Lead Toxicity in Mute Swans

Comp. Biochem. Physiol. Vol. 868, No. 4, pp. 701- 704, 1987
Printed in Great Britain
0305-0491 /87 S3.00 + 0.00
Pergamon Journals Ltd
DETERMINATION OF HAEMOGLOBIN IN BIRDS
BY A MODIFIED ALKALINE HAEMATIN
(D-575) METHOD
(Received 3 June 1986)
Abstract-1. A recently published method for measuring human haemoglobin based on alkaline haematin
(Zander et al. , Clin. chem. Acta 136, 83- 93 , 1984) has been adopted for bird samples.
2. The new method yields comparable haemoglobin values with that of a previously used alkaline
haematin method.
3. Levels of haemoglobin estimated using alkaline haematin were higher than for cyanhaemiglobin,
the reference method for human haemoglobin. This difference is due to the loss of haemoglobin in the
cyanhaemiglobin procedure due to insolubility.
4. The values for haemoglobin found by the alkaline haematin method did not vary significantly
between a range of bird species.
5. The method overcomes some important deficiencies of the cyanhaemiglobin method, in particular,
problems of turbidity and quality control assessment.
INTRODUCTION
Haematology plays an important diagnostic role in
human and veterinary medicine and modern haematological
methods are now being increasingly used
in avian research and wildlife investigations. The
direct application of methods developed for mammalian
studies is, however, problematical in birds due
to the presence of red blood cell nuclei, in addition
to other cell debris on haemolysis. This debris is liable
to lead to substantial error due to turbidity of the
specimen. The accuracy and precision of the various
methods for estimating haemoglobin differs markedly
(Van Assendelft, 1972; Rick, 1976; Bankowski, 1942).
The spectrophotometric method using cyanbaemiglobin
has been the most widely accepted (Spaander,
1964; Saundermann, 1956) and was recommended
by the International Committee for Standardisation
in Haematology (ICSH) in 1965 and in 1967. Since
cyanhaemiglobin is considered the only stable derivative
(Richterich, 1971) all other methods were
rejected (Rick, 1976). Thus the cyanhaemiglobin
method is considered the reference method against
which all others must be tested.
While the use of cyanhaemiglobin has many
significant advantages it also has a number of
deficiencies which were recently restated by Zander et
al. ( 1984). These can be summarised as follows:
(1) The cyanhaemiglobin reagent contains cyanide,
is toxic and thus hazardous.
(2) The reaction solution is light labile.
(3) The concentration of the reaction components,
especially that of cyanide and the buffer have to be
carefully chosen and kept constant.
(4) Standardization of the method is based on
purified cyanhaemiglobin solutions, the quality of
which is controlled only indirectly by spectrophotometry.
(5) The reaction times of the different haemoglobin
species and derivatives differ markedly.
While the above disadvantages are important for
mammalian studies their significance becomes even
greater for animals with nucleated red blood cells.
Thus the following disadvantage can be added to the
above:
(6) The reaction end-product is turbid and requires
centrifugation before reading spectrophotometrically.
With centrifugation, the supernatant becomes
clear and a residue forms as a pellet at the
bottom of the tube.
A further disadvantage of the cyanhaemiglobin
method which is important for the field biologist is
the fact that the reaction end-product is light labile
which imposes time constraints on field biologists, in
remote areas wishing to study blood parameters.
Information on the precise haemoglobin levels in
healthy birds and birds contaminated by environmental
toxins, such as lead, is needed but to what
extent cyanhaemiglobin may assay actual avian
haemoglobin is not known (Archer, 1977).
This investigation therefore examined a new
method described for estimating haemoglobin in humans
(Zander et al. , 1984) to see if current difficulties
in estimating non-mammalian haemoglobin could be
resolved. This method is based on the conversion of
all haeme, haemoglobin, and haemiglobin species
into stable end-products by an alkaline solution of a
non-ionic detergent. The reaction product, designated
as alkaline haematin D-575, is extremely stable
and shows an absorption peak at 575 nm. This paper
describes the results obtained from using this
modified alkaline haematin method for avian blood.
These results were also compared with the reference
method (cyanhaemiglobin), and a previously used
alkaline haematin method of Bell et al. (1965).
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