Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

IV. Porphyrias
247
6. Uroporphyrins are insoluble in ether. Therefore,
fluorescence in the aqueous urinary phase indicates the
presence of uroporphyrins.
B . Fecal Porphyrins
The screening test as described for urine can also be used
with fecal samples after prior extraction with strong acid.
First, 5 g of a fecal sample is emulsified with 10 ml 95%
ethanol. Then 25 ml of concentrated HCl and 25 ml water
are added to the emulsion, and the mixture is kept overnight
at room temperature. The mixture is next diluted to
200 ml with water, filtered, and the filtrate examined for
porphyrins as described for urine.
C . Blood Porphyrins
Almost all porphyrins in blood are present in the erythrocytes,
and only trace amounts are in plasma. Therefore, it is
important to use whole blood in the test and to understand
that the test is a measure of the porphyrin content of the
erythrocytes. Mix 2 ml heparinized whole blood and 6 ml
ethyl acetate:glacial acetic acid (4:1) in a glass centrifuge
tube. Stir thoroughly, centrifuge, and pour off the supernate
into a second centrifuge tube. Add 1 ml 3 N HCl, vortex,
and allow the phases to separate. Fluorescence under UV
light in the aqueous layer indicates the presence of porphyrins
in the erythrocytes. Normally, there will only be
a trace of fluorescence so that more than a trace indicates
an abnormal concentration.
D . Porphobilinogen
The Watson and Schwartz test for porphobilinogen is reliable
for screening ( Watson and Schwartz, 1941 ) . For this
test, 3 ml fresh urine is mixed with 3 ml Ehrlich’s aldehyde
reagent (0.7 g p-dimethylaminobenzaldehyde, 150 ml concentrated
HCl, and 100 ml water), the reagent that is used
for the urine urobilinogen test. Next, 5 ml chloroform
are added, shaken vigorously in a separatory flask, and
allowed to separate. The porphobilinogen aldehyde formed
in the test is insoluble in chloroform and will remain in
the lower aqueous phase. If the pink color is due to urobilinogen,
it will be extracted into the chloroform phase.
Porphobilinogen is found characteristically in the urine of
patients with hepatic forms of porphyria, forms that have
not been reported in animals.
IV . PORPHYRIAS
A . Classification
By convention, the term porphyria is used to define those
disease states that have a hereditary basis and increased
urinary or fecal excretion of uroporphyrins and coproporphyrins.
Depending on the fundamental biochemical
defect, the porphyrias can be broadly classified on the
basis of their tissue of origin, the erythropoietic system, or
the liver. The term porphyrinuria is used to define those
acquired conditions in which the principal, if not the sole,
porphyrins being excreted are the coproporphyrins. Excess
coproporphyrin excretion is observed in a wide variety of
conditions including infections, hemolytic anemias, liver
disease, and lead poisoning. The screening test for coproporphyrinuria
has been especially useful for detecting
exposure to lead.
Many systems for classifying porphyrias have been
devised, and most are based on the defect in the tissue of
origin, the erythropoietic system, or the liver. There is general
agreement on the classification of the erythropoietic
forms, but there is still some uncertainty as to the classification
of the hepatic forms. These have been classified
on the basis of their clinical manifestations, heredity, porphyrins
excreted, and their enzymatic defects (Elder and
Sheppard, 1982; Hindmarsh, 1986 ; Sassa, 2006 ; Sassa and
Kappas, 2000 ) . Each of the enzymatic defects has now
been described on a molecular level in humans, and they
have been summarized by Sassa and Kappas (2000) . The
genes have been cloned, and it is now known that there is
a great deal of genetic heterogeneity, which presumably
accounts for the wide variety of disease manifestations in
each of the porphyrias. A useful system of classification is
given in Table 8-3 .
Methods are also available for the experimental production
of the two major types of porphyria. In lead or
TABLE 8-3 Classification of the Porphyrias a
Porphyria Type Inheritance Enzyme
Deficiency
I. Erythropoietic porphyries
A. Congenital erythropoietic
porphyria
B. Erythropoietic
protoporphyria
AR
AD
UROgenIII-Cosyn
FER-Ch
II. Hepatic porphyrias
A. ALA-D deficiency AR
ALA-D
porphyria
B. Acute intermittent AD
PBG-D
porphyria
C. Porphyria cutanea tarda AD UROgen-D
D. Hepatoerythropoietic AR
UROgen-D
porphyria
E. Harderoporphyria AR COPROgenIII-Ox
F. Hereditary coproporphyria AD COPROgenIII-Ox
G. Variegate porphyria AD PROTOgen-Ox
a
Inheritance: A autosomal; R recessive; D dominant; see Table 8-2 for enzyme
abbreviations.