**2.2.3 Development: Results**

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

Atomic Absorption Spectroscopy: Fundamentals and Applications in Medicine 15

Men (n = 223) 2.96 ± 1.30 0.41 ± 0.42 Women (n = 102) 2.42 ± 1.17 0.35 ± 0.35 Adults (n = 325) 2.79 ± 1.28 0.39 ± 0.40 Children (n = 36) 2.84 ± 0.91 0.46 ± 0.43

Men (n = 223) 11.09 ± 6.00 101.95 ± 55.46 Women (n = 102) 9.08 ± 4.95 64.34 ± 35.07 Adults (n = 325) 10.46 ± 5.76 90.15 ± 52.88 Children (n = 36) 4.18 ± 2.98 56.59 ± 34.03

Table 5. Normal values of uroporphyrins and coproporphyrins in 24 hours urine

provide a more complete picture of potential changes in Pb poisoning.

hours) of Pb, 5-ALA, PBG, uroporphyrins and coproporphyrins.

The values of Pb in blood and urine (Table 1) for the different population groups studied are within the range of those reported by other authors of the time the study was conducted (Carton, 1985; Carton, 1988). Because of that environmental improvements and labor have reduced Pb concentrations in the environment, the serum concentration of Pb have been also

With regard to the values of the biomarkers of Pb poisoning in blood analyzed (Tables 2, 3, 4 and 5), all of them are within the range reported by other authors (Goldberg, 1972; Tomokuni, K. & Ogata, 1976; Campbell et al., 1977; Goldberg et al. 1978; Granick et al., 1978; Meredith et al. 1979; Sakai et al., 1982; Barbosa et al., 2005). Although the standard deviation values can be considered high for some parameters, this should not be attributed to the methodology used given the biological variability that is observed in the study population. There have also been included the values for Hb in the blood and the ratio of protoporphyrin IX/g of Hb. This last value increases in the Pb poisoning (protoporphyrin increased and decreased hemoglobin) while in the protoporphyria the Hb did not decrease and consequently the ratio does not increase as much as in the Pb poisoning. Likewise the values of 5-ALA, PBG and porphyrins (uro-and copro-) are included in the study as they

**I.B Pb poisoning.** In table 6 and 7 are presented the statistical tests of comparison of means observed in large samples with independent data and their degree of significance, performed for each biomarker of Pb poisoning analysed in each group studied with respect to the control group. Specifically, in table 6 are presented the results in blood of Pb, ALA-D, protoporphyrine IX and Protoporphyrine IX/g Hb and in table 7 the results in urine (24

Table 4. Normal values of 5-ALA and PBG in urine of 24 hours

reduced (Trasobares, 2010).

**5-ALA PBG mg/24 hours mg/24 hours x ± SD x ± SD** 

**Uroporphyrins Coproporphyrins μg/24 hours μg/24 hours x ± SD x ± SD** 

the porphyrin biosynthesis pathway. Moreover, it was also included the results of a second study from some years later, presenting the normality values of Pb in blood as an update, observing the difference of Pb concentration with respect to the earlier study.

**I.A Control group.** The results of the control group for Pb in blood and in 24 hours urine are shown in Table I. In Table 2 are presented the values of ALA-D and protoporphyrin IX in erythrocytes for the same population and in Table III those of the hemoglobin concentration and protoporphirin IX/g of haemoglobin coefficient. In table 4 are indicated the values of 5- ALA and porphobilinogen (PBG) in 24 hours urine and in the last (table 5) are presented the results found in urine of 24h of uroporphyrins and coproporphyrins.


Table 1. Values (x ± SD) of Pb in blood and in 24 hours urine


(\*): Units of the European Standards Committee

Table 2. Values (x ± SD) of ALA-D and protoporphyrine IX in erythrocytes


Table 3. Values of Hb in blood and protoporphyrin IX/g of Hb coefficient

the porphyrin biosynthesis pathway. Moreover, it was also included the results of a second study from some years later, presenting the normality values of Pb in blood as an update,

**I.A Control group.** The results of the control group for Pb in blood and in 24 hours urine are shown in Table I. In Table 2 are presented the values of ALA-D and protoporphyrin IX in erythrocytes for the same population and in Table III those of the hemoglobin concentration and protoporphirin IX/g of haemoglobin coefficient. In table 4 are indicated the values of 5- ALA and porphobilinogen (PBG) in 24 hours urine and in the last (table 5) are presented the

> **Pb in blood Pb in 24 hours urine µg/dL µg/dL x ± SD x ± SD**

**ALA-D Protoporphyrin IX** 

**U. of CEE/mL erythrocytes\* µg/dL of blood** 

**x ± SD x ± SD** 

**Hemoglobin Protoporphyrin IX g/dL of blood µg/g Hb x ± SD x ± SD** 

observing the difference of Pb concentration with respect to the earlier study.

Men (n = 104) 17.72 ± 6.01 45.10 ± 39.85 Women (n = 61) 14.00 ± 3.86 35.78 ± 27.26 Adults (n = 165) 16.54 ± 5.49 40.96 ± 35.01 Children (n = 36) 14.58 ± 2.79 -

Men (n = 104) 44.70 ± 13.94 28.92 ± 11.50 Women (n = 61) 50.41 ± 17.27 26.80 ± 9.66 Adults (n = 165) 46.81 ± 15.45 28.13 ± 10.87 Children (n = 36) 64.42 ± 13.39 28.53 ± 9.28

Table 2. Values (x ± SD) of ALA-D and protoporphyrine IX in erythrocytes

Table 3. Values of Hb in blood and protoporphyrin IX/g of Hb coefficient

Men (n = 104) 15.27 ± 1.54 1.92 ± 0.84 Women (n = 61) 13.39 ± 1.16 2.02 ± 0.78 Adults (n = 165) 14.57 ± 1.68 1.96 ± 0.82 Children (n = 36) 12.94 ± 0.71 2.19 ± 0.65

results found in urine of 24h of uroporphyrins and coproporphyrins.

Table 1. Values (x ± SD) of Pb in blood and in 24 hours urine

(\*): Units of the European Standards Committee


Table 4. Normal values of 5-ALA and PBG in urine of 24 hours


Table 5. Normal values of uroporphyrins and coproporphyrins in 24 hours urine

The values of Pb in blood and urine (Table 1) for the different population groups studied are within the range of those reported by other authors of the time the study was conducted (Carton, 1985; Carton, 1988). Because of that environmental improvements and labor have reduced Pb concentrations in the environment, the serum concentration of Pb have been also reduced (Trasobares, 2010).

With regard to the values of the biomarkers of Pb poisoning in blood analyzed (Tables 2, 3, 4 and 5), all of them are within the range reported by other authors (Goldberg, 1972; Tomokuni, K. & Ogata, 1976; Campbell et al., 1977; Goldberg et al. 1978; Granick et al., 1978; Meredith et al. 1979; Sakai et al., 1982; Barbosa et al., 2005). Although the standard deviation values can be considered high for some parameters, this should not be attributed to the methodology used given the biological variability that is observed in the study population.

There have also been included the values for Hb in the blood and the ratio of protoporphyrin IX/g of Hb. This last value increases in the Pb poisoning (protoporphyrin increased and decreased hemoglobin) while in the protoporphyria the Hb did not decrease and consequently the ratio does not increase as much as in the Pb poisoning. Likewise the values of 5-ALA, PBG and porphyrins (uro-and copro-) are included in the study as they provide a more complete picture of potential changes in Pb poisoning.

**I.B Pb poisoning.** In table 6 and 7 are presented the statistical tests of comparison of means observed in large samples with independent data and their degree of significance, performed for each biomarker of Pb poisoning analysed in each group studied with respect to the control group. Specifically, in table 6 are presented the results in blood of Pb, ALA-D, protoporphyrine IX and Protoporphyrine IX/g Hb and in table 7 the results in urine (24 hours) of Pb, 5-ALA, PBG, uroporphyrins and coproporphyrins.

Atomic Absorption Spectroscopy: Fundamentals and Applications in Medicine 17

The identification and quantification of high levels of porphyrins in erythrocytes, mainly protoporphyrin IX and its chelated form, Zn-protoporphyrin, are essentials in the diagnosis

The results of the above table present significant differences in the Pb urinary elimination in Pb poisoning both severe as slight in relation to the control group. Moreover, increases in the elimination of all parameters (biomarkers) analyzed in severe poisoning, being the most important those of the 5-ALA and coproporphyrins. In contrast, in slight poisoning has only significantly increased the elimination of 5-ALA. After treatment of severe cases there is a significant decrease in excretion of 5-ALA and coproporphyrins, keeping the levels still increased. Of the above comments it can be seen that the most effective urinary biomarkers

These results agree, in the literature review performed, with the studies published by numerous authors and refered in the Doctoral Thesis of González-López (1992) and others (Sakai, 2000; Gurer-Orhan et al., 2004). All of them showed the influence of Pb on the heme biosynthesis pathway and the effects produced in some of the enzymes which take part in the biosynthesis of porphyrins, phenomena demonstrated in clinical research, *in vitro*

In the diagnosis and evolutionary control of Pb poisoning, the study of the biomarkers of the hem biosynthesis pathway is very efficient, even more so than that demonstrated by the concentration of Pb in blood and urine due to the susceptibility of these to hormonal influences as well as metabolic ones such as the pH of the internal medium and the activity of bone turnover. According to this, Pb in blood means the degree of uptake; Pb in urine, the degree of elimination; Pb in urine provoked by EDTA or some other chelating agent, the degree of accumulation; ALA-D, the degree of exposure and is directly related to Pb in blood; erythrocyte free protoporphyrin IX, especially Zn-protoporphyrin IX, the degree of intake and chronic evolution; and the coproporphyrins (coproporphyrin III) the severity of

**II.** In the second study was performed a review of the values of Pb in blood in the present. The values were obtained by EASS in a graphite furnace with correction of the Zeeman

> **Pb in blood μg/dL x ± SD**

effect, in a sample of 156 individuals. The results obtained are shown in Table 8.

Table 8. Normal values (x ± SD) of healthy individuals of Pb concentration in blood

These results indicate a decrease of the concentration obtained in 1989 which was 13.17 ± 3.47 μg/dL, attributed to the improvement of environmental and working conditions as well as to the suppression of Pb as antiknock agent in petrol (Izquierdo-Álvarez et al., 1985;

Men (n = 83) 3.51 ± 2.16 Women (n = 73) 2.29 ± 1.64 Adults (men + women) (n = 156) 2.94 ± 2.02

of Pb poisoning and in erythropoietic porphyrias (Meyer et al., 1980b).

are the urinary elimination of 5-ALA and coproporphyrins (type isomeric III).

studies and in experiments on animals.

the poisoning.

Trasobares, 2010).


Control group: 14.57±1.68;

Severe poisoning: 11.46±1.98b Slight poisoning: 13.59±1.46a;

Post treatment: 14.12±1.17

Degree of statistical significance: a (p < 0.050); b (p < 0.001);

Table 6. Values (x ± SD) of Pb, ALA-D, protoporphyrine IX and Protoporphyrine IX/g Hb in blood of the indicated group

The above table shows that in both, severe and slight Pb poisoning, the increases in the concentration of blood Pb are associated with significant inhibition of ALA-D activity (Campbell et al., 1977; Goldberg et al., 1978; Sakai et al., 1982) and with significant increases of protoporphyrin IX concentrations (Goldberg et al., 1978; Meredith et al., 1979). After treatment the values still remain significantly altered, indicating that the patients need a new series of treatment, because of the guidelines therapy is performed during five days with breaks in which they carry out checks on biomarkers. The fact of treating patients with different series of treatment would explain the dispersion of the results.


Degree of statistical significance: a (p<0.002); b (p<0.001).

Table 7. Values (x ±SD) of Pb, 5-ALA, PBG, uroporphyrins and coproporphyrins in 24 hours urine

**Pb ALA-D Protoporphyrine IX Protop. IX/Hb μg/dL U/mL μg/dL μg/g Hb x ± SD x ± SD x ± SD x ± SD** 

16.54 ± 5.49 46.81 ± 15.45 28.13 ± 10.87 1.96 ± 0.82

 **BLOOD** 

Severe pois. 101.04±58.03b 12.00 ± 5.56b 198.49 ± 89.51b 17.24 ± 8.74b Slight pois. 41.60±15.86b 26.47 ± 20.50b 26.57 ± 92.15b 9.65 ± 7.35b Post Treatment 40.92±18.74b 37.08 ± 14.20a 116.94 ± 106.21b 8.70 ± 8.26b

Table 6. Values (x ± SD) of Pb, ALA-D, protoporphyrine IX and Protoporphyrine IX/g Hb in

The above table shows that in both, severe and slight Pb poisoning, the increases in the concentration of blood Pb are associated with significant inhibition of ALA-D activity (Campbell et al., 1977; Goldberg et al., 1978; Sakai et al., 1982) and with significant increases of protoporphyrin IX concentrations (Goldberg et al., 1978; Meredith et al., 1979). After treatment the values still remain significantly altered, indicating that the patients need a new series of treatment, because of the guidelines therapy is performed during five days with breaks in which they carry out checks on biomarkers. The fact of treating patients with

**Pb 5-ALA PBG UroporphyrinsCoproporphyrins** 

**μg/24h mg/24h mg/24h μg/24h μg/24h x ±SD x ±SD x ±SD x ±SD x ±SD** 

41±35 2.79±1.28 0.39±0.60 10.46±5.76 90.15±52.88

Severe pois. 361±189b 47.3±21.8b 2.21±1.84a 26.4±18.8b 1612.9±682.8b

Post treatment 197±167b 5.43±3.52b 0.42±0.62 9.67±6.50 148.9±119.1b

Table 7. Values (x ±SD) of Pb, 5-ALA, PBG, uroporphyrins and coproporphyrins in 24 hours

Slight pois. 114±81b 4.89±1.96b 0.49±0.62 5.61±2.25 87.7±54.2

different series of treatment would explain the dispersion of the results.

 **URINE** 

Concentration of Hb (g/dL blood) in each group studied:

Degree of statistical significance: a (p < 0.050); b (p < 0.001);

Degree of statistical significance: a (p<0.002); b (p<0.001).

Control group (n = 165)

Control group: 14.57±1.68; Severe poisoning: 11.46±1.98b Slight poisoning: 13.59±1.46a; Post treatment: 14.12±1.17

blood of the indicated group

Control group (n = 325)

urine

The identification and quantification of high levels of porphyrins in erythrocytes, mainly protoporphyrin IX and its chelated form, Zn-protoporphyrin, are essentials in the diagnosis of Pb poisoning and in erythropoietic porphyrias (Meyer et al., 1980b).

The results of the above table present significant differences in the Pb urinary elimination in Pb poisoning both severe as slight in relation to the control group. Moreover, increases in the elimination of all parameters (biomarkers) analyzed in severe poisoning, being the most important those of the 5-ALA and coproporphyrins. In contrast, in slight poisoning has only significantly increased the elimination of 5-ALA. After treatment of severe cases there is a significant decrease in excretion of 5-ALA and coproporphyrins, keeping the levels still increased. Of the above comments it can be seen that the most effective urinary biomarkers are the urinary elimination of 5-ALA and coproporphyrins (type isomeric III).

These results agree, in the literature review performed, with the studies published by numerous authors and refered in the Doctoral Thesis of González-López (1992) and others (Sakai, 2000; Gurer-Orhan et al., 2004). All of them showed the influence of Pb on the heme biosynthesis pathway and the effects produced in some of the enzymes which take part in the biosynthesis of porphyrins, phenomena demonstrated in clinical research, *in vitro* studies and in experiments on animals.

In the diagnosis and evolutionary control of Pb poisoning, the study of the biomarkers of the hem biosynthesis pathway is very efficient, even more so than that demonstrated by the concentration of Pb in blood and urine due to the susceptibility of these to hormonal influences as well as metabolic ones such as the pH of the internal medium and the activity of bone turnover. According to this, Pb in blood means the degree of uptake; Pb in urine, the degree of elimination; Pb in urine provoked by EDTA or some other chelating agent, the degree of accumulation; ALA-D, the degree of exposure and is directly related to Pb in blood; erythrocyte free protoporphyrin IX, especially Zn-protoporphyrin IX, the degree of intake and chronic evolution; and the coproporphyrins (coproporphyrin III) the severity of the poisoning.

**II.** In the second study was performed a review of the values of Pb in blood in the present. The values were obtained by EASS in a graphite furnace with correction of the Zeeman effect, in a sample of 156 individuals. The results obtained are shown in Table 8.


Table 8. Normal values (x ± SD) of healthy individuals of Pb concentration in blood

These results indicate a decrease of the concentration obtained in 1989 which was 13.17 ± 3.47 μg/dL, attributed to the improvement of environmental and working conditions as well as to the suppression of Pb as antiknock agent in petrol (Izquierdo-Álvarez et al., 1985; Trasobares, 2010).

Atomic Absorption Spectroscopy: Fundamentals and Applications in Medicine 19

In addition, analysis was made of the CSF of 136 individuals from the Services of Neurology,

The analysis of Ca, Mg, Zn, Cu, Fe and Mn was performed by flame atomic absorption using the corresponding hollow cathode for each metal. The wavelengths for the reading of the absorbance corresponding to each cation analysed were the indicated by the manufacturer. The values (mean and standard deviation) obtained in the control group for each metal analysed were as follows: Ca (mg/dL), 4.95 ± 0.70; Mg (mg/dL), 2.74 ± 0.10; Zn (µg/dL), 17.40 ± 7.50; Cu (µg/dL), 15.70 ± 4.50; Fe (µg/dL), 13.10 ± 3.60; and Mn (µg/dL), 2.50 ± 0.70. These values agree with the findings published by other authors (Hazell, 1997; Kapaki et al.,

With regard to the results obtained in the pathological CSF, significant increases were found (p<0.05) in the concentrations of Ca, Cu, Fe, Zn and Mn in the groups classified with cell and protein increase in CSF in comparison to the control group. It was also see that the significant increase of the Ca, Zn and Cu concentrations is greater in those groups which present a higher concentration of proteins, while the increase of Mn corresponds to the increase of cell count. With regard to magnesium, it was seen that the significant reduction of its concentration in relation to the control group corresponds equally both if the cell count

Interest in analysing these metals in CSF is directed to explaining the pathogenesis of some dysfunctions of the CNS. Clinical and experimental studies reviewed in the literature confirm the influence that these metals have on the pathogeny of some CNS dysfunctions

In this way the Ca and Mg ions play an important role in the action of glutamate, which is one of the most important neurotransmitter of vertebrates in the brain. The receptor of glutamate N-methyl-D-aspartate (NMDA) can only be activated in certain conditions of depolarisation of the membrane (Johnson & Ascher, 1988). The Mg ion blocks the channel, not being permeable to the Ca ions**.** When the receptor of glutamate are activated, the receptor reduces its affinity for Mg and the channel becomes permeable, permitting the entry of Ca ions to the neurone, a phenomenon which has been related to memory and the learning process (Hammond & Tritsch, 1990; Thomson, 1986). A reduction has been found in the glutamate and

Some studies have found an increase of Cu in serum and CSF and an increase of Mn in the spinal cord in Amyotrophic Lateral Sclerosis (ALS), which suggests that this metal plays a

The clinical association of Pb poisoning and ALS with an increase of Pb in blood and bone has been reported (Kamel et al., 2006). It has been suggested that patients with polymorphism in the gene of the ALA-D enzyme might be more at risk of presenting ALS in exposure to Pb (Kamel et al., 2003). Other studies have found no association between

It has been suggested that, regarding Mn, the binding of this metal in the basal ganglions of the brain may contribute in the pathogeny of the symptomatology of hepatic encephalopathy (Kuliseusky & Puyols, 1992; Weissenborn, 1995; Noremberg, 1998).

Mg concentration in the CSF in schizophrenic patients (Levine et al., 1996).

exposure to metals and ALS (McGuire et al., 1997; Bergomi et al., 2002).

role in the pathogeny of this disease (Kapaki et al., 1997).

Neurosurgery and Emergency. The CSF was extracted by lumbar puncture in all cases.

1997; Levine et al., 1996).

and diverse neuropathies.

or the protein concentration was increased.

In the review of results for 2008 (results not published), the values for ALA-D in the adult sample were 60.59 ± 16.49 (x ± SD), higher than those obtained in 1990: 46.81 ± 15.45 (x ± SD), which is logical if we take into account the fact that the activity of ALA-D in erythrocytes has a negative or inverse correlation with respect to the concentration of Pb in blood. In the 1990 study, Pearson's coefficient of linear correlation obtained was: r = -0.568 (p<0.001).
