Protocol:Zinc-protoporphyrin (ZnPP)

Principle
ZnPP is a metabolite normally produced in trace amounts during heme biosynthesis. In the presence of iron deficiency or impaired iron utilisation, zinc becomes the alternative to iron for ferrochelatase-mediated chelation by protoporphyrin, leading to increased ZnPP formation (21). The substitution of zinc for iron is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes.

Method
ZnPP is a fluorescent compound and haematofluorometry is the fastest and easiest method of determining ZnPP in blood specimens (in any anticoagulant). Dedicated haematofluorometers can measure the ratio of ZnPP fluorescence to haem (haemoglobin absorption) directly in whole blood or in washed erythrocytes. ZnPP is determined in oxygenated blood by illuminating the surface of a glass slide containing a layer of blood with light (wavelength 415nm) and by measuring the emitted fluorescence at 596 nm.

Currently, two haematofluorometers are commercially available (Aviv Associates, Inc., Lakewood NJ, and the ProtoFluor Z, by Helena Laboratories, Beaumont, Tx). The methodology for both instruments is cheap, fast and requires only one drop of whole blood.


 * 1) 50 &mu;l of blood is placed and spread on the 25 x 25 mm2 cover glass.
 * 2) The instrument should be adequately calibrated with 3 control blood of different known protoporphyrins values.

Results
In normal non iron deficient individuals, the ZnPP values are lower than 30 &mu;g/dl whole blood.

In non iron deficient &beta;-thalassaemia carriers, the ZnPP values are lower than 40 &mu;g/dl.

Comments
One advantage of determining ZnPP is that it can be performed from the same sample used for the complete blood count. The ZnPP concentration in a blood sample is stable for several days (unless haemolysis is present). ZnPP is increased also in conditions of relative iron deficiency, when iron is delivered to the marrow at a rate inadequate to meet the requests of increased erythropoiesis. Examples are conditions associated with ineffective erythropoiesis. Conditions of impaired iron utilization, including sideroblastic anaemia and anaemia of chronic disease, lead to increased ZnPP as well. ZnPP is also increased in lead intoxication because of the inhibition of ferrochelatase. Haemolysis and hyperbilirubinaemia may give false increased values.

Despite this low specificity ZnPP determination is a very good screening tool for iron deficiency and is considered a suitable screening test also for lead intoxication. Transferrin saturation should always be determined to confirm the presence of iron deficiency.