Determination of Iron, Folic Acid Determination of Iron, Folic Acid and Vitamin B12and Vitamin B12

541CLS-M. Sc program541CLS-M. Sc program

Lab 7Lab 7

Serum IronSerum Iron Iron is largely mediated by three proteins:

transferrin, transferrin receptor and ferritin.Iron is carried in the plasma bound to the

protein transferrin. This molecule binds two atoms of F+3 and delivers iron to cells by interaction with the membrane transferrine receptor.

Normal range for serum iron 10-30 µmol/l Specimen: sufficient blood to provide 3.5ml serum or

heparinized plasma.

Principle of serum iron assay

Transferrin-Fe + trichloracetic acid Fe+transferrin

Fe(III) + thioglycolic Fe(II)

Fe(II) +bathophenanthroline

Bathophenanthrolin-Fe(II) Colored

serum iron (µmol/l)= A test -A blank XCst

A st - A blank

Total iron-binding capacity

ONLY about one-third of the iron-binding sites of transferrin are occupied by Fe (III), serum transferrin has considerable reserve iron-binding capacity. This called the serum unsaturated iron binding capacity (UIBC)

TIBC is a measurement of the maximum concentration of iron that serum proteins, particularly transferrin, can bind.

It increased in iron deficiency and decreased in chronic inflammatory disorders or malignancies.

Excess iron as ferric chloride is added to serum. any iron which dose not bind to transferrin is removed with excess magnesium carbonate.the iron concentration of the iron-saturated serum is then measured.

Normal range for TIBC= 40-75 µmol/lTIBC (µmol/l) =serum iron +UIBC

Serum ferritin The quantity of iron in the storage compartment is

reflected in plasma ferritin concentration. Serum ferritin is a very sensitive indicator of a iron

deficiency. A large number of chronic disease result in increased

in serum ferritin concentration. Can measured by radioimmunoassay (RIA) ,ELISA

kits. Normal range=40-340µg/l in males. Normal range=14-150µg/l in females.

Folic Acid and Vitamin B12

Two types of methods : microbiological assays and competitive protein binding assay

Several commercial kits are available for the competitive protein binding assay (CPB) of vitamin B12 and folic acid.

Most provide for the simultaneous measurement of both substances in same reaction tube by using 125I- labeled folate and 57Co-labeled cobalamin.

Serum specimen are recommended for this procedure also plasma specimen collected in EDTA may also be used (from fasting individuals).

Normal range of serum vitamin B12 = 160-925ng/l.

Normal range of serum folate 3-15µg/l.In folate deficiency usually <1µg/l.

Normal range of red cell folate= 160-640µg/l.

Schiling test Principle: the vitamin B12 absorption test permits

differentiation between pernicious anemia and intestinal malabsorption as causes of vitamin B12 deficiency.

Orally administered 57Co or 58Co-labeled vitamin B12 can be measured by determining the radioactivity in faces, urine, or serum(0.5µg).

In pernicious anemia <7% is excreted in urine. In normal, excrete in urine 8% or more. Also injected intramuscularly with a “flushing dose”

of 1000µg unlabeled vitamin B12 .