**2.2.2 Development: Subjects and methods**

Subjects: The study of the biomarkers of Pb poisoning was performed in 377 adults (30-65 years old) and in 36 healthy children aged between 6 and 14. The adults were distributed as follows: 325 healthy (control group) and 52 cases of Pb poisoning: 24 severe, 15 slight and 13 treated with chelating agents. The children were another control group.

For inclusion in the group of healthy people, control groups, were required to:


The group of the 13 patients treated with chelating agents were integrated as patients with severe poisoning and they were treated with CaNa2-EDTA at doses of 50-75 mg/Kg body weight per day for five days, not exceeding the amount of 500 mg. It may be given an additional set after two days of interruption. This treatment requires hospitalization and

Atomic Absorption Spectroscopy: Fundamentals and Applications in Medicine 13

The method used allows us to obtain a type of chromatogram in which the separation can be observed and the identification performed of the carboxylic acids of free porphyrins, from the octacarboxylic porphyrins (8-COOH, uroporphyrins) followed by the heptacarboxylic porphyrins (7-COOH), hexacarboxylic (6-COOH) and pentacarboxylic (5-COOH) and the tetracarboxylic porphyrins (4-COOH, coproporphyrins). The dicarboxylic form (2-COOH,

As standard the Porphyrin acids chromatographic marker kit is used (Porphyrin products,

Analysis of type I and II isomers of uroporphyrins and coproporphyrins in urine by means of HPLC/FD. According to the disposition the substitutes may adopt around the tetrapyrrol ring of the porphyrin molecule, there are only four possible types of uroporphyrinogens (I, II, III and IV), but in nature only the type I and III uroporphyrinogens exist and,

The use of high pressure liquid chromatography (HPLC) with fluorimetric detection (FD) allows to separate and identify the isomer forms of type I and III uro- and coproporphyrins as metabolytes derived from the oxidation of the corresponding porphyrinogens, produced in the process of natural metaloporphyrin biosynthesis. With this aim, the application

With this method, a chromotagram is obtained in which the separation and identification can be observed of the isomers of uroporphyrins I and III and coproporphyrins I and III

The following standards are used: Uroporphyrin fluorescence standard, Uroporphyrin I (Porphyrin products, INC. UFS-I), Uroporphyrin III octamethyl ester (Sigma), Coproporphyrin I (Sigma), Coproporphyrin fluorescence standard, Coproporphyrin III

The alteration of the enzymatic activity of ferrochelatase due to the inhibition of this enzyme by the effect of Pb produces an increase in the protoporphyrin IX concentration in erythrocytes. This increase of protoporphyrin IX produces and accumulation of Zn-

The method described by Meyer et al. (1980) was used for the analysis of porphyrins in erythrocytes. They developed a procedure for the separation of porphyrins from erythrocytes in blood with HPLC/FD in reverse phase by formation of the ionic pair (Meyer et al., 1982). The standards used were Coproporphyrin fluorescente Standard (Porphyrin products, INC. CFS-3, Logan, UTA, USA), Protoporphyrin fluorescent Standard (Porphyrin products, INC. PFS-9, Logan, UTA, USA) and Mesoporphyrin IX (Porphyrin products, INC. M 566-9,

The first group of values (results not published) were obtained in an early study and show the concentrations of Pb in blood and urine, as well as the values of various biomarkers of

protoporphyrin I due to the complexation formation of this porphyrin with Zn2+.

consequently, decarboxylation will only produce coproporphyrinogens I and III.

method described by Jacob et al. (1985) was used.

Analysis of protoporphyrin-Zn of erythrocytes in blood.

which are eluated and detected in this order.

(Porphyrin products, INC. CFS-3).

Logan, UTA, USA).

**2.2.3 Development: Results** 

protoporfirin) is not excreted in urine (Meyer et al., 1980a; Meyer, 1985).

INC., CMK-IA).

clinical management with special attention to controlling renal function due to its nephrotoxicity.

Methods: The parameters analyzed were: Pb in blood and urine and the biomarkers of hem characteristic of Pb poisoning, ALA-D and protoporphyrin IX in blood and 5-ALA and coproporphyrins in urine.

The methods used were as follows:

Lead was analyzed in heparinized whole blood and 24-hour urine collection in container without additives. The first results of Pb were performed in both blood and urine by FAAS, using a modification of the method of Hessel (1968) by extraction into n-butyl acetate of the complex formed by the Pb with dithiocarbamate ammonium pyrrolidine (Pb-APDC).

Subsequently, the latest determinations were obtained by EAAS with Zeeman correction spectrometry, given the recent introduction of this technique to the laboratory. Blood was used diluted to 0.2% nitric acid (Pearson & Slavin, 1993).

Biomarkers: ALA-D of erythrocytes (in total blood with heparine) was determined applying the method of the European Standards Committee (Sibar Diagnostici) (Berlin & Schaller, 1974; Schaller & Berlin, 1984).

Erythrocyte-free Protoporphyrin IX (in total blood with heparine) was performed applying the method of Piomelli (1977), (Sibar Diagnostici).

At present, both products are manufactured by Immuno Pharmacology Research (IPR) Diagnostics.

ALA/PBG in urine was determined by means of column chromatography. For analysis a modification of the method of Mauzerall & Granick (1956), manufactured by Bio-Rad, was applied.

Determination of porhyrins in urine was performed after the separation of uro- and coproporphyrins by means of ethyl acetate which extracts the coproporphyrins from the aqueous phase and later absorption of uroporphyrins from the aqueous phase with activated Al2O3. Finally, the uroporphyrins are extracted from the Al2O3 activated and the coproporphyrins from the ethyl acetate by means of HCl 1.5 N, performing a fluorimetric reading of the extracts obtained (Schwartz et al., 1960).

In cases of the increased excretion of porphyrins, it is important to analyze the porphyrin biosynthesis pathway, applying the following analytic methods:

Analysis of carboxylic acids of free porphyrins in urine, by means of HPLC/FD. The chemical structure of the porphyrins presents the natural property of being fluorescent compounds, which makes them detectable by spectrofluorimetry.

The application of high pressure liquid chromatography (HPLC), constitutes a valuable resource in research applied to the study of the porphyrin biosynthesis pathway.

The analysis of the carboxylic acids of free porphyrins does not require any prior treatment of the samples (urine or faeces) to be chromatographed. They are only passed through a 22 μm Millex-GS (Millipore) filter. If the quantity of porphyrins contained in the sample is very high, it will be diluted with the eluyent.

clinical management with special attention to controlling renal function due to its

Methods: The parameters analyzed were: Pb in blood and urine and the biomarkers of hem characteristic of Pb poisoning, ALA-D and protoporphyrin IX in blood and 5-ALA and

Lead was analyzed in heparinized whole blood and 24-hour urine collection in container without additives. The first results of Pb were performed in both blood and urine by FAAS, using a modification of the method of Hessel (1968) by extraction into n-butyl acetate of the complex formed by the Pb with dithiocarbamate ammonium pyrrolidine (Pb-APDC).

Subsequently, the latest determinations were obtained by EAAS with Zeeman correction spectrometry, given the recent introduction of this technique to the laboratory. Blood was

Biomarkers: ALA-D of erythrocytes (in total blood with heparine) was determined applying the method of the European Standards Committee (Sibar Diagnostici) (Berlin & Schaller,

Erythrocyte-free Protoporphyrin IX (in total blood with heparine) was performed applying

At present, both products are manufactured by Immuno Pharmacology Research (IPR)

ALA/PBG in urine was determined by means of column chromatography. For analysis a modification of the method of Mauzerall & Granick (1956), manufactured by Bio-Rad, was

Determination of porhyrins in urine was performed after the separation of uro- and coproporphyrins by means of ethyl acetate which extracts the coproporphyrins from the aqueous phase and later absorption of uroporphyrins from the aqueous phase with activated Al2O3. Finally, the uroporphyrins are extracted from the Al2O3 activated and the coproporphyrins from the ethyl acetate by means of HCl 1.5 N, performing a fluorimetric

In cases of the increased excretion of porphyrins, it is important to analyze the porphyrin

Analysis of carboxylic acids of free porphyrins in urine, by means of HPLC/FD. The chemical structure of the porphyrins presents the natural property of being fluorescent

The application of high pressure liquid chromatography (HPLC), constitutes a valuable

The analysis of the carboxylic acids of free porphyrins does not require any prior treatment of the samples (urine or faeces) to be chromatographed. They are only passed through a 22 μm Millex-GS (Millipore) filter. If the quantity of porphyrins contained in the sample is very

resource in research applied to the study of the porphyrin biosynthesis pathway.

nephrotoxicity.

coproporphyrins in urine.

1974; Schaller & Berlin, 1984).

Diagnostics.

applied.

The methods used were as follows:

used diluted to 0.2% nitric acid (Pearson & Slavin, 1993).

the method of Piomelli (1977), (Sibar Diagnostici).

reading of the extracts obtained (Schwartz et al., 1960).

high, it will be diluted with the eluyent.

biosynthesis pathway, applying the following analytic methods:

compounds, which makes them detectable by spectrofluorimetry.

The method used allows us to obtain a type of chromatogram in which the separation can be observed and the identification performed of the carboxylic acids of free porphyrins, from the octacarboxylic porphyrins (8-COOH, uroporphyrins) followed by the heptacarboxylic porphyrins (7-COOH), hexacarboxylic (6-COOH) and pentacarboxylic (5-COOH) and the tetracarboxylic porphyrins (4-COOH, coproporphyrins). The dicarboxylic form (2-COOH, protoporfirin) is not excreted in urine (Meyer et al., 1980a; Meyer, 1985).

As standard the Porphyrin acids chromatographic marker kit is used (Porphyrin products, INC., CMK-IA).

Analysis of type I and II isomers of uroporphyrins and coproporphyrins in urine by means of HPLC/FD. According to the disposition the substitutes may adopt around the tetrapyrrol ring of the porphyrin molecule, there are only four possible types of uroporphyrinogens (I, II, III and IV), but in nature only the type I and III uroporphyrinogens exist and, consequently, decarboxylation will only produce coproporphyrinogens I and III.

The use of high pressure liquid chromatography (HPLC) with fluorimetric detection (FD) allows to separate and identify the isomer forms of type I and III uro- and coproporphyrins as metabolytes derived from the oxidation of the corresponding porphyrinogens, produced in the process of natural metaloporphyrin biosynthesis. With this aim, the application method described by Jacob et al. (1985) was used.

With this method, a chromotagram is obtained in which the separation and identification can be observed of the isomers of uroporphyrins I and III and coproporphyrins I and III which are eluated and detected in this order.

The following standards are used: Uroporphyrin fluorescence standard, Uroporphyrin I (Porphyrin products, INC. UFS-I), Uroporphyrin III octamethyl ester (Sigma), Coproporphyrin I (Sigma), Coproporphyrin fluorescence standard, Coproporphyrin III (Porphyrin products, INC. CFS-3).

Analysis of protoporphyrin-Zn of erythrocytes in blood.

The alteration of the enzymatic activity of ferrochelatase due to the inhibition of this enzyme by the effect of Pb produces an increase in the protoporphyrin IX concentration in erythrocytes. This increase of protoporphyrin IX produces and accumulation of Znprotoporphyrin I due to the complexation formation of this porphyrin with Zn2+.

The method described by Meyer et al. (1980) was used for the analysis of porphyrins in erythrocytes. They developed a procedure for the separation of porphyrins from erythrocytes in blood with HPLC/FD in reverse phase by formation of the ionic pair (Meyer et al., 1982).

The standards used were Coproporphyrin fluorescente Standard (Porphyrin products, INC. CFS-3, Logan, UTA, USA), Protoporphyrin fluorescent Standard (Porphyrin products, INC. PFS-9, Logan, UTA, USA) and Mesoporphyrin IX (Porphyrin products, INC. M 566-9, Logan, UTA, USA).
