Pier Luigi Dodi1
1Dipartimento di Salute Animale - Sezione di Clinica Medica Veterinaria
The eye presents an anatomic and physiological structure which is particular and complicated with a very high metabolism. Its position in contact with the outside agents, as environmental pollution and sun rays, and its very wide vascularization makes it considered a target organ of many sistematic diseases, whether contagious or toxic.
In these last few years, the lifetime of carnivorous domestic animals has increased considerably, thanks to the progress carried out in the veterinarian field. Even though, it is possible for a dog to reach a miximum age of 27 years, in reality, the average lifetime, taken into consideration all the breeds, is of 13 years (Dairin, 1997). As it grows older, the organs ability to maintain the homeostasis and to adapt to the aggressions of the external elements alter progressively, in this way, eye diseases and alterations increase.
The crystalline is undoubtedly, the easiest structure to study in its ageing progress. As its test is relatively simple and its ophthaimological study has been strongly helped by the growth of ocular surgery (Chaudieu et al, 1997). Senile alternations of the crystalline are common in old dogs. The most frequent are nuclear sclerosis, the formation of cataract and the crystalline luxation. The cataract in particular senile type, represents a model to study in the ageing pathology.
Among the most important causes, up till now recognized, of the damage caused by the ageing of oculare structure. A position of relief is kept without doubt by the oxidation produced by luminous radiation.
These processes of oxidation, take place due to the fact that the eye is chronicley exposed to luminous radiation, able to trigger a chain of harmful chemical reactions (Orzalesi e coll., 1998) with free radical synthesis produced by lipidic peroxidation, which reacting with biological molecules, determines serious cellular damages. The effectiveness of the enzymatic defenive systems and the anti-oxident activity substances is reduced with ageing.
It is supposable that environmental pollution plays an important role in the human being, in etiology of sinile cataract. In this pathology it has been observed in the human being, a high concentration of cadmium and lead and a slight increase of copper concentration (Racz and coll., 1988). Moreover, the polluting substances can determine oxidative stress. Cekie, 1998, has studied the effects caused by smoke in the cataractous lenses and has pointed out a significant increase of copper, lead and cadmium in cataractous lenses of smokers. These heavy metals, which are present in the environment, due to pollution, could re-enter among causes of cataract. The mechanism of etiopathogenesis, of which these elements cause cataract is not yet totally clear.
In order to carry out the above ideas, a research having the following aims was done on a dog:
· To characterize the physiopathologist metabolic aspects, significant in the cataract of a dog on the bases of the information supplied by the dosage of toxic minerals (cadmium and lead) and nutritions (calcium, chlorine,phosphur, magnesium potassium, sodium, copper and zinc) present in the lenses and in the plasma.
·To evaluate the possible role of the lens which indicates the state of oxidation in the dog, based on the amount of plasmatic concentration of vitamin A, E and C.
Animals
The present study has been conducted on a sample of randomized dogs of various breeds, consisting of a total of 30 subjects, of varied ages, weight and sexes, which come from north Italy.
Lenses
The 20 samples in which the concentrations were determinated of macro and microelements were made up of 14 lenses cataractized, 7 males and 7 females and 6 normal lenses of 2 males and 4 females, from different breeds. The ages of the dogs with cataract varied from 4 to 7 years, whereas, the group under inspection aged from 1 to 10 years, all the cataract lenses were classified as mature.
All the subjects, before being surgically treated, were subjected to an exam with a general objective, a hematic biochemical – clinical (formula with hemochrome and liverwort, renal and muscular functionality) exam and an exam, which is particularly aimed at an eye covered in electroretinalgraghy and ocular ecography.
As there hasn't been found any contraindications, the subjects have been ideal candidates for the operation. The pathological lenses have been removed through an extracapsule surgical technique. The normal lenses removed with the same technique have been obtained within 6 hours, after the death of the subjects.
The lenses removed before being analized have been placed in plastic test tubes and frozen at –20°.
On the day of the tests, the crystallines have been weighed, and after, dried in an oven at the temperature of 100°C for 24 hours. The weight of the samples has been taken after. The moisture of the samples has been calculated. To determine the concentrations of the metals in atomic absorption a preventive breakdown is necessary of the organic matrix. The method chosen was the wet mineralization.
This technique requires the use of strong acids, such as nicric, perchloric, hydrochloric and their mixtures which should be able to completely breakdown the organic material through oxidative reactions. The speed of the reaction can be increased by the temperature and by the pressure.
In our case, the dried samples have been placed in teflon capsules together with 3ml of HNO³ (65%) Suprapur® (Merck) and 0.5ml H²O² (volume 110). The capsules have been placed in a microwave oven (Milestone 1200 Mega) where the complete mineralization takes place in 20' with a program of 6 increasing steps, at a maximum temperature 180°C and a pressure of 20 bar. After the cooling, the liquid was brought to a final volume of 10ml with hexastyled water.
The spectrophotometric definitions have been executed with a Perkin-Elmer Mod.305/B apparatus, equipped with lamps charged with radiofrequency and corrector at the base of the lamp with deuterium. The vaporization was obtained by means of a oxyacetylene flame of Ca, K, Na, Mg, and Mn and by means of a grahite oven (HGA500) for Cd, Cu, Pb. In order to guarantee accuracy of the preparation and of the analytics definition, controls on the materials in reference to NBS (SRM 1566 and SRM 1577) were done.
Plasma
The 10 older dogs or equal to 8 years of age, therefore old, whom which were determined the plasmatic values, came from a kennel in the north of Italy. The subjects were male, 4 with mature cataract and 6 normal and they had been kept for years with the same diet and environmental condition and at the time of the hematic removal, sistemic pathology was not present in course, as confirmed by the objective exam and from the results of the laboratory exams.
The blood samples were placed in test tubes containing ETDA, for the dosage of the vitamins, and Eparina, for the minerals, and have been analized the same day when taken.
The dosage of the vitamins A and E has been done with the technique HPLC. Acetate-alpha-tocoherol has been added to the plasma with EDTA, used as internal standard. Later the sample is dissolved in chloroform and diluted in ethanol. The function of ethanol is to get the plasmatic proteins to precipitate. The vitamins have been extracted twice with hexane through vaporization under nitrogeno. Finally, the determination of the liposolubile vitamins takes place through HPLC.
The determination of vitamin C in the blood is done with the HPLC technique by electrochemistry of 650 nA. 220 microlitres have been added at four parts at 6% of metaphosphoric acid, and in this way it acids the plasma and it stabilizes the ascorbic acid. After the samples are whirled at 2500 rpm. The supernatant obtained is added to a solution containing trisodiumphosphate and dichloride, in order to reduce the dihydroascorbic, formed from the previous reaction, to ascorbic. The minerals are determinated by a spectrophotometer of COBAS MIRA Plus chemical clinic.
Statistical methods
Among the 20 dogs of which the crystallines have been analized, different groups based on the normal or pathologic eye, sex, age, right or left eye.
For all the groups each element's media and deviation has been calculated.
The 10 subjects in which the plasma level of some minerals has been determinated, have been subdivided into two groups, pathological and normal. Media and standard deviation have been calculated.
The values of the concentrations in healthy dogs and in dogs with cataract have been statistically compared by student test. The differences have been considered when p<0.5.
Moreover, the factors of correlation of the different determinations have been calculated.
Correlated differences of sex or age, in the concentration of cadmium, lead, copper and magnesium in cataractous lenses have not been found. In the cataractous lenses the level of calcium, sodium and zinc is very high if compared to normal lenses. The level of calcium is higher in the cataractic lenses of young subjects, age less than 7.
Magnesium doesn't seem to be correlated with the process of cataract in the dog because there aren't any significant variations.
In the 10 subjects in which sodium, potassium, chlorine, calcium, phosphor and magnesium and vitamin A, E and C have pointed out an increase of plasmatic sodium in subjects with total senile cataract and an increase of chlorine in the normal subjects of the controlled group. Potassium, calcium, phosphor and magnesium do not have a significant variation.
Vitamin E is mainly high in the plasma of subjects with cataract, in respect of the controlled group, while, vitamin A and C do not present significant variations.
All the results can be examined in the appendix tables.
The bibliography of veterinary science regarding micro-macroelements and vitamins in lenses and in plasma in dogs with cataract is poorly pubblished.
LENSES
Cadmium, lead, and copper
These elements are considered biomarkers. Biomarkers represent the level of contamination in a certain area and, consequently, establish an indicator of the toxic risk level to which a certain population can be subject to (Fossi, 1994). These contaminated elements induce biochemical variations or cells which are able to speed up the cataractic process in the lens. In the present research there hasn't been found any significant difference correlated to the sex and age in the concentration of cadmium, lead, copper in the cataractous lenses compared to the normal ones.
These results are in contrast with the esisting work done with the human being. As a matter of fact, in the senile cataract of the human being the concentration of cadmium, lead and copper are high, if compared to normal ones. Lead is the element with the highest concentration, followed by cadmium and copper.
The concentration of these elements, moreover, increases with the age of the subjects (Racz and coll., 1998).
Other studies confirm these facts. In 1998 Cekic has determined the concentration of the three previous elements of lenses with cataract of different stages of the human being. In the group of pathologic lenses belonging to male subjects, high concentrations of cadmium have been registered compared to the female group. In the human being, as in amimals, the origin of cadmium, lead and copper is tied to the environmental issue of the contaminated factors coming from different industrial production stages. Cadmium, in the human being, is mainly from cigarette smoke and fertilizers used in agriculture, while lead is caused by the vehicles' exhaust pipes (Racz and coll., 1998).
Moreover, alimentation cannot be excluded, infact, through a diet (mainly cereals) the most largest quantities of cadmium and lead are taken (Galal-Gorchev, 1993, FAO/WHO, 1998/a/b).
The role of cadmium in the cataract pathogenesis of the human being is mainly in chronic smokers (Ramakrishnan and coll., 1995). This would confirm the pathogenic role of metal, only in certain conditions. It cannot be excluded that in lenses, in which a cataract is present at an incipient stage, modifications of the crystalline capsule help the entry of the metals, that could speed up the degenerating process, but not to set it off.
Copper is important for many physiological functions and among these it is essential for many enzymes (Underwood, 1977). The concentration of copper in the normal dog's lenses decreases with age (Cook and coll., 1985), as in human beings (Shlopak, 1962).
In the dog, with a mature cataract, the amount of copper is significantly low even though a high level has been noted in the hypermature stage (Cook and coll., 1985).
Therefore, the results obtained in the present research suggest some theories on the low concentration of heavy metals in the toxic actions, cadmium and lead, in the dog's cataractous lenses:
Lead is the heavy metal which is mainly present in nature. In mammals it is easily absorbed. Infact, it is possible to find it in different organs, like kidneys, bones and nerve tissues.
Cadium is considered one of the heavy metals, mostly dangerous for the health due to its high environmental dispirsion, it is possible to find in the air at high concentration. Infact, one of the main ways of absorbing it, is through the respiratory system.
As to regards to copper, it is possible to absorb it through a diet, therefore, it is extremely difficult to establish the toxic level of the lens.
Calcium, Sodium, Zinc, Potassium and Magnesium
In the present research of the cataractous lenses the level of calcium, sodium and zinc is significantly high if compared to normal lenses.
The level of calcium results to be high in the cataractous lenses of young subjects, under the age of 7 years. The average age statistically significant, is of 5.33 ± 1.03 and 12.9 ± 3.1 years. Respectively in the young group and in that of the old.
This confirms a part of the studies completed in human medicine that calcium is very high in cataractous lenses (Cekic, 1998), mainly in the case of senile cataract (Burge, 1909; Adams, 1929; Kraus, 1934). Also, cigarette smoke has a cataractic action on calcium. An increase of calcium was noted in the normal lenses of mice, exposed to cigarette smoke (Avunduk and coll., 1997). These variations seem to be caused by morphological modifications of the lens epithelium (Avenduk and coll., 1999).
Based on these last researches it is possible to assume that dogs which live with their owners that smoke feel the effects of it in their eyes.
In the dog's lenses examinated calcium variations are higher in pathologic lenses of young subjects compared to adult subjects. For this assumed difference the pathogentic role of calcium in dogs is particularly evident in young subjects. Infact, the concentration of this element results to be about four times higher (1453,78 ppm against 345.1 ppm of the old group) than in dogs with cataract at a mature stage.
In the human being you can assist of an increase in calcium as cataract matures (Cristini and coll., 1983).
The porportionality between calcium tension and its maturing level results to be so much that authors have suggested it for classifying cataracts (Saraux and coll., 1986). The levels of calcium which have been discovered in this research enable to state that the membrane is more permeable in young subjects and this can speed up the pathologic process.
The potassium level in examined dogs is high in normal lenses. Magnesium doesn't seem to be involved in the dog's cataractic process because there aren't any significant variations.
This is confirmed by the esisting bibliography. Researches done on laboratory animals, in cataractic lenses of mice, potassium and magnesium decrease,while sodium and calcium increase (Dilsiz and coll., 1999). Also, in the human being you can assist at a decrease of potassium in the lenses (Stanojevic and coll., 1987; Rasi and coll., 1992).
The increase of zinc and sodium in dog's pathologic lenses is indicated also in the human being. Infact, zinc increases all types of cataract (Srivastava and coll., 1992; Rasi and coll., 1992), while the second one (sodium) is found in high concentrations only in senile cataracts (Stanojevic and coll., 1987)
Hematic plasma
In the second study regarding dogs with senile cataract, a small number of dogs was studied and this due to that fact, that a group of harmonious subjects in age, environment, alimentation. The ten dogs came from a kennel from the north of Italy and have been feed for years the same alimentation, they are old subjects (over 8 years of age), all males: four with senile cataract at a mature stage and six present normal controls.
The necessity of a sample, the most homogeneous possible, is binded to the strong influence to the type of alimentation in the variations of the minerals and plasmatic vitamins.
Vitamins A, E and C.
The result of the dosage of the vitamins of the dogs examined, has not pointed out significant differences in comparision with the 2 groups, except for vitamin E, which is the major in the group of the subjects with senile cataract.
This last result is in contrast with the scientific work done, which has demonstrated the importance of this anti-oxidant in the prevention of cataract.
In different researches it has been observed that additional vitamin E, both in the human being and in animals of laboratory reduce significantly the effect of cataract, because high dosage of vitamin E slow down or eliminate the oxidative stress, which is the main cause of cataract (Lyle, 1999).
The result obtained in the dogs of the present research can be referred to two factors.
High mobility of vitamin E in the organism and low concentrations that limit a precise dosage at a plasmatic level. The tissue deposits mainly at a hepatic level, are able to supply enough provision for long periods and so the plasmatic concentrations vary from subject to subject, generally, the plasmatic levels of vitamin E seem to be correlated with the absorbed quantity with the diet and able to absorb lipidic compound for these reasons it is not possible to evaluate the anti-oxidant effect in a subject of vitaman E based only on the plasmatic concentration.
The low number of the examined subjects does not enable to draw final considerations. This fact, as already confirmed is due to the need to have a calculation on samples of subjects the most homogeneous possible, which would not be possible with others. Only through a dosage of more homogeneous groups, a significant result will be possible. During this study there hasn't been pointed out differences of ascorbic acid and vitamin A.
An English research, on a sample group of 23 poodle dogs with cataract, has pointed out a significant decrease of ascorbic acid at a plasmatic level, compared to the controlled group, even if the difference in concentration at level of aqueous humour, in the same animals, has not resulted to be significant (Barros and coll., 1999).
Other studies have established that the concentration of vitamin C in the aqueous humour, is correlated at the same level of vitamin C in the plasma. This would confirm the importance of the additional alimentation with ascorbic acid (Haung and coll., 1997).
From the different bibliograpy, the role of vitamin C does not appear clear in the pathogenisis of cataract. Moreover, the dog holds a high endogenous production and does not need this type of vitamin. Even, additioned high quantities result to be contraindicated because they are cause of hypervitaminic pathologies.
With vitamin C, also, for vitamin A there hasn't been found a difference in the 2 groups of dogs.
Researches done by Lyle, 1999, in the human being have established that the plasmatic level of vitamin A does not seem to influence the development of cataract.
Sodium, Potassium, Chlorine, Calcium, Potassium and Magnesium.
The present research has underlined an increase of plasmatic sodium in subjects with mature senile cataract and an increase of chlorine in normal subjects of the controlled group. Phosphor presents an increasing trend in the controlled group. Potassium, calcium and magnesium have not gotten a significant variation.
Researches done on human plasma in patients with senile cataract, have underlined a significant lower level of calcium, while, phosphor is higher compared to the normal controlled group (Chen, 1992).
The emersed datas must be interpretated with caution, considering the limits of the present study.
A first potential limit able to influence the result obtained is the geographic one. Infact, all dogs that from the north of Italy, could not be the representatives of the whole Italian dog population.
The possibility of minium outside contaminations of the examined lenses is not to be undervalued, because eventhough, sterile containers and standard laboratory methods are used, it is impossible to avoid them in particular for calcium.
However, even considering the limits as indictated above from the obtained data, it's possible to arrive to some important preliminary conclusions:
Basic studies of bibliography suggest that the oxidative mechanisms, mainly with the ageing, can play a very important role in the pathogenisis of cataract.
Many results seem to agree on the positive effect of the vitamins and the traced elements in the progression of cataract, even, if the result of the epidemic observation in the human being remain however, not totally clear and inconclusive (Christen, 1999).
Therefore, only through double blind, randomized on large scale it will be possible to define the potentiality and the benefits of the vitamins and the micro and macroelements.
At the present time, the datas which are at disposition and the limited number of observations of the present research do not enable to draw definate considerations on the existence and entity of such differences.
In any case, the obtained results represent a clear indication in this sense and lead to further examinations, on which the author is already involved with.
Parole chiave: cane, cataratta, minerali, vitamine.
Key words: dog , cataract, mineral, vitamins,.
RIASSUNTO - L'occhio presenta una struttura anatomica e fisiologica
complessa e particolare con un metabolismo tra i più elevati. La sua
posizione a contatto con gli agenti esterni, quali inquinanti ambientali e raggi
solari, nonché la sua estesa vascolarizzazione, lo portano ad essere
considerato un organo bersaglio di molte malattie sistemiche sia di natura infettiva
che tossica. Finalità della ricerca:
a) Caratterizzare aspetti fisiopatologici metabolici significativi della cataratta
nel cane, sulla base delle informazioni fornite dal dosaggio dei minerali tossici
(cadmio e piombo) e nutrizionali (calcio, cloro, fosforo, magnesio, potassio,
sodio, rame e zinco) presenti nella lente e nel plasma.
b) Valutare il possibile ruolo della lente quale indicatore dello stato di ossidazione
del cane in base alla determinazione della concentrazione plasmatica delle vitamine
A, E e C.
I 20 campioni in cui sono state determinate le concentrazioni macro e microelementi
erano composti provenienti da 14 lenti catarattose, 7 maschi e 7 femmine, e
6 lenti normali, da 2 maschi e 4 femmine di razza differente. L'età dei
cani con cataratta variava da 4 a 17 anni mentre il gruppo di controllo da 1
a 10 anni. Tutte le lenti catarattose sono state classificate come mature.
I 10 cani con età maggiore o uguale ad 8 anni, quindi anziani, in cui
sono stati determinati i valori plasmatici provenivano da un canile del Nord
Italia.
Non è stata rilevata nessuna differenza, correlate a sesso ed età,
nelle concentrazioni in cadmio, piombo, rame e magnesio nelle lenti catarattose
rispetto alle normali. Nelle lenti catarattose il livello di calcio, sodio e
zinco è significativamente alto se comparato con le lenti normali. Mentre
il livello di potassio risulta elevato nelle lenti normali. Il livello di calcio
risulta maggiormente elevato nelle lenti catarattose dei soggetti giovani, con
una età inferiore ai 7 anni.
Il magnesio non sembrerebbe correlato al processo della catarattogenesi nel
cane in quanto non vi sono variazioni significative.
Nei 10 soggetti in cui si è determinato il sodio, potassio, cloro, calcio,
fosforo e magnesio e le vitamine A, E e C ha evidenziato un aumento di sodio
plasmatico nei soggetti con cataratta totale senile ed un aumento del cloro
nei soggetti normali del gruppo di controllo. Il potassio, calcio, fosforo e
magnesio non hanno una variazione significativa.
Le vitamine E è maggiormente elevata nel plasma dei soggetti con cataratta
rispetto al gruppo di controllo, mentre le vitamine A e C non presentano delle
variazioni significative.
Attualmente i dati a disposizione e il ristretto numero di osservazioni del
presente ricerca non consentono di trarre considerazioni definitive sulla esistenza
e entità di tali differenze.
In ogni caso il risultati ottenuti rappresentano un chiaro indizio in questo
senso e spingono ad ulteriori approfondite verifiche.
SUMMARY - The eye presents an anatomic and physiological structure
which is particular and complicated with a very high metabolism. Its position
in contact with the outside agents, as environmental pollution and sun rays,
and its very wide vascularization makes it considered a target organ of many
sistematic diseases, whether contagious or toxic.
The purpose of the research are: 1) To characterize the physiopathologist metabolic
aspects, significant in the cataract of a dog on the bases of the information
supplied by the dosage of toxic minerals (cadmium and lead) and nutritions (calcium,
chlorine,phosphur, magnesium potassium, sodium, copper and zinc) present in
the lenses and in the plasma;2) To evaluate the possible role of the lens which
indicates the state of oxidation in the dog, based on the amount of plasmatic
concentration of vitamin A, E and C.
The 20 samples in which the concentrations were determinated of macro and microelements
were made up of 14 lenses cataractized, 7 males and 7 females and 6 normal lenses
of 2 males and 4 females, from different breeds.
The 10 older dogs or equal to 8 years of age, therefore old, whom which were
determined the plasmatic values, came from a kennel in the north of Italy.
Correlated differences of sex or age, in the concentration of cadmium, lead,
copper and magnesium in cataractous lenses have not been found. In the cataractic
lenses the level of calcium, sodium and zinc is very high if compared to normal
lenses. The level of calcium is higher in the cataractous lenses of young subjects,
age less than 7.
Magnesium doesn't seem to be correlated with the process of cataract in the
dog because there aren't any significant variations.
In the 10 subjects in which sodium, potassium, chlorine, calcium, phosphor and
magnesium and vitamin A, E and C have pointed out an increase of plasmatic sodium
in subjects with total senile cataract and an increase of chlorine in the normal
subjects of the controlled group. Potassium, calcium, phosphor and magnesium
do not have a significant variation.
Vitamin E is mainly high in the plasma of subjects with cataract, in respect
of the controlled group, while, vitamin A and C do not present significant variations.
At the present time, the datas which are at disposition and the limited number
of observations of the present research do not enable to draw definate considerations
on the existence and entity of such differences.
In any case, the obtained results represent a clear indication in this sense
and lead to further examinations
References