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

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Chapter | 8 Porphyrins and the Porphyrias
7 . Heme
Within the mitochondria, ferrous iron (Fe 2 ) is chelated
with PROTO IX to form heme and is catalyzed by the
enzyme ferrochelatase (FER-Ch). Iron can also be incorporated
with relative ease by a nonenzymic method, but
the enzymic iron incorporation is more than 10 times that
of the nonenzymic route ( Labbe and Hubbard, 1961 ).
Conditions that help to maintain iron in its ferrous form
including the presence of reducing agents (ascorbic acid,
cysteine, glutathione) enhance both enzymic and nonenzymic
iron incorporation. Because iron is incorporated into
heme as an integral part of the molecule, labeled iron can
also be used as a cohort label to determine the life span of
the erythrocyte ( Table 8-1 ).
8 . Hemoglobin
Heme next moves into the cytosol where it is linked to
the heme pocket of globin in a precise and stable position
that permits binding of oxygen to the heme. Hemoglobin
consists of four moles of this heme-globin moiety linked
together as a globular tetramer. It is this globular tetrameric
form of the hemoglobin molecule that permits the cooperative
interaction of oxygen binding, which gives the familiar
sigmoid oxygen-hemoglobin saturation curve.
9 . Summary
In summary, the synthesis of porphyrins, heme, and globin
can only occur in those respiring cells with full complements
of mitochondrial and cytosolic enzymes. The TCA
cycle is an aerobic cycle, and therefore a lack of oxygen
would preclude synthesis of succinyl-CoA and hence of
heme. PROTO IX formation and the chelation of iron to
form heme are also oxygen-requiring systems. Therefore,
the reticulocyte with its residual complement of enzymes
can synthesize hemoglobin, but the mature erythrocyte,
which is devoid of mitochondrial enzymes, cannot.
III . METHODOLOGY
The principal method now employed for the detection of
porphyrins in biological materials in the clinical laboratory
is based on the characteristic red fluorescence observed
when acidic solutions of the porphyrins are exposed to
ultraviolet light. The color of the fluorescence cannot be
used to distinguish between the uroporphyrins and the
coproporphyrins, and, therefore, these must be separated
before examination for fluorescence. The separation procedures
are based on the solubility differences of the porphyrins
in various organic solvents. In general, the following
solubility properties are used in the separation of the uroporphyrins
from the coproporphyrins:
1. The coproporphyrins are soluble in diethyl ether,
whereas the uroporphyrins are not, and therefore uroporphyrins
remain in the aqueous phase.
2. Both uroporphyrin and coproporphyrin are soluble
in strong acid, 1.5 N HCl. Coproporphyrins are therefore
extracted from the organic phase with 1.5 N HCl. The uroporphyrins
in the aqueous phase are absorbed with aluminum
trioxide and subsequently eluted with 1.5 N HCl.
The acidic solutions of the porphyrins are then observed
visually for fluorescence or examined in a sensitive fluorometer.
The most suitable condition for the excitation of
fluorescence is the use of ultraviolet light in the near visible
range using aqueous solutions of the porphyrins at
pH 1–2. Further means of identification include spectrophotometric
examination, melting points of their methyl
esters, and high-performance liquid chromatography
(HPLC). The quantitative methods employing HPLC with
fluorometric detection have been described in detail by
Hindmarsh et al. (1999) . The HPLC method now appears
to be the most accurate method for urine and fecal porphyrins
( Zuijderhoudt et al. , 2000, 2002 ). The following
screening procedures are guides for further laboratory
examinations.
A . Urinary Porphyrins
The urine for porphyrin examination must be alkalinized
because porphyrins readily precipitate in acid urine.
Addition of 0.5 g of sodium bicarbonate to the collecting
bottle for each 100 ml of urine will keep the urine alkaline.
The alkalinized urine can be stored at 4°C for 2 to 3 days
before analysis. All contact of the urine with metal must
be avoided and unfiltered urine is used. The following is a
simplified screening procedure:
1. Place 5ml urine in a 250-ml separatory funnel and
add 5 ml acetate buffer (four parts glacial acetic acid: 1 part
saturated sodium acetate) and adjust pH to 4.6 to 5.0.
2. Add 15 ml cold water.
3. Extract the mixture with two 50 ml aliquots of
diethyl ether (or until the ether phases show no fluorescence
under ultraviolet [UV] light) and pool the aliquots.
The coproporphyrins will enter the ether phase.
4. Most of the porphyrins in urine are in the form of
their nonfluorescent precursors. Storage of the urine for
24 h in a refrigerator will enhance their conversion to the
fluorescent pigments. If fresh urine is used, the ether phase
is gently shaken with 5 ml of fresh 0.005% iodine solution
(dilute 0.5 ml of a stock 1% iodine in ethanol solution
to 100 ml with water) to convert the precursors to the
porphyrins.
5. Extract the pooled ether phases with 20 ml 5%
HCl (1.5 N) and examine for fluorescence under UV light.
Fluorescence indicates the presence of coproporphyrins.