Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis.

Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.

The role of iron in metal-metal interactions in hard tissues of roe deer (Capreolus capreolus L.).

INTRODUCTION: The toxicity of heavy metals in humans and animals has been attributed to their potential for accumulation in solid tissues and interference with other vitally important elements present in the body. We have devised this study to determine the content of some metals in bone and to determine with the Statistica Neural Networks program whether the content of one metal has any effect on the content of other metals. MATERIAL AND METHODS: Our material consisted of mandibles obtained from deer aged 2 to 6 years. Using a dental drill, pulverized bone was obtained for measurements of calcium, copper, and zinc with atomic absorption spectrometry and magnesium, chromium, manganese, and iron with inductively coupled plasma atomic emission spectometry. Statistical analysis was performed with the Statistica software and its Statistica Neural Network program. RESULTS: We found that the concentration in bone of one metal affects the concentrations of other metals. CONCLUSIONS: Iron seems to play a special role in the accumulation of other metals by bone. The use of artificial neural networks was helpful in identifying candidate elements (like iron) which are active in controlling concentrations of other metals in bone.

Guanine and inosine nucleotides, nucleosides and oxypurines in snail muscles as potential biomarkers of fluoride toxicity.

The aim of the present study was to determine the toxicity of fluorides on energy metabolism in muscles of the Helix aspersa maxima snail. Qualitative and quantitative analysis of purine compounds was performed in slices of foot from mature snails with high-performance liquid chromatography. Fluoride concentrations were measured using an ion-selective electrode and gas chromatography. The results show that exposure to fluoride pollution was accompanied by a statistically significant increase in fluoride concentrations in soft tissues. This effect was already noticeable with the smallest fluoride dose. Accumulation was greatest in the shell. There is a significant and positive correlation between fluoride concentrations in foot muscles and guanine and inosine nucleotides or uridine content. The content of low-energy guanylate, inosylate and oxypurine in foot muscles significantly increased with rising dose of fluoride. The difference as compared with controls was significant only for the highest dose of fluoride. Interestingly, uric acid, the final product of purine catabolism, dominated quantitatively in the foot muscles of snails. In conclusion, increased low-energy guanylate and inosylate as well as decreased xanthine concentrations in snail muscle can be indicators of the toxic influence of fluoride on the organism. The measuring of fluoride accumulation in the shell is the most suitable bioindicator of fluoride pollution in the environment.

[Current trends in fluorine research]. / Aktualne kierunki badaan nad fluorem.

PURPOSE: Current topics in fluorine research are presented with emphasis on findings by researchers in Szczecin and Poland, as well as in the world. Reports are cited on the distribution of fluorine compounds in the environment, routes of penetration into living organisms, and analytical methods for the quantitative determinations of fluorine content in air, water, soil, and foods. Important contributions have been made by Polish researchers on the role and patterns of fluorides in body fluids, soft and hard tissues, which remain in direct relationship to accumulation and elimination of fluorine. So far, comprehensive studies on mutagenic effects of fluorine and its potential role in bone neoplasms, Down syndrome, and other genetic disorders have not been carried out in Poland. Worthy of mention are reports on mechanisms of action of fluorine compounds on the cellular and subcellular level. CONCLUSIONS: Finally, two achievements of recent years in the field of fluorine research are discussed briefly. The first is concerned with the use in dentistry of chemical analysis for studying mineral reconstruction of teeth throughout the lifetime of an individual. The second is in the field of medicine where molecular modeling has been applied to explain the mechanism of action of aluminofluoride complexes (AlFx) as a messenger of false information during protein biosynthesis and their apparent role in the etiology of Alzheimer's disease.

[Calcium, magnesium, fluoride, and phosphorus content in antlers and skull bones of roe deer (Capreolus capreolus) in relation to age]. / Porównanie zawartosci wapnia, magnezu, fluorków i fosforu w porozach i kosciach czaszek saren (Capreolus capreolus) w zaleznosci od wieku.

PURPOSE: This study was undertaken to compare calcium, magnesium, fluoride, and phosphorus content in antlers and skull bones of roe deer in relation to their age and habitat. MATERIAL AND METHODS: The study was done in 22 samples originating from two Forestry Districts of West Pomerania. The study population was divided into two age groups: young animals (aged 1-2 years) and mature animals (aged 4-8 years). Calcium and magnesium was measured with atomic absorption spectrometry, phosphorus with a spectrophotometric method, and fluoride with an ion-selective electrode. CONCLUSIONS: Statistical analysis demonstrated differences in the content of the elements depending on age and environmental pollution. Correlations between the content of elements in skull bones and antlers were found.

[Interactions of aluminofluoride complexes (ALFx) with high-energy compounds (ADP, ATP) as revealed by molecular modeling]. / Oddzialywanie kompleksów glinowo-fluorkowych (ALFXx) na zwiazki wysokoenergetyczne (ADP, ATP) oceniane przy zastosowaniu modelowania molekularnego.

INTRODUCTION: Aluminofluoride complexes (AlFx) are toxic substances at the cellular and subcellular level, apparently implicated in degeneration of the nervous system. The present study focused on direct interactions with A1Fx of two common adenine nucleotides: adenosine diphosphate and adenosine triphosphate, both of which play an important role in many biochemical processes. The aim was to search for interactions between ADP-AlIF and ATP-AlFx complexes depending on the environment of the reaction. MATERIAL AND METHODS: Modern HyperChem computer software was used allowing for the computation of heat of formation and bond energy. Results. The strongest bonds in vacuum were calculated for ADP-AIF4 ', ADP-AIF3 and ATP-AlF3. In water, the strongest bonds were found for ADP-AF3 and ATP-AlF3. Due to inhibition of metabolic processes, stable bonds between those molecules are less advantageous than labile ones. Nevertheless, strong bonds seem to be associated with reduced toxicity of AlFx complexes.

The role of bivalent metals in hydroxyapatite structures as revealed by molecular modeling with the HyperChem software.

Hydroxyapatite, the major component of bone, demonstrates significant reactivity with metals. Knowledge of spatial structure and energy data of the molecule helps understand the binding of metals by hydroxyapatite and elucidate the chemical and physical properties of such complexes. We used HyperChem software (Hypercube Inc.) to analyze the structure of hydroxyapatite when the central calcium atom is replaced by one of the metal ions (Mg, Cu, Zn, Fe, Cr, Mn) marked by us in bone. Our results show that hetero-ionic exchange affects composition and leads to deformation of hydroxyapatite crystals. Replacement was accompanied by changes in bond lengths between oxygen and calcium atoms in the hydroxyapatite molecule and by displacement of groups of atoms surrounding the central calcium atom. The use of molecular modeling as a computational tool enabled a preliminary and theoretical understanding of chemical structure without the need for laboratory tests.

Comparison of rat and human alkaline phosphatase isoenzymes and isoforms using HPLC and electrophoresis.

Total activity of alkaline phosphatase (ALP) in serum represents the sum of activities of some isoenzymes and their numerous isoforms derived from various tissues of the organism. The aim of this work was to separate ALP isoenzymes and their isoforms in rat and human serum, compare the properties of serum ALP isoforms in rats and humans, and determine the usefulness of some analytical methods for specific measurements of ALP isoenzyme and isoform activities. Two methods of separation, i.e. high-performance liquid chromatography (HPLC) and agarose gel electrophoresis were chosen. The combination of HPLC with electrophoresis of the eluted fractions and with ALP inhibition methods (urea, l-phenylalanine), inactivation (heat) and precipitation (wheat-germ lectins) enabled the identification of isoenzymes and isoforms of ALP in serum. Using chromatography and a post-column reactor, three isoforms of the intestinal isoenzyme and one bone isoform of a tissue non-specific isoenzyme were detected. Rat serum differs significantly from human as regards activities of intestinal and hepatic isoforms, whereas the properties of the bone isoform are similar in both species. Our HPLC method offers a higher resolution than agarose gel electrophoresis with respect to ALP subfractions in rat serum.

Adenine nucleotides in snail muscles as one of biomarkers of fluoride toxicity.

The aim of this work was to determine the extent of bioaccumulation of fluorides in tissues of Helix aspersa maxima. The toxicity of fluorides administered orally on the energy balance of the snail's foot was investigated based on measurements of concentrations of adenine nucleotides and their metabolism degradation products. Quantitation of fluoride levels was done in soft tissues (foot, hepatopancreas) and shells of mature snails. Qualitative and quantitative analysis of purine compounds was performed in slices of foot from mature snails. Fluoride concentrations in pulverized shells were measured using an ion-selective electrode. Gas chromatography was used to determine fluoride concentrations in soft tissues (hepatopancreas and foot). Purines were measured in foot muscle slices with high-performance liquid chromatography (HPLC). Fluoride levels in soft tissues of the snail cannot serve as an indicator for biomonitoring purposes as no significant accumulation was observed during exposure to maximum allowable concentrations of fluoride in drinking water. Contrary to this, levels of fluoride in the shell rose significantly with this concentration of fluoride in drinking water. The effect of fluorides on energy metabolism of foot muscle was evidenced by elevated AMP levels, increased adenine nucleotide pool and reduced conversion of ADP to ATP. Exposure to rising F(-) concentrations was accompanied by decreasing values of the adenylate energy charge AEC. Determination of AMP or AEC in foot muscle of exposed snails seems to be a useful indicator of fluoride effects on metabolic activity.

Methylated purines in urinary stones.

BACKGROUND: The aim of the study was to measure the content of methylated purines that appear as admixtures in uric acid stones. METHODS: We analyzed urinary calculi from 48 residents of Western Pomerania who underwent surgery at the urology ward in Szczecin. Stone samples were dissolved in 0.1 mol/L NaOH. Extracts were diluted in 50 mmol/L KH(2)PO(4) and analyzed by reversed-phase HPLC with ultraviolet detection and use of a gradient of methanol concentration and pH. RESULTS: Uric acid was the main component of 9 stones. All 9 showed admixtures of 9 other purine derivatives: endogenous purine breakdown products (xanthine, hypoxanthine, and 2,8-dihydroxyadenine) and exogenous methyl derivatives of uric acid and xanthine (1-, 3-, and 7-methyluric acid; 1,3-dimethyluric acid; and 3- and 7-methylxanthine). Amounts of these purine derivatives ranged from the limit of detection to 12 mg/g of stone weight and showed a strong positive correlation (Spearman rank correlation coefficients, 0.63-0.94) with the uric acid content of the samples. The main methylated purine in the stones was 1-methyluric acid. CONCLUSIONS: Urinary purines at concentrations below their saturation limits may coprecipitate in samples supersaturated with uric acid and appear as admixtures in urinary stones. The amount of each purine depends on its average urinary excretion, similarity to the chemical structure of uric acid, and concentration of the latter in the stone. These findings suggest that purines in stones represent a substitutional solid solution with uric acid as solvent. Methylxanthines, which are ubiquitous components of the diet, drugs, and uric acid calculi, may be involved in the pathogenesis of urolithiasis.

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection.

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.

Estimation of fluoride distribution in the mandible and teeth of the red deer (Cervus elaphus L.) from industrially polluted areas in Poland.

MINI SUMMARY: Animals from areas contaminated by industrial emissions containing fluoride may accumulate it predominantly in hard tissues. Therefore, an analysis was performed for the determination of fluoride content in mandibles and teeth of deer living at large in different areas in Western Pomerania (Poland). Samples of hard tissues obtained with a dental drill, were dissolved in perchloric acid and analyzed for fluoride content with an ion-selective electrode. Using the Generalized Regression Neural Network (GRNN), it was possible to arrange definite variables according to their order of importance concerning the fluoride mean content in investigated bone material. Parametric analysis revealed in which sites of mandibles and teeth the fluoride accumulation was the highest. In our calculation, the traditional statistic as well as artificial neural network was applied. OBJECTIVE: One of the interesting properties of fluoride is its balancing accumulation in the hard and soft tissues. In this paper, we decided estimate how the changeable doses of fluoride in natural environment influence on fluoride distribution and accumulation in the examined sites of mandible and teeth. MATERIAL AND METHOD: The material consist of 103 mandibles of red deer (Cervus elaphus L.) from five forestry districts of Western Pomerania in Poland, which are located near or far away from two major industrial plants: Police Chemical Work and the Dolna Odra Coal Power Plant. Samples (10 mg of powdered bone or tooth) were dissolved in perchloric acid and the fluoride content was determined with an ion-selective electrode. Comparisons were done with the Statistica 5.5 and Statistica Neural Networks. RESULTS: Animals living in Western Pomerania (Poland) have high fluoride contents in their bones. Fluoride accumulation in mandibles and teeth is irregular. The higher content of fluoride show incisal area, but coronoid process of the mandible was not sensitive to different exposure of environmental fluoride at all. Parametric analysis revealed that the accumulation pattern of fluorine was different in the two groups of deer. CONCLUSIONS: Many biological and environmental factors may influence on incessant fluoride accumulation in osseous tissue of ruminants living at large. The use of artificial neural networks enables a more accurate insight into the process of fluorine accumulation in the mandible and teeth and helps in the ranking of factors influencing this process.

Shell of snail Helix aspersa maxima (Helicidae) as a protection of bioaccumulation toxic sodium fluoride in soft tissue.

The aim of this work was to determine the extent of bioaccumulation of sodium fluorides in tissues of snails under strictly controlled conditions, and also to determine resistance and tolerance to sodium fluoride load in these organisms. The study was performed on snails removed from aestivation. Quantitation of fluoride levels was done in soft tissues (foot, hepatopancreas) and shells of mature snails. Results show that long exposure to sodium fluoride pollution at a low level results in accumulation principally in the soft tissues of the snails. Because of the possibility of fluoride accumulation in the foot, the number of snails used for culinary purposes must be controlled, as it can potentially cause chronic toxemia caused by this trace element. Results also show that the shells of snails offer protection against the bioaccumulation of toxic fluoride in the soft tissue. The Helix aspersa maxima snail is characterised by high resistance and tolerance to fluoride load. Fluoride levels in soft tissues of the snail cannot serve as an indicator for biomonitoring purposes. In contrast, levels in the shell rose significantly with the concentration of fluoride and can be used in biomonitoring of sodium fluoride pollution.

[A comparative analysis of occupational risk in industry employees based on concentrations of some elements in teeth and body fluids]. / Ocena zagrozen zdrowia pracowników zatrudnionych w wybranych zakladach przemyslowych na podstawie oznaczenia zawartosci niektórych pierwiastków w zekbach i plynach ustrojowych.

Work safety and hygiene is a set of basic measures to protect workers from the negative effects of manufacturing processes. So far, numerous procedures for assessment of occupational health risk have been described. We measured the concentrations of some elements in teeth and body fluids of employees working in three different industries with an established production profile: Zaklady Naprawcze Taboru Kolejowego (repairs of rail vehicles), Philips Lighting Poland (production of lighting systems) and Metalplast (build ing furbishing factory). Different technologies were in place at each of these plants. Basing on laboratory analyses, the risk of exposure to chemical substances was evaluated. The study material included 100 extracted teeth, as well as body fluid samples (saliva, urine and blood) collected during routine health checks. Whenever possible, concentrations of the following elements were measured: calcium, magnesium, fluorine, phosphorus in the form of phosphates, potassium, sodium, iron, zinc, copper, cadmium, and lead. Metal elements were measured spectrophotometrically (ASA), fluorine with an ion-selective electrode, and phosphates with a colorimetric method. We found that concentrations of the elements in teeth and body fluids differed depending on the industry. For teeth, statistically significant differences applied to magnesium, phosphates, zinc, sodium, and potassium. In body fluids, statistically significant differences were found for calcium (blood and urine), magnesium (blood, urine and saliva), zinc (blood, urine and saliva), iron, lead and copper (urine). In conclusion, our findings may be helpful for monitoring safety at work in industrial plants.

[Living conditions of deer in the provinces of Western Pomerania and Lubuskie as revealed by mandibular content of fluoride, calcium, and magnesium. I. Inter-relations between fluoride, calcium, and magnesium content in mandible]. / Ocena warunków bytowania zwierzyny plowej z Pomorza Zachodniego i Ziemi Lubuskiej na podstawie zawartosci fluorków, wapnia i magnezu w zuchwach. I. Zaleznosci pomiedzy zawartosciami pierwiastków w zuchwach zwierzat.

The south-western regions of Poland are more exposed to industrial emissions containing fluorine than eastern and northern regions. Fluoride is known to accumulate in hard tissues of animals and humans. This paper describes the content of fluoride in mandibles (lower jaws) of deer from two provinces: Western Pomerania and Lubuskie. The fluoride content in lower jaws of deer from Lubuskie was statistically higher compared with fluoride content in Western Pomerania. We concluded that the living conditions of animals in Western Pomerania are more favorable than in Lubuskie. When the concentration of fluoride in bone becomes too high, mineralization defects can occur causing major loss in mechanical resistance despite an increase in bone mass.

[Living conditions of deer in the provinces of Western Pomerania and Lubuskie as revealed by mandibular content of fluoride, calcium and magnesium. II. Detailed statistical analysis with the statistica neural networks software]. / Ocena warunków bytowania zwierzyny plowej z Pomorza Zachodniego i Ziemi Lubuskiej na podstawie zawartosci fluorków, wapnia i magnezu w zuchwach. II. Poglebiona analiza statystyczna przy wykorzystaniu programu statistica neural networks.

Animals from areas contaminated by industrial emissions containing fluoride may accumulate it predominantly in hard tissues. The stable composition of the mandible makes it suitable for the study of accumulation of fluorides and other elements (Mg, Ca). It is also possible to determine the extent of pollution with fluorine compounds around industrial plants and to reveal some of the features of the natural habitat of wild animals. We applied the advanced technique of neural networks to investigate the simultaneous influence of multiple parameters on the accumulation of fluorine, magnesium, and calcium in the mandible of deer and possible interactions between these elements. The first step involved the generation of a neural network. In the present work, networks were created with the Intelligent Problem Solver (IPS). Input data were divided into three sets. One was used for training (Tr) of the network, another for validation (Ve) and control over the training process and the last one for testing (Te). Network quality was checked with the following parameters: Data Mean--average value of variable; Data SD--standard deviation of variable; Error Mean--average value of error; Abs. Mean--average error modulus; Error SD--standard deviation of error; SD Ratio--ratio of standard deviation of error to standard deviation of variable (Error SD/Data SD); Correlation--Pearson's correlation coefficient. For the model to be satisfactory, Pearson's correlation coefficient should be close to 1. In the case of complex problems with an unclear influence of parameters on the process, an analysis of parametric sensitivity must be performed. This task was accomplished automatically with the computer program. It was found that the effect of the same parameters on the accumulation of fluorine, magnesium and calcium varied. The results suggest interactions between the examined elements.

Changes in the content of zinc and fluoride during growth of the femur in chicken.

Zinc and fluorides are capable of modifying the process of bone formation and mineralization. Statistically significant differences have been revealed in the content of zinc and fluorides between structures of the femur in chicken. The content of zinc in compact bone remained constant during the first 50 d of life. Lower and less stable contents were found in spongy bone and bone marrow. The content of fluorides in compact bone was higher than in spongy bone. The lowest concentrations of zinc and fluorides were found in articular cartilage and were further reduced at the end of observation. Correlations revealed between the content of zinc and fluorides point to structural and functional relationships between these elements in various parts of the bone.