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

VII. Tests for Evaluating Iron Metabolism
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growing dogs ( Fry and Kirk, 2006 ), but serum TIBC is
generally normal in dogs with naturally occurring iron deficiency
anemia ( Harvey et al ., 1982 ; Weiser and O’Grady,
1983 ). TIBC may be increased in some animals with iron
overload ( House et al ., 1994 ; Sprague et al ., 2003 ) and in
dogs with chronic hepatopathy ( Soubasis et al ., 2006 ).
D. Serum Ferritin
Serum ferritin concentration correlates with tissue iron
stores in humans and domestic animals ( Andrews et al .,
1994 ; Smith et al ., 1984a ; Smith et al ., 1984b ; Weeks
et al ., 1989b ). Increased serum ferritin occurs in animals
with increased storage iron as reported in dogs with chronic
hemolytic anemia (e.g., pyruvate kinase and phosphofructokinase
deficiency) ( Harvey and Smith, 1994 ), malignant
histiocytosis ( Newlands et al ., 1994 ), and hemochromatosis
secondary to repeated blood transfusion ( Sprague
et al ., 2003 ). Serum ferritin is also increased in cattle with
increased iron stores secondary to Theileria sergenti—
induced hemolytic anemia ( Watanabe et al ., 1998 ). Serum
ferritin is transiently increased in horses after moderate to
severe exercise ( Hyyppa et al ., 2002 ) and in foals following
consumption of colostrum, which contains high ferritin concentrations
compared to milk ( Harvey et al ., 1987a ). Serum
ferritin is decreased in animals with iron deficiency ( Harvey
et al ., 1987a ; Smith et al ., 1986a ; Weeks et al ., 1990 ).
Serum ferritin is an acute phase protein; consequently,
increased values are expected in inflammatory conditions,
in addition to conditions with increased iron stores
( Ottenjann et al ., 2006 ; Smith et al ., 1986a ; Smith and
Cipriano, 1987 ). The proinflammatory cytokine tumor
necrosis factor- α (TNF α ) stimulated sustained ferritin
secretion in cultured human hepatocytes ( Torti and Torti,
2002 ). Serum iron concentration may be decreased in both
iron deficiency and in inflammatory conditions ( Andrews
and Smith, 2000 ). Consequently, serum ferritin concentration
can help differentiate true iron deficiency (serum ferritin
is low) from the anemia of inflammatory disease (serum
ferritin is normal or high). It should be remembered that
true iron deficiency could be missed if concomitant inflammation
was present and resulted in increased ferritin secretion
into blood. Commercial assay kits are not available for
serum ferritin assays in animals, but ferritin assays may be
performed for several species at Kansas State University.
E. Bone Marrow Iron
Prussian blue stain is used to evaluate bone marrow hemosiderin
stores. Smears may be sent to a commercial laboratory
for this stain, or a stain kit can be purchased and applied
in-house (Harleco Ferric Iron Histochemical Reaction
Set, #6498693, EM Diagnostic Systems, Gibbstown, New
Jersey). When this stain is applied, iron-positive material
stains blue, in contrast to the dark pink color of the cells and
background. A good-quality marrow aspirate smear with at
least nine particles has been recommended to adequately
access marrow hemosiderin stores in macrophages using
the Prussian blue stain ( Hughes et al ., 2004 ). Determination
of stainable iron in bone marrow is used as a measure of
total body iron stores ( Blum and Zuber, 1975 ; Franken
et al ., 1981 ). A lack of stainable iron is consistent with iron
deficiency; however, negative iron staining is not necessarily
predictive of iron deficiency ( Ganti et al ., 2003 ). Cats
normally lack stainable iron in the marrow ( Harvey, 1981 ).
In addition, some cattle (especially younger animals) lack
stainable iron in the marrow even though marrow iron
can be demonstrated by chemical assay ( Blum and Zuber,
1975 ). Cattle that lack stainable iron generally have lower
marrow iron concentrations when measured chemically than
cattle with a positive iron stain. Similarly, recently weaned
dogs have little or no stainable iron in marrow, presumably
reflecting low iron stores at the end of the nursing period
( Fry and Kirk, 2006 ). Stainable iron in the marrow tends to
increase with advancing age in humans, horses, and cattle
( Blum and Zuber, 1975 ; Franken et al ., 1981 ). Stainable
iron in bone marrow is generally increased in animals with
hemolytic anemia and dyserythropoiesis, in which phagocytosis
of erythroid cells is increased ( Canfield et al ., 1987 ;
Holland et al ., 1991 ; Steffen et al ., 1992 ; Weiss and Lulich,
1999 ), and in animals with anemia resulting from decreased
erythrocyte production, including the anemia of inflammatory
disease ( Feldman et al ., 1981b ).
Some nucleated erythrocytes in Prussian blue-stained
smears from normal animals may contain one to three
small, blue granules in their cytoplasm ( Deiss et al ., 1966 ;
Feldman et al ., 1981a ). When heme synthesis is impaired
(other than by iron deficiency), mitochondria accumulate
excess amorphous iron aggregates, and increased siderotic
granules are present that may form a ring around the
nucleus (generally called a ringed sideroblast in human
hematology) ( Bottomley, 2004 ). Read Section VIII.F in
this chapter for more information.
F. Erythrocyte Zinc Protoporphyrin
Heme is formed inside mitochondria when an Fe 2 ion is
inserted into protoporphyrin IX in a reaction catalyzed by
ferrochelatase. The ferrochelatase enzyme also catalyzes zinc
chelation with protoporphyrin IX to form zinc protoporphyrin
(ZnPP) in trace amounts in normal animals. When iron is
deficient or iron utilization is impaired, zinc becomes a prominent
substrate for ferrochelatase, leading to increased ZnPP
formation and accumulation in erythroid cells ( Labbe et al .,
1999 ). ZnPP is a stable chelate that remains in erythrocytes
throughout their life spans. It can be extracted from erythrocytes
and measured using fluorometry or spectrophotometry.
Unfortunately, ZnPP was unknowingly converted to free protoporphyrin
during analysis for many years; consequently,