**2.1.2.2 Hormonal regulation of strontium**

From a general point of view, this research with Sr was designed to find the hormonal regulatory mechanisms in order to be able to act on them, provoking or forcing its elimination. This approach was based on several facts: a) The chemical similarity between Ca and Sr and their common participation in certain physiological processes lead one to think that both elements share a hormonal regulatory mechanism; b) The contribution of Chausmer et al. (1965) who had demonstrated the existence of specific action of calcitonin (CT) on the distribution of Zn, contrary to that exercised in the distribution of Ca, reducing the levels of this element in different tissues -thymus, testicles- seemed guiding; and c) Comar's idea (1967) that the plasmatic levels of Ca could participate in the regulation of Sr metabolism was particularly attractive. Taking these facts into account and with the possibility of a shared hormonal regulation for different trace elements (Zn and Sr among others), with specific properties for each one, the first steps in this approach were addressed to finding out more about serum Sr.

In the first study (Escanero, 1974), Comar's idea was proved since, at the same time as the concentration of Sr in bovine blood increased, so did that of Ca in total proteins, the contrary phenomena occurring with inorganic phosphorus and Ca in albumin.

In a later study (Alda & Escanero, 1985), the association constant and the maximal binding capacity for Ca, Mg and Sr to human serum proteins taken as a whole were determined. For Sr, a maximal binding capacity of 0.128 mmol/g of proteins and the association constant (Kprs) of 49.9 ± 16 M-1 were obtained. These values were not far from those of Ca (0.19 mmol/g of proteins and 55.7 ± 18 M-1).

Later, Córdova et al. (1990) studied the regulating hormones of Ca and the effects of the administration of parathormone (PTH) and CT in rats and after thyroparathyroidectomy (TPTX) were analyzed. The PTH excess and defect (TPTX treated with CT + T4) showed plasmatic increases in Sr. However, CT excess provokes decreases while the defect (administration of PTH + T4 to TPTX rats) causes increases. Consequently, CT may be the hormone that plays a regulating role in the plasmatic Sr concentrations.

In this line, a study (Escanero and Córdova, 1991) was conducted in order to known the effect of the administration of glucagon on the serum levels of the alkaline earth metals since the phosphocalcic response to glucagon was already known in different animal species and it had been reported that, in mammals, glucagon triggered the release of CT by the thyroid gland. The study was completed with the analysis of the changes induced in these metals by the administration of CT. The effect (reduction) was observed two hours after administration and in daily administration the effect peaks on the 3rd day and CT significantly reduces the serum levels of these metals up to the 3rd day of treatment, just when the glucagon effect is highest.

#### **2.1.2.3 Continuing with serum proteins**

While the alkaline metals hardly bond in proteins and the alkaline earth metals do so in a proportion of 50% or slightly less, the trace elements do so almost completely. Zinc is

real intestinal absorption were analysed at those moments when their activity was maximum. Different concentrations of Sr were used and it was concluded that the discrimination occurred

From a general point of view, this research with Sr was designed to find the hormonal regulatory mechanisms in order to be able to act on them, provoking or forcing its elimination. This approach was based on several facts: a) The chemical similarity between Ca and Sr and their common participation in certain physiological processes lead one to think that both elements share a hormonal regulatory mechanism; b) The contribution of Chausmer et al. (1965) who had demonstrated the existence of specific action of calcitonin (CT) on the distribution of Zn, contrary to that exercised in the distribution of Ca, reducing the levels of this element in different tissues -thymus, testicles- seemed guiding; and c) Comar's idea (1967) that the plasmatic levels of Ca could participate in the regulation of Sr metabolism was particularly attractive. Taking these facts into account and with the possibility of a shared hormonal regulation for different trace elements (Zn and Sr among others), with specific properties for each one, the first steps in this approach were addressed

In the first study (Escanero, 1974), Comar's idea was proved since, at the same time as the concentration of Sr in bovine blood increased, so did that of Ca in total proteins, the contrary

In a later study (Alda & Escanero, 1985), the association constant and the maximal binding capacity for Ca, Mg and Sr to human serum proteins taken as a whole were determined. For Sr, a maximal binding capacity of 0.128 mmol/g of proteins and the association constant (Kprs) of 49.9 ± 16 M-1 were obtained. These values were not far from those of Ca (0.19

Later, Córdova et al. (1990) studied the regulating hormones of Ca and the effects of the administration of parathormone (PTH) and CT in rats and after thyroparathyroidectomy (TPTX) were analyzed. The PTH excess and defect (TPTX treated with CT + T4) showed plasmatic increases in Sr. However, CT excess provokes decreases while the defect (administration of PTH + T4 to TPTX rats) causes increases. Consequently, CT may be the

In this line, a study (Escanero and Córdova, 1991) was conducted in order to known the effect of the administration of glucagon on the serum levels of the alkaline earth metals since the phosphocalcic response to glucagon was already known in different animal species and it had been reported that, in mammals, glucagon triggered the release of CT by the thyroid gland. The study was completed with the analysis of the changes induced in these metals by the administration of CT. The effect (reduction) was observed two hours after administration and in daily administration the effect peaks on the 3rd day and CT significantly reduces the serum levels of these metals up to the 3rd day of treatment, just

While the alkaline metals hardly bond in proteins and the alkaline earth metals do so in a proportion of 50% or slightly less, the trace elements do so almost completely. Zinc is

phenomena occurring with inorganic phosphorus and Ca in albumin.

hormone that plays a regulating role in the plasmatic Sr concentrations.

in the passive rather than in the vitamin-dependent transport (Escanero et al., 1976).

**2.1.2.2 Hormonal regulation of strontium** 

to finding out more about serum Sr.

mmol/g of proteins and 55.7 ± 18 M-1).

when the glucagon effect is highest.

**2.1.2.3 Continuing with serum proteins** 

deposited in a proportion of more than 99% and the proteins involved with albumin and the alpha2 macroglobulin (α2MG). The α2MG bound tightly to the metal is responsible for 30- 40%. The aminoacids are only responsible for about 2% of bonded Zn (Giroux & Henkin, 1972). The serum levels of Zn vary in different pathological symptomatologies and in relation to the amount of exercise. Several studies have shown these variations are related to the percentage of the element bound to albumin and another one established this relation between total serum Zn and α2MG-bound Zn, in athletes after exercise (Castellano et al., 1988). This last study was aimed at analysing the variations in the serum levels of Zn after acute and chronic overload of this element and verifying whether these variations may be correlated to changes in the percentage of the element bound to albumin, α2MG or both.

After a single intragastric administration of 0.5 mL of a solution containing 1000 ppm of Zn, the levels of this metal increased significantly (p<0.01) in serum 30 min after the beginning of the experiment, reaching maximum values at one hour and returning to normal levels 24 h later. It should be noted, however, that 8 h after administration the increases were no significant (p>0.05). The concentration of Zn bonded to albumin varied in parallel to the total serum levels of Zn. The values of bonded Zn in the globulins α2MG also varied but the values returned to basal concentrations 4 h after the beginning of the experiment.

The chronic overload was performed with different groups who underwent a daily intragastric dosis of 0.5 mL of the same solution used for the acute overload. Different group of animals were sacrificed on days 7, 14, and 30 after the beginning of the experiment, with the aim of collecting values at different times of the overload. The control group and one other were used for recuperation and were kept for ten days with no treatment after day 30. Chronic overload of Zn caused significant increases in the serum levels of Zn throughout all the experiments. The highest values were found on the 14th day. The amount of Zn bonded to albumin varied in parallel with the total serum Zn; however, the concentrations of Zn bonded to globulins (α2MG) showed a significant decrease (p<0.05) on the seventh day, increasing significantly (p<0.01) on the 14th day and the 30th day and returning to normal values 10 days after Zn overload was interrupted.

The results of the acute overload suggest a correlation with the proportion of element bonded to the albumin and those of the chronic overload showed a rapid response of the albumin while the increases of element bonded to the α2MG responded slowly and remained constant until the end of the experiment. However, at this time the total serum Zn and the Zn bonded to albumin decreased.

These results suggest that albumin may play a new physiological role by adjusting its binding capacity to the serum Zn levels.

In the same line the levels and distribution of serum Cu and Zn were studied in patients diagnosed with acute myocardial infarction from the day of admission to the Cardiovascular Intensive Care Unit until the 10th day following the attack (Gómez et al., 2000). The results obtained showed that Cu increased significantly after the 5th day after the myocardial infarction, while Zn decreased significantly (p<0.01) with relation to the control group after the 1st day, the lowest values being found on the 3rd day after the attack.

Later, the total serum Cu showed an excellent correlation with the Cu bonded to the albumin and to the globulins (ceruloplasmin), as well as with the concentration of both

Atomic Absorption Spectroscopy: Fundamentals and Applications in Medicine 11

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

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.

phenomenon observed over the last twenty years (González-López, 1992).

treated with chelating agents. The children were another control group.

c. Normal values of biomarkers characteristic of lead poisoning.

a. No symptoms of lead poisoning and other diseases, b. No changes in routine biochemical parameters and

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

with CaNa2EDTA as chelating agent.

**2.2.2 Development: Subjects and methods** 

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

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

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

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

urine and the coproporphyrins in urine (Meredith et al., 1979).

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 albumin concentration.

These findings suggest the existence of some kind of relationship between the two fractions of the element bonded to proteins, which is probably different for each metal.

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 transport and anionic exchanger.

Other properties of the uptake of Zn by erythrocytes were published in previous reports (Galvez et al., 1992, 1996a, 1996b):

