The Measurement of Ionised Calcium in Grey Parrots

Michael Stanford BVSc MRCVS

Birch Heath Road

Tarporley

Cheshire

CW6 9UU

United Kingdom

Key words:

Introduction

Calcium in blood is distributed as free calcium ions (50%), bound to proteins, mostly albumin (40%) and 10% bound to anions such as bicarbonate, citrate, phosphate and lactate. Only ionised calcium can be used by the body to perform such vital processes as muscular contraction, cardiac function and blood clotting.

It has become apparent that the measurement of total calcium in many exotic species does not give the submitting clinician an accurate picture of the calcium status of a particular patient as the results obtained, though methodology wise are correct, quantitively are inaccurate. Inaccurate results are often due to protein binding of the calcium in the plasma or serum sample. The need to assess accurately the calcium status of patients was required and the measurement of ionised calcium is considered more important and accurate than that of total calcium.

Equipment and methodology employed:

Ionised Calcium:

The study was conducted using an AVL 9181 analyser. The methodology employed by the analyser is based on the ion-selective electrode (ISE) measurement principle to precisely determine the measurement values. The analyser is fitted with three ISE electrodes, Sodium, Potassium and Ionised Calcium. Each electrode has an ion selective membrane that undergoes a specific reaction with the corresponding ions contained within a particular sample being tested. The membrane is an ion exchanger reacting to the electrical charge of the ion causing change in the membrane potential or measuring voltage, which is built up in the film between the sample and the membrane. A galvanic measuring chain within the electrode determines the difference between two potential values on either side of the membrane of the active electrode. The potential is conducted by a highly conductive, inner electrode to an amplifier. The ion concentration in the sample is then determined by using a calibration curve determined by measured points of standard solutions with precisely known ion concentration.

Measurement principle

The AVL 9181 is a sophisticated piece of medical equipment that uses ISE measurement principle to precisely determine electrolyte values. Although the technology employed is quite complicated, understanding how the instrument performs sampling analysis is relatively simple. In short, the 9181 compares an unknown value against a known value to compute the electrolyte, or in this case, ionised calcium value.

The physical principle employed is:

An ion selective electrode is connected with a reference electrode to form a measuring system. When immersed in a solution that contains a relative ion the Nernst equation applies:

^1.                     E = E’ ± {R.T – n.F} . In a_i

Or

_2.                     E = E’± {R.T– n.F} . In (f_{I .} c_I)

_{(+) for cations}

_{(-) for anions}

The equation can also be written:

         E=E’ ± S.log (f_{I .} c_I)

          E          the measured electric potential

E’         the e.m.f. of the system in a standard solution

a_I                    activity of the ion measured

R          the general gas constant (8.31 J/Kmol)

T          temperature

            N          valence of measured ion

            F          faraday constant 96.496 A.s/g

            f_I                     the activity coefficient

            c_I                    the concentration of measured ion

            S          the slope of the electrode.

If the ion concentration of one of the measuring solution is known, the ion concentration of the sample can be determined on the basis of the difference of two measured potentials.

            4.         E _sample =            E’ + S . log (f_i.C_{I sample})

            5.         E _standard =           E’ + S . log (f_i.C_{I standard})

            6.         LE = E_sample – E _standard = S . log (C_{i sample} – C_{i standard})

LE                    the difference between the measured potentials of the  sample and standard.

S                      the potential difference of the electrode, determined from the potential difference of two measured standard solutions.

C_{i sample}     concentration of the measured ions in the sample

C_{I standard}                           concentration of measured ions in the standard solutions

The unknown concentration in the sample can now be determined by:

 7.         C_{I sample} = C_{i standard} . 10 ^(LE/S)

As demonstrated by these equations, the ion selective electrodes do not measure the ion concentration but the activity of the ions concerned. This activity of the ions concerned. The activity is a criterion of the ion’s ability to interact with other ions, in which each ion binds a proportion of its energy.

Total Calcium:

Total Calcium estimations were measured using the RANDOX Arsenazo method. The samples were assayed on the spACEÔ Analyzer.

Principle:

Arsenazo III specifically binds to calcium forming a coloured complex at 600 nm.

Ca⁺⁺ + Arsenazo III                    Coloured complex.

The amount of calcium present in the sample is directly proportional to the intensity of the coloured complex formed. 

Due to non specific absorbance interference these were corrected by the addition of EDTA reagent which removes calcium from the calcium Arsenazo III coloured complex allowing accurate sample blank measurement.

Handling and storage of samples for ionised calcium

All samples should be stored and handled as far as possible in anaerobic conditions with minimal expose with ambient air. Contact with ambient air will cause a loss of C0₂ in the sample and the subsequent rise in pH will cause a reduction in
ionised calcium. If determination was used by using whole blood, then heparin samples were required, these were filled as much as possible so that the residual air space was minimal.

Limitations of procedure

Limitations of ionised calcium depend greatly on whether the sample has been exposed to significant pH changes prior to sampling. As with all clinical reactions, the laboratory was told of any substances and medications used on the patient prior to examinations. No significant effects on serum or heparin samples has been demonstrated from bromide, ammonium and iodide.

Samples received for analysis

Samples from 40 grey parrots received into the laboratory were measured for total and ionised calcium. All samples received were initially screened both for biochemical and haematological parameters prior to being assayed for Ionised Calcium, where possible, clearly abnormal results were taken away from the core values used for the final guide range production. All birds assayed had undergone veterinary inspection as part of their annual veterinary health check prior to being sampled. Any bird which was obviously not healthy so far as the inspecting veterinary surgeon was concerned were not sampled. For the purpose of the production of guide ranges, only, as far as clinical and laboratory examination could state, were healthy birds tested as part of a routine health screen.

Results of Analysis:

40 Grey Parrots as described were analysed for ionised and total calcium as detailed their results are listed below:

The results were subjected to statistical analysis using a modified t test to produce a normal range for ionised calcium of 0.96 – 1.20 mmol/litre.