such as nitrogenase and hydrogenase, and some coenzymes require iron for their proper functioning. Iron is also the main ingredient of hemoglobin – the oxygen-carrying molecule in the blood, and is needed for red blood cell formation. In other words, blood owes its red color to iron. Because free iron is toxic to the cells, iron circulates within the body bound to the protein transferrin and accumulates within the cells in a protein complex as ferritin. The primary storage place of iron is the liver and the normal serum iron levels in the human body range between 10 and 40 µM.
Low serum iron levels are characteristic for hemorrhagic and hypochromic anemia, while pernicious anemia is associated with high serum iron values. Hepatic and renal disease, lead poisoning and blood transfusion can also cause higher iron levels. Therefore, monitoring the blood iron level is important for the differential diagnosis of many disorders, including anemias.

The determination of the iron concentration of a blood sample is straightforward. In the presence of acid buffer, transferrin dissociates ferric ions (Fe3+) into solution. Ferric ions are then reduced to ferrous ions (Fe2+) that react with a chromogenic substance to form a colored complex. The complex absorbs light at a certain wavelength and absorbance is directly proportional to the serum iron concentration.

A serum iron test is usually accompanied by a total iron binding capacity (TIBC) test. TIBC is the maximum concentration of iron that serum proteins can bind and is determined indirectly through unsaturated iron binding capacity (UIBC). UIBC is measured by incubating serum with a known ferrous standard - the iron saturates all the transferrin available binding sites and the excess iron is measured with a chromogenic substance.

   UIBC = total iron added – exess iron  

   TIBC = UIBC + serum iron

Reagents used to determine the iron concentration

Bathophenanthroline used to be the standard chromogenic compound for iron detection. With the advancement of science a list of chromogenic compounds with better characteristics were discovered. Sulfonation of Bathophenanthroline produces a water-soluble derivative which retains the chromogenic characteristics of its parent compound. Next, phenyl-substituted triazines were synthesized. Those compounds exhibit chromophoric enhancement, probably due to the phenyl groups favoring the charge transfer and intensifying the color of the iron(II) complex.

B-0400_Bathophenanthroline
  Bathophenanthroline

Ferene S

Similar chromophoric enhancement is observed in a molecule containing two furyl groups - 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5',5''-sulfonic acid. Its disodium salt, better known as Ferene S, is a highly sensitive, water-soluble colorimetric reagent for iron. The iron - ferene complex is a stable, water-soluble, blue-colored complex. The intense blue of the complex fully develops in 3 minutes at 25° C and is stable over a pH range of 2 - 7.5 in acetate buffer. The complex absorbs light at 593 nm exhibiting a single sharp peak.

Ferene-S_method
Interference studies have shown that apart from iron, the only other metal found in serum which forms a colored complex with Ferene S is copper. Copper interference with Ferene S is similar to this with Ferrozine and can be almost entirely eliminated by the use of thiourea or thioglycolic acid, which preferentially bind to copper.

Until today, Ferene S is one of the most sensitive colorimetric reagents for iron(II) - it is 25% more sensitive than Ferrozine and 50% more sensitive than Bathophenanthroline sulfonate. Moreover, both Ferene S and the iron-ferene complex are water-soluble, which makes Ferene S perfect for automated analyses.
Since 1990, Ferene S is the recommended chromogenic substance for iron detection by the International Committee for Standardisation in Haematology.

The history of iron detection

Symptoms of iron deficiency were described way back in the 16th century as a disease called chlorosis. Along with epilepsy and paranoia, chlorosis was classified as a ‘hysterical disease’ - a disease with no physical explanation, yet characterized by severe symptoms such as painful cramps or paralysis. What we now diagnose as hypochromic anemia was once thought to be the result of a nervous disorder. Other hypotheses were that the disease is caused by reabsorption of menstrual blood, wearing corsets, bad morals or increased libido. Suggestions to treat chlorosis with iron supplements were neglected and incidence of the disease increased by the end of the 19th century. Although chlorosis ceased to be reported in the 1930s people were treated inappropriately for centuries, due to a lack of methods for iron detection and improper diagnosis in the past.

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Ferene S (Cat.No. P-8300)

CAS No. [79551-14-7]

Ferene_S_P-8300


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