**2.2.1 Introduction**

Lead produces interferences in the hem biosynthesis pathway (Cambell et al., 1977), inhibiting the enzymatic effects of ALA-Dehydratase (ALA-D, EC 4.2.1.24) in cytosol and coproporphyrinogen oxydase (EC 1.3.3.3) and ferrochelatase (EC 4.99.1.1), both in mitochondria (González-López, 1992).

In the exposed organism, Pb produces affection of the target organs and critical effects which are characteristic of the disease known as saturnism since the days of Ancient Rome. Lead poisoning is diagnosed in the clinic and is shown by the analysis of Pb in blood. However, the concentration of Pb in blood depends on the metabolic condition of the individual (pH of the internal medium, bone activity, etc.) as well as on interactions with

fractions of serum proteins. In contrast, the total serum Zn only presented this correlation with the Zn bonded to the albumin, but not with the Zn bonded to the globulin or the

These findings suggest the existence of some kind of relationship between the two fractions

A further step was taken when wanting to analyse the possible role of albumin in the uptake of Zn by erythrocytes (Gálvez et al., 2001). Zinc is incorporated to erythrocytes by several mechanisms: i) passive transport, ii) anionic exchanger, iii) amino acid transport and iv) especially in the efflux, the Zn2+-Ca2+ exchanger. In accordance with the free-ligand hypothesis only the free fraction could be used for the erythrocyte uptake. The results showed a significantly higher uptake (p<0.01) of Zn in the absence than in the presence of albumin for equimolar concentrations in both cases. However, the uptake of Zn in an albumin-free medium with a similar free-Zn concentration to Zn ultrafiltrable (20%) to another with albumin, a significantly (p<0.01) greater Zn uptake was observed in the latter. The DIDS (4-4´-diidothiocyanatostilbene-2.2´-disulphonic acid), that inhibits an important fraction of the Zn bonded to the anion carrier, also triggered a greater inhibition in the uptake of Zn when the albumin was present. Consequently, it was suggested that albumin must be directly or indirectly involved in Zn capture, facilitating the processes of passive

Other properties of the uptake of Zn by erythrocytes were published in previous reports


These studies are shown as an example of integration of the analysis provided for Pb by FAAS and EAAS with the results of the biomarkers of hem obtained with other laboratory

Lead produces interferences in the hem biosynthesis pathway (Cambell et al., 1977), inhibiting the enzymatic effects of ALA-Dehydratase (ALA-D, EC 4.2.1.24) in cytosol and coproporphyrinogen oxydase (EC 1.3.3.3) and ferrochelatase (EC 4.99.1.1), both in

In the exposed organism, Pb produces affection of the target organs and critical effects which are characteristic of the disease known as saturnism since the days of Ancient Rome. Lead poisoning is diagnosed in the clinic and is shown by the analysis of Pb in blood. However, the concentration of Pb in blood depends on the metabolic condition of the individual (pH of the internal medium, bone activity, etc.) as well as on interactions with



**2.2 Study of the effects of lead in hem biosynthesis** 

concentrations of Zn the value was 15,3 μM (unpublished data).

of the element bonded to proteins, which is probably different for each metal.

albumin concentration.

transport and anionic exchanger.

(Galvez et al., 1992, 1996a, 1996b):

techniques.

**2.2.1 Introduction** 

mitochondria (González-López, 1992).

other metals such as Ca, Fe, Zn, Cu and Mg, among others. In order to ascertain the intensity and degree of affection of the Pb intoxication, it is necessary the study the so-called biomarkers of Pb exposure and poisoning. The most frequently analysed are the enzyme ALA-D in erythrocytes, protoporphyrin IX in erythrocytes, 5-aminolevulinic acid (5-ALA) in urine and the coproporphyrins in urine (Meredith et al., 1979).

The extreme sensitivity of ALA-D to divalent Pb ions has resulted in the measurement of its activity, as an indirect measurement of Pb in human blood (Berlin et al., 1977). Of all the enzymes involved in the hem biosynthesis pathway, it is the one which has been most studied due to the inhibiting effect that Pb has on its activity and the practical importance of the measurement of the enzymatic activity of ALA-D is considered to be of interest as a bioanalytic marker of environmental exposure to Pb. This has been assisted by the development of a method which has been standardised at the proposal of the executive council of the European Union. Hemberg & Nikkanen (1972) have published an extensive report on the biological meaning of ALA-D inhibition and its use as an exposure test.

As well as because of the effect of Pb, the activity of ALA-D may be also reduced by the effect of ethanol in alcoholics and by carbon monoxide in smokers although, in both cases, the reduction of activity is slight. In this line, porphyria of Doss is a recessive autonomous hereditary disease produced by the alteration of the gen which codifies the synthesis of ALA-D located in allele q34 of chromosome 9. It is a rarely presented porphyria, characterised by a deficit of ALA-D. In the homozygote form, a great reduction of ALA-D activity is observed in erythrocytes (2% of the control mean value), while in the heterozygotes, the enzymatic activity of ALA-D is reduced to 50%, being asymptomatic but especially sensitive to the toxic effect of Pb even with scarcely increased levels of Pb. The improvement of environmental and working conditions as well as the use of unleaded petrol, has led to the reduction of the concentration of Pb in blood in the population, a phenomenon observed over the last twenty years (González-López, 1992).

Taking into account the precedent facts, this section will analyse the effects of Pb poisoning in the biomarkers of hem studied in order to evaluate the recovery in the post-treatment with CaNa2EDTA as chelating agent.
