Measurement of Ionised Calcium in Captive Grey Parrots




Дата канвертавання25.04.2016
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Measurement of Ionised Calcium in Captive Grey Parrots (Psittacus e. erithacus).
Michael Stanford BVSc MRCVS Birch Heath Veterinary Clinic, Birch Heath Road, Tarporley, Cheshire, UK.
Keywords: Ionised Calcium, Grey Parrot, Total Calcium, Albumin, Ion-selective electrode.
Summary

Investigating any disorder of calcium metabolism requires an accurate assay to assess the calcium stasis of the subject. The measurement of serum ionised calcium is the most accurate reflection of the calcium status of an individual. Captive grey parrots are considered to be susceptible to hypocalcaemia and disorders of calcium metabolism could be under diagnosed if laboratories measure total blood calcium concentrations rather than ionised blood calcium. The study developed a normal range of 0.96-1.22moml/l for ionised calcium from a population of 80 healthy captive grey parrots. Ionised calcium concentrations were within a narrower range compared with total calcium concentrations in the same birds. The study showed that there was no significant correlation between albumin and total calcium concentrations and it was not possible to derive a formula that could compensate for fluctuations in albumin levels. The study indicated that delays of up to 48 hours in analysing blood samples would not significantly effect the ionised calcium concentrations so that posting blood samples to external laboratories would not be expected to adversely effect the results.


Introduction
The control of calcium metabolism in birds has developed into a highly efficient homeostatic system able to quickly respond to sudden demands for calcium required for both their ability to produce hard shelled eggs and their rapid growth rate when young (Whittow 2000). Although the hormones parathyroid hormone (PTH), metabolites of vitamin D3 and calcitonin, regulate calcium in a similar manner to mammals by acting on the main target organs of liver, kidney, gastrointestinal tract and bone, there are distinct phyletic differences (Bentley 1998). The most dramatic difference between the calcium metabolism of mammals and birds is related to the rate of skeletal calcium metabolism at times of demand. These differences enable the domestic chicken to respond to hypocalcaemic challenges within minutes compared to similar challenges in mammals, which can take over 24 hours (Koch and others 1984). For example, egg-laying birds can require the mobilisation of 10% of the total body calcium reserves in a 24 hour period. The calcium required for egg shell production is mainly obtained from increased intestinal absorption and a highly labile reservoir found in the medullary bone, which is normally visible radiographically in the medullary cavities of the bones of female birds (Hurwitz 1989). Hypocalcaemia is a common clinical presentation in captive grey parrots although the aetiology is still unconfirmed (Hochleithner 1989). It is thought that the syndrome could be due either to primary hypoparathyroidism or a secondary hyperparathyroidism due to inadequate husbandry. Seed based diets contain low levels of calcium and vitamin D3 and these are traditionally fed to grey parrots in captivity. It has been postulated that this contributes to a nutritional secondary hyperparathyroidism (Klasing 1998). However this does not explain why other psittacine species, despite being fed the same seed diet, rarely develop signs of hypocalcaemia. An alternative hypothesis is that lack of adequate ultraviolet light (UVb spectrum 285-315nm) in captive birds may lead to a functional vitamin D deficiency and subsequent problems with calcium metabolism. The role of UVb (285-315nm) radiation in the control of vitamin D metabolism has not been researched in this species at the present time although it has been extensively studied in poultry (Edwards and others 1994, Tian and others 1994)

Grey parrots affected by hypocalcaemia present with a variety of neurological signs ranging from slight ataxia to seizures. These signs resolve with calcium therapy (Hochleithner 1989). In young captive grey parrots osteodystrophy is a common clinical sign (Harcourt-Brown 2003, Rosskopf and others 1985). Reproductive conditions such as egg binding or thin shelled eggs are common presenting disorders of calcium metabolism in adult birds. Calcium exists as three fractions in the avian serum: the ionised salt, calcium bound to proteins and as complexed calcium bound to a variety of anions (citrate, bicarbonate and phosphate). Ionised calcium is the physiologically active fraction of serum calcium with a role to play in bone homeostasis, muscle and nerve conduction, blood coagulation, and the control of hormone secretion such as vitamin D3 and parathyroid hormone (Flavus and others 1999). Ionised calcium concentrations are regulated by the interaction of PTH, vitamin D3 metabolites and calcitonin in response to changing demands (Whittow 2000). The ionised calcium level would be expected to be maintained within a narrow range in the normal individual compared with the total calcium level and any major change in the serum ionised calcium level is likely to be of pathological significance. Ionised calcium levels are measured by ion selective electrodes. The protein bound calcium fraction is physiologically inactive and any increase would not be considered to have a pathophysiological significance (Bush 1991); this calcium is bound mainly to albumin. Any physiological or pathological condition affecting serum albumin values in the blood will affect the total calcium concentration leading to an imprecise result. The binding reaction between the calcium ion and albumin is strongly pH dependent so that acid base imbalances will affect ionised calcium levels. An increase or decrease in pH will respectively increase or decrease the protein bound calcium fraction.

The significance of the small complexed calcium fraction is unknown at the present time but any disease state that affects the levels of the binding anions lactate, bicarbonate and citrate would be expected to alter calcium levels significantly. Most veterinary pathology laboratories will report total calcium serum concentration which is the sum of all three calcium fractions. Total calcium levels are measured routinely by spectrophotometer.

Measurement of total calcium in an avian patient with abnormal protein levels or acid-base abnormalities would not be an accurate assessment of the calcium available to the animal. Ionised calcium levels are considered a more appropriate reflection of the patient’s calcium status especially in a diseased animal.


In mammals positive correlations have been found between albumin and total calcium levels, which have allowed formulae to be developed to “correct” total calcium levels when there are fluctuations in albumin levels (Torrance 1998). However, recent research has suggested these corrected estimates of free calcium are inaccurate in 20-30% of cases in human medicine and that ionised calcium should be measured wherever possible (Flavus et al.1999). Previous research in psittacine birds found a positive correlation between albumin and total protein in grey but not in amazon parrots (Lumeij 1990). Blood samples for ionised calcium assays should be taken into heparin and analysed as soon as possible after venepuncture. Changes in the blood pH would be expected to affect the accuracy of the ionised calcium levels sample, for example contact with carbon dioxide in the air will reduce the pH of the sample and affect the protein binding reaction to calcium (Scenck and others 1995).

Materials and Methods
A group of 100 grey parrots was used for this study. The birds were kept indoors at the premises of a parrot breeder in the UK. The study group consisted of healthy adult grey parrots of known sex that had been fed on a seed diet (Tidymix, John Heath, Hull, UK) for 12 months prior to the start of the study. Faecal samples were taken from each bird and examined for parasites by direct smears and worm egg count (McMaster method). Blood samples taken from each bird under isoflurane anaesthesia were subject to standardised haematological and biochemical analysis. Prior to their selection for the group all birds had been found to be negative for circovirus, polyoma virus and chlamydophila psittaci using polymerase chain reaction (PCR) tests. Each bird was examined clinically and by laparoscopy. On the basis of these tests only healthy sexually mature birds were included in the study. Additional blood was taken during the health examination with the informed consent of the owner from 80 birds to collate data to develop a normal range for ionised calcium in this species. The blood was taken into heparin and was analysed within 60 minutes of venepuncture using an AVL 9181 analyser. Blood was taken from the remaining 20 birds to assess the effect of sample handling on ionised calcium assays. The blood was divided into three separate samples that were analysed for ionised calcium at 24 hour intervals. The blood samples were stored at 18 degrees Celsius until analysis was carried out.

The biochemical analysis of the samples including the total calcium concentrations were performed on the Space analyser (Randox) using standard spectrophotometer techniques. The albumin values were measured by serum protein electrophoresis ( Helena Bioscience SAS2 analyser) due to the known problems with protein measurements in the avian subject using the bromol-cresol green method (Lumeij 1991).



Results
Figure 1. indicates the results obtained from 80 healthy captive grey parrots. The normal range of ionised calcium was found to be 0.96-1.22mmol/l with 95% confidence limits.

Figure 2 indicates the total calcium concentrations in the 80 healthy captive grey parrots in 2001. The laboratory’s normal range for total calcium was 2.0-3.0mmol/l.


Figure 3 indicates the relationship between albumin and total calcium. Using a Pearson correlation test no statistically significant relationship could be found between total calcium and albumin concentrations.
Figure 4 indicates the effects on sample handling on ionised calcium concentrations. No statistically significant difference with a 95% confidence limits (using t2 comparison of the means) was found between samples processed immediately, 24 hours or 48 hours post venepuncture.

Discussion
The author considers that the measurement of the physiologically active ionised calcium rather than total calcium levels is important when investigating disorders of calcium metabolism in captive grey parrots. The study confirmed that in the grey parrot ionised calcium is kept within a narrow range compared with total calcium levels which fluctuate with both protein levels and blood pH changes. The laboratory normal range for total calcium concentrations in captive grey parrots is 2-3mmol/l. Measurement of the total blood calcium in this group of 80 healthy captive grey showed that five birds appeared to be hypercalcaemic and 19 birds appeared hypocalcaemic. This suggests using total calcium values in isolation would lead to the misdiagnosis of disorders of calcium metabolism in this species even in healthy individuals. Of 394 samples submitted from grey parrots into a practice laboratory (MEDLAB, Tarporley,UK) in 2001, 54 samples (13.17%) had a low ionised calcium level despite having normal total calcium concentrations. In the diseased grey parrot the significance of measuring ionised calcium rather than total calcium concentrations would be expected to become more apparent. A grey parrot with metabolic acidosis would be expected to show an ionised hypercalcaemia level due to decreased protein binding. An alkalotic patient would be expected to exhibit an ionised hypocalcaemia as the protein binding reaction increases. In both cases measurement of total calcium levels alone would not reflect these pathologically significant changes in ionised calcium concentrations. Any increase in the serum albumin level would be expected to increase the protein bound calcium fraction but not the ionised calcium fraction. This is best seen in laying female birds where the serum albumin levels may rise by up to 100% (Bentley1998). This would produce an inflated total calcium concentration due to an increased protein bound calcium fraction whilst not affecting the ionised calcium level and clinically hypocalcaemia could be missed. Conversely in a grey parrot with hypoalbuminaemia a reduction in the total calcium levels would be expected due to a low protein bound calcium fraction but the ionised fraction would not be significantly affected. These simple common clinical situations indicate the importance of the use of the ionised calcium levels in grey parrots.

A statistically significant correlation between albumin and total calcium was not present so a formula could not be developed as in mammals to adjust for variation in protein levels. In humans the correlation between total calcium and albumin levels increases in disease states perhaps explaining why no significant relationship could be found in the captive healthy grey parrots. Recent research in humans advises against the use of such adjustment formulae suggesting the use of ion selective electrodes to measure ionised calcium wherever possible (Flavus et al 1999). Analysers designed to measure ionised calcium concentrations are increasingly available for use in veterinary clinics such as the I-stat (Heska Industries) and measurement of ionised calcium should become commonplace (Feldman 1995).

Although it is preferable to analyse samples immediately a delay of 48 hours did not give a statistically significant difference in the ionised calcium levels. This suggests that posting samples to external laboratories is acceptable. These results are similar to findings in canine samples (Schenck and others 1995).

Acknowledgements

The author is grateful for financial assistance for the study from The Royal College of Veterinary Surgeons Small Grants Trust and Harrison’s Bird Foods International (Nebraska, U.S.A.) Thank you to N.H. Harcourt-Brown, Dr. J. Elliot, Dr. G. Harrison, Dr. S Baker and all colleagues at Birch Heath Veterinary Clinic for their help and support. The author is indebted to Mr and Mrs Taylor (Becks Bird Barn, Windy Oak Farm, Ellerdine,Telford, Shropshire UK), the owners of the birds, for their unwavering support.


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