Energy Metabolism of Erythrocytes in Lambs Chronically Exposed to Fluorine Compounds

Suska M.: Energy Metabolism of Erythrocytes in Lambs Chronically Exposed to Fluorine Compounds. Acta Vet. Brno 2002, 71: 313-317. Studies were performed to test the effect of environmental fluorine compounds on the energy metabolism of erythrocytes in lambs (Merino × Kent). The concentration of fluorine in serum, ATP, ADP and AMP content in blood and erythrocytes, adenine nucleotide pool (TAN) and adenylate energy charge (AEC) of erythrocytes were determined. A significant decrease in ATP concentration and a significant increase in ADP concentration (p ≤ 0.05) were observed compared to control group. The exposure of lambs to environmental fluorine compounds also caused a significant increase in the content of fluorine in serum. Moreover, a negative linear correlation (r = -0.836) between the erythrocyte ATP concentration and the serum fluorine ion concentration was found. AEC level also correlated linearly (r ≤ -0.949) with the concentration of fluorine ions in serum. These observations suggest that exposure of lambs to environmental fluorine compounds resulted in impaired energy metabolism of their erythrocytes. Adenine nucleotides, adenine nucleotide pool, adenylate energy charge, fluorine, blood Fluorine is known for its exceptional biological activity (Guminska 1994). Chronic environmental exposure of animals to fluorine compounds results in bone fluorosis and accumulation of the element in tendons, articular capsules and muscles (Guminska 1981; Pirog and Socha 2000). The accumulation of fluorine in the body continues during the whole lifespan and constitutes a serious threat to health (Mokrzynski et al. 1994; Chlubek et al. 1994; Machoy 1995). In animals, tenderness of long bone epiphyses, pareses, demineralisation and bone fragility have been observed. Apart from the relatively late anatomical lesions, fluorine is responsible for metabolic disorders in systems, organs, tissues and individual cells (Chlubek et al. 1995; Ignacak and Guminska 1991; Machoy 1995). Fluorine enters the bloodstream through the intestines and lungs. According to Guminska (1990), approx. 75% of fluorine appears in plasma, the remaining part in erythrocytes. Apparently, red blood cells possess a special mechanism protecting them against entry or facilitating elimination of fluorine (Korkmaz 2000). Yamamoto et al. (1989) studied the distribution of this element in blood and found that fluorine exists as a free ion, in inorganic and organic compounds. It also binds to plasma proteins, mainly albumin, and as such is biologically inactive (Guminska 1981). Free ions are the active form of fluorine, inhibiting approximately 70 enzymes (Guminska 1994), among them magnesium-dependent transport enzymes (Grabowska et al. 1991; Morris 1992; London and Gabel 1995) and several metabolic pathways (Guminska 1985; Machoy 1987) in red blood cells. Fluorine is a halogen with the lowest mass and ion radius and the highest electronegative potential and reactivity (Wakselman 1999). It may act directly on the enzyme protein, leading to structural changes through disruption of hydrogen bonds, or indirectly by ACTA VET. BRNO 2002, 71: 313–317 Address for correspondence: Department of Biochemistry, University of Szczecin 3a Felczaka St. 71-412 Szczecin, Poland Phone: + 48 91 444-15-51 Fax: + 48 91 444-15-50 E-mail: tarasiuk@univ.szczecin.pl http://www.vfu.cz/acta-vet/actavet.htm removing cations necessary for enzyme activity (Machoy 1987). Studies on the influence of fluorine compounds on magnesium-dependent enzymes have convincingly shown that fluorine ions inhibit the activity of enzymes participating in glycolysis and the pentose cycle in red blood cells (Guminska 1981, 1985, 1994), indirectly affecting their adenine nucleotide pool. Following this line of reasoning, we investigated the concentration of ATP, ADP and AMP in erythrocytes, to determine the adenylate energy charge of these cells and relate the results to the concentration of fluorine in serum. We used lambs bred near a major chemical plant in Police and thus chronically exposed to fluorine. Materials and Methods Animals The study was performed during autumn and winter 1999 in 24 lambs of Merino × Kent strain aged approx. 12 months. The animals were bred in farms located within 2-3 km from the Police Chemical Plant. This plant annually releases some 46.5 tons of fluorine compounds into the atmosphere (Zab∏ocki 1998). The animals were fed hay and silage of corn and sugar beet leaves. The fodder originated from fields polluted with fluorine compounds. Veterinary examination of the animals did not reveal any signs of fluorosis. Nevertheless, the lambs were apparently weaker, weighed less and consumed less food than controls. The control group consisted of 15 healthy lambs of the same strain, bred at least 70 km south-west of Police. They were fed the same type of fodder coming from unpolluted fields. Analyt ical procedure Blood was obtained from the jugular vein between 08.00 and 09.00 h. A 7 ml sample was transferred to a heparinized (250 IU heparin from Polfa, Poland), and 5 ml to a standard plastic test tube. Blood was delivered to the laboratory in ice flasks and immediately analyzed. Biochemical analyses Heparinized blood served to measure hematocrit. Following deproteinization with perchloric acid, ATP, ADP and AMP were measured in the acid-soluble fraction of erythrocytes with test kits (Biochemica Test Combination, Boehringer Mannheim). From absorbance differences determined according to the Schmidt method (1974) the adenine nucleotides concentration (μmol/l whole blood) in whole blood was calculated. The adenine nucleotides concentration in erythrocytes was calculated using hematocrit. The value of the nucleotide pool (TAN) and the adenylate energy charge (AEC) was calculated according to the formulas (Atkinson D.E. 1968): TAN = (ATP) + (ADP) + (AMP); AEC = Blood in plastic test tubes was centrifuged for 15 min at the RCF value 720 g and fluorine level in serum was determined according to Maruta’s method (1978). Stat is t ical analysis The results were expressed in SI units. Statistical analysis was performed using Student’s t-test (Statgraphics v. 50 software). Pearson’s correlation coefficient (r) was determined for fluorine levels in serum vs. erythrocyte energy parameters in each study group. The significance level of 0.05 was implemented to serve as a criterion for approving or discarding hypotheses for drawing the conclusion, relying on statistical analytic data. Results and Discussion Adenine nucleotides play a key role in the energy metabolism of cells. Levels of ATP, ADP and AMP reflect the rate of energy transformations in mature red blood cells, fueled mainly by glycolysis and the pentose cycle (Kwiatkowska 1989). The adenine nucleotide pool necessary for normal activity of erythrocytes remains under the control of an elaborate set of enzymes (Ataullakhonov et al. 1981), which includes three magnesium-dependent regulatory enzymes: hexokinase (EC.2.7.1.1), phosphofructokinase (EC.2.7.1.11) and pyruvate kinase (EC.2.7.1.40), all inhibited by fluorine ions (Machoy 1987; Guminska 1994). Minor shifts in the level of allosteric activators and inhibitors may grossly affect the rate of enzyme reactions, among them glycolysis, leading indirectly to changes in the level of ATP, ADP and AMP (Dabrowska 1997) and in the adenylate energetic charge of erythrocytes (Atkinson 1968). 314

removing cations necessary for enzyme activity (Machoy 1987).Studies on the influence of fluorine compounds on magnesium-dependent enzymes have convincingly shown that fluorine ions inhibit the activity of enzymes participating in glycolysis and the pentose cycle in red blood cells (Guminska 1981(Guminska , 1985(Guminska , 1994)), indirectly affecting their adenine nucleotide pool.
Following this line of reasoning, we investigated the concentration of ATP, ADP and AMP in erythrocytes, to determine the adenylate energy charge of these cells and relate the results to the concentration of fluorine in serum.We used lambs bred near a major chemical plant in Police and thus chronically exposed to fluorine.

Materials and Methods Animals
The study was performed during autumn and winter 1999 in 24 lambs of Merino × Kent strain aged approx. 12months.The animals were bred in farms located within 2-3 km from the Police Chemical Plant.This plant annually releases some 46.5 tons of fluorine compounds into the atmosphere (Zab∏ocki 1998).The animals were fed hay and silage of corn and sugar beet leaves.The fodder originated from fields polluted with fluorine compounds.Veterinary examination of the animals did not reveal any signs of fluorosis.Nevertheless, the lambs were apparently weaker, weighed less and consumed less food than controls.
The control group consisted of 15 healthy lambs of the same strain, bred at least 70 km south-west of Police.They were fed the same type of fodder coming from unpolluted fields.

Analytical procedure
Blood was obtained from the jugular vein between 08.00 and 09.00 h.A 7 ml sample was transferred to a heparinized (250 IU heparin from Polfa, Poland), and 5 ml to a standard plastic test tube.Blood was delivered to the laboratory in ice flasks and immediately analyzed.

Biochemical analyses
Heparinized blood served to measure hematocrit.Following deproteinization with perchloric acid, ATP, ADP and AMP were measured in the acid-soluble fraction of erythrocytes with test kits (Biochemica Test Combination, Boehringer Mannheim).From absorbance differences determined according to the Schmidt method (1974) the adenine nucleotides concentration (µmol/l whole blood) in whole blood was calculated.The adenine nucleotides concentration in erythrocytes was calculated using hematocrit.The value of the nucleotide pool (TAN) and the adenylate energy charge (AEC) was calculated according to the formulas (Atkinson D.E. 1968): TAN = (ATP) + (ADP) + (AMP); AEC = Blood in plastic test tubes was centrifuged for 15 min at the RCF value 720 g and fluorine level in serum was determined according to Maruta's method (1978).

Statistical analysis
The results were expressed in SI units.Statistical analysis was performed using Student's t-test (Statgraphics v. 50 software).Pearson's correlation coefficient (r) was determined for fluorine levels in serum vs. erythrocyte energy parameters in each study group.The significance level of 0.05 was implemented to serve as a criterion for approving or discarding hypotheses for drawing the conclusion, relying on statistical analytic data.

Results and Discussion
Adenine nucleotides play a key role in the energy metabolism of cells.Levels of ATP, ADP and AMP reflect the rate of energy transformations in mature red blood cells, fueled mainly by glycolysis and the pentose cycle (Kwiatkowska 1989).The adenine nucleotide pool necessary for normal activity of erythrocytes remains under the control of an elaborate set of enzymes (Ataullakhonov et al. 1981), which includes three magnesium-dependent regulatory enzymes: hexokinase (EC.2.7.1.1),phosphofructokinase (EC.2.7.1.11)and pyruvate kinase (EC.2.7.1.40),all inhibited by fluorine ions (Machoy 1987;Guminska 1994).Minor shifts in the level of allosteric activators and inhibitors may grossly affect the rate of enzyme reactions, among them glycolysis, leading indirectly to changes in the level of ATP, ADP and AMP (Dabrowska 1997) and in the adenylate energetic charge of erythrocytes (Atkinson 1968).
Our lambs were exposed to fluorine compounds during approximately 12 months.The serum content of fluorine in the study group was found to be significantly higher (157.1 ± 12.94 µM) than in controls (116.3 ± 15.35 µM).This was accompanied by reduced ATP levels in erythrocytes and elevated ADP levels in blood (p ≤ 0.05) (Table 1).Veterinary examination revealed no signs of fluorosis in the study animals.The inhibitory effect of fluorine on magnesium-dependent enzymes found by other authors (Guminska 1981;Machoy 1987) was confirmed in our study in the form of significantly lower levels of ATP.Furthermore, serum levels of fluorine in animals exposed to fluorine correlated inversely with erythrocyte levels of ATP and adenylate energetic charge of these cells (Table 2).The energy charge is a useful marker of shifts between consumption of energy stored in ATP and its production in metabolic processes (Atkinson 1968).The adenine nucleotide pools in both groups of animals did not differ significantly (Table 3).Moreover, changes in the energy metabolism of erythrocytes are frequently associated with disorders of mineral balance (Kedryna et al. 1991;H∏ynczak et al. 1980;Guminska and Skowron-Sula 1985).
Qualitative and quantitative distribution of electrolytes in the red blood cell varies, depending on species and strain of animal.For example, sheep erythrocytes differ according to levels of sodium and potassium.Two inheritable forms have been described.HK (high potassium) cells have high levels of potassium and low levels of sodium, while the inverse is true of LK (low potassium) cells (Baranowski 1998).HK lambs usually have a higher body mass than their LK counterparts (Atroshi 1979) and are more valuable as reproductors (Kmieç 1989;Lipecka et al. 1994).Other mammals, including cats, dogs and cows belong to the LK type (De Meydonca et al. 1970).Differences between animal species concerning magnesium content in erythrocytes and plasma have been reported.Sheep usually have low levels of this element in red blood cells and plasma (Torrance 1972) and in consequence are more susceptible to fluorine than other mammals.Guminska (1985) has observed a strong correlation between magnesium content in erythrocytes and potency of fluorine to inhibit various enzymes.The antagonism between fluorine and magnesium is well known and has been traced in vivo to the loss of magnesium from erythrocytes in the presence of fluorine ions (Marier 1980b;Stachurska and Guminska 1991;Guminska 1994).
Reduced levels of ATP in erythrocytes have been observed in adults and children residing in environments contaminated with fluorine (H∏ynczak et al. 1980).Changes in levels of adenine nucleotides depend not only on the exposure period but also on the dietary content of magnesium (Guminska 1985;Guminska 1994).Our results concerning ATP, ADP and AMP levels, as well as the adenylate energy charge in lambs seem to be the consequence of elevated levels of fluorine in serum.As a countermeasure against the negative effects of fluorine on energy metabolism (Guminska 1985), the fodder fed to animals in contaminated regions should be enriched with magnesium.
Lambs have relatively low levels of magnesium in plasma and erythrocytes and in consequence are more susceptible to toxic effects of fluorine.
Exposure of animals to fluorine compounds leads to abnormalities in energy metabolism of erythrocytes, presumably due to the inhibitory effect of fluorine on magnesium-dependent enzymes, catalyzing the process of glycolysis.

Table 1
Concentration of adenine nucleotides (µmol/l) in erythrocytes and whole blood of lambs chronically exposed to fluorine compounds (mean ± SD)