2-year-old male Labrador retriever dog weighing 16 kg (Canis familiars).This patient was one of two Labrador retriever dogs from the same litter that were noted to have persistently pale mucous membranes and a grade I/VI systolic heart murmur. Serial complete blood counts revealed a chronic, moderate to severe, macrocytic, and strongly regenerative anemia. Extensive diagnostic workup to rule out conditions leading to a strongly regenerative anemia was performed, including those causing acquired intermittent or chronic hemolytic anemia (immune-mediated, infectious, toxic), or blood loss anemia, all of which were negative. Additional studies led to the diagnosis of hereditary erythrocytic pyruvate kinase (PK) deficiency.
Over the next several months this dog developed mildly icteric mucous membranes with progression of the heart murmur to IV/VI, although otherwise remained clinically well despite persistent anemia and few crises. Three days prior to death he became icteric and lethargic and started vomiting. His clinical condition deteriorated rapidly, and aspiration pneumonia was strongly suspected. Due to a grave prognosis he was humanely euthanized.
The subcutaneous tissues, mucous membranes and fat were diffusely discolored yellow. The femoral bone marrow cavities were filled with dense bone matrix and small amounts of bone marrow, which sank in water.
Approximately 200 ml of yellow-red serosanguineous fluid with fibrin tags was present within the abdominal cavity. The liver weighed 1070 g (6.7% of body weight), was moderately enlarged and firm, with a dark red-brown granular capsular surface and multifocal 1- 2 mm in diameter circular firm tan foci within the parenchyma. The hepatic lymph nodes were prominent with normal architecture. The spleen was dark pink and enlarged, measuring 26 x 9 x 2.5 cm. On the capsular surface, there were multifocal 2-3 mm blue to green plaques (siderofibrotic plaques).
There was 100 ml of red serosaguineous fluid in the thoracic cavity. The sternal lymph nodes were prominent with normal architecture. There was 20 ml of red serosaguineous fluid within the pericardium, and the heart weighed 180 g (1.1% of body weight). The ventral portions of the right cranial, middle and caudal lung lobes were red brown and firm. The left cranial lung lobe contained a 4-5 cm long depressed firm tan streak on the serosal surface. The right middle lung lobe sank in formalin.
Liver: Multifocally within the hepatic parenchyma, portal areas are moderately to markedly expanded by increased numbers of macrophages that contain abundant dense dark brown to black globular pigment (hemosiderin), increased bile duct profiles, and bands of loose fibrous connective tissue that occasionally bridge adjacent portal areas, and less frequently, centrilobular regions. Fibrous connective tissue multifocally surrounds and individualizes periportal hepatocytes, which often contain intracytoplasmic finely granular dark brown to black pigment. Hepatocytes in the surrounding lobules also contain variable amount of a similar cytoplasmic pigment. Hepatic lobules are irregular in size and there are multifocal nodules of regeneration. Frequently within sinusoids and portals areas there are scattered aggregates of primarily erythroid and fewer myeloid precursors (extramedullary hematopoiesis), as well as rare individual hepatocytes which are shrunken with hypereosinophilic cytoplasm and pyknotic nuclei (necrotic). Occasionally within connective tissue in portal areas and within blood vessel walls there is granular to globular basophilic material (mineral). Multifocally on the capsular surface and extending into the underlying hepatic parenchyma, there are regionally extensive areas of fibrosis with increased bile duct profiles (post-necrotic scarring).
Liver (Perls iron, Figures 1A and 1B): Globular pigment within hepatocyte, macrophage and Kupffer cell cytoplasm in portal areas and extending into adjacent hepatic lobules stains positively, confirming the presence of iron.
Lymph node (hepatic, Figure 2): Multifocally, medullary cords are moderately expanded by large numbers of macrophages that contain abundant dense dark brown to black globular pigment (hemosiderin), intracytoplasmic fragments of erythrocytes (erythrophagocytosis) and scattered foci of extramedullary hematopoiesis. In these regions, connective tissue and blood vessel walls often contains mineral. A mild drainage reaction is present.
Liver: Moderate multifocal chronic periportal and hepatocellular hemosiderosis, portal-portal and portal-central bridging fibrosis with nodular regeneration and bile duct hyperplasia (secondary hemochromatosis, cirrhosis); extramedullary hematopoiesis, connective tissue mineralization and post-necrotic scarring.
Serial CBCs: PCV/Hct persistently ranging from 15-20%, with reticulocyte counts >170 K/uL.
- Direct antiglobulin (Coombs) test: Negative
- Erythrocytic osmotic fragility testing: Normal
- Postmortem liver iron (ICP/MS): 37,300 ppm dry weight (reference range canine: 350-1200 ppm dry weight, 100-300 ppm wet weight). Most published reference ranges are reported on a wet weight basis, dry weight results are expected to be 3.5 4 X higher than wet weight.
- DNA-based screening test for known PK mutations in Basenjis, Beagles and West Highland White terriers (8): Negative
- DNA sequencing and mutation results: Homozygous single base missense mutation of PK-LR gene (8)
Iron overload disorders are defined as either increased deposition of iron in parenchymal organs without clinical manifestations (hemosiderosis) or by organ dysfunction secondary to iron-induced injury (hemochromatosis).(11,16) Hemochromatosis was first described in humans in the 19th century based on the classical clinical presentation of bronze pigmentation of the skin, cirrhosis and diabetes mellitus.(14) It is common in humans and some species of birds, but rare in domestic animals.(17) Primary or hereditary hemochromatosis is due to autosomal recessive disorders in iron metabolism. In humans, most are associated with HFE gene mutations. The most common is caused by a single mutation in HFE gene; however, other gene mutations have also been described.(1,2,14) A hereditary form of hemochromatosis has also been described in Salers cattle and is similar to what is seen in humans.(10) Hereditary hemochromatosis is more common in humans than secondary; however, causes of secondary include hemolytic anemias +/- chronic transfusions (thalassemia major, sickle cell anemia, and others), dietary iron overload, chronic liver diseases (hepatitis C and B, alcohol-induced liver disease, porphyria cutanea tarda, and fatty liver disease), and other miscellaneous causes.(3,18) In domestic animals, secondary hemochromatosis is rarely seen and has been attributed to repeat blood transfusions, excessive dietary iron, parenteral iron supplementation, primary liver disease and hereditary erythroenzymopathies.(6,10,11,14,16,17)
In the dog of this report, the hemochromatosis is most likely due to the chronic severe hemolytic anemia caused by hereditary PK deficiency.(8) Pyruvate kinase deficiency is an autosomal recessive trait described most commonly in Basenjis, Beagles and West Highland White and Cairn terriers. Affected dogs present with decreased exercise tolerance, tachycardia, systolic heart murmur, pale mucous membranes and splenomegaly.(4,9) In the past the diagnosis was confirmed by measuring erythrocytic enzyme activity. This can be problematic, as erythrocytes of affected dogs lack the normal adult R-PK isoenzyme, but persistently express the M2-PK isoenzyme, which is present in many fetal and adult tissues.(9,17) Testing for known PK -LR gene mutations is simpler and now available for Basenjis, Beagles, West Highland White and Cairn terriers, and Pugs.(7,8,15) The Labrador in this report did not have any of the known mutations but was homozygous for a single base substitution in exon 7 of the PK-LR gene; this mutation renders the protein dysfunctional.(8)
Pyruvate kinase is primarily responsible for catalyzing the last step in anaerobic glycolysis. A lack of this enzyme greatly impairs ATP-generation in affected erythrocytes, resulting in decreased lifespan and an extravascular hemolytic anemia which is strongly regenerative, with a marked peripheral reticulocytosis.(9) In normal healthy Basenjis, the apparent erythrocyte half-life is approximately 10-28 days, but only 5.8 days in affected dogs.(6) It is estimated that this rapid erythrocyte turnover results in a plasma iron turnover rate that is twice normal. Prolonged and progressive iron overload frequently results in hemosiderosis and may lead to bridging portal fibrosis (cirrhosis) and thus hemochromatosis in the liver of affected dogs.(6,11,12)
Tissue is damaged by excessive uptake of iron as non-transferrin bound iron (NTBI), which, if it accumulates at sufficient concentrations, results in free radical production, iron-induced lipid peroxidation and organelle dysfunction, such as mitochondrial death.(13,14) The organs most commonly affected due to excessive cytoplasmic iron deposition include the liver and pancreas, but can also occur in lymphoid organs, as well as myocardial and skeletal muscles.(10,14,16) Fibrosis and cirrhosis develop in people at a threshold level of approximately 22,000 ppm per dry weight matter (6600 wet weight), which is 12 x normal.(10) The postmortem liver iron level in this dog was 31 x the normal upper reference range limit for dogs, resulting in the changes observed within the hepatic parenchyma.
The lesions associated with hemochromatosis occur when there is loss of equilibrium in systemic iron homeostasis, which is a fine balance between absorption and loss. Control of intestinal iron absorption is tightly regulated by multiple genes and proteins, as there is apparently no regulated mechanism for hepatic or renal excretion of iron in mammals, which occurs only through loss of body secretions, desquamation of intestinal and epidermal cells, or bleeding.(13) Absorption occurs in the duodenum and proximal jejunum across the basolateral membrane of enterocytes where it is taken up as two forms: heme iron from the digestive breakdown of hemoglobin and myoglobin, and non-heme iron released from vegetarian food sources. One of two proteins then sequester the iron to keep it non-reactive: ferritin, which stores iron in cells and is the precursor to hemosiderin, or transferrin, which is the principal iron carrying protein in plasma that distributes iron among tissues for use in biosynthesis of hemoglobin and other iron-containing proteins, and transports to hepatocytes for storage or to tissue macrophages that phagocytize senescent erythrocytes and recycle iron.(1,11,13)
Much recent work has shown that the liver plays a central role in sensing iron needs, which is modulated in part by hepcidin, an acute phase protein which is produced in the liver and secreted into the circulation.(1,3) It is thought that hepcidin responds to two independent signals, iron level and inflammatory status.(13) Hepcidin is upregulated when increased plasma iron levels are sensed, and inhibits export of iron by enterocytes and iron laden macrophages, thus participating in a negative feedback mechanism of regulation.(11,13) It functions by binding to the iron export protein ferroportin, and induces its degradation.(5) Macrophage-based cytokines IL-6, IL-1 and tumor necrosis factor-α also stimulate hepatocellular synthesis of hepcidin.(13) This mechanism is proposed to limit the availability of iron to infectious agents and tumor cells, which require iron to proliferate.(13) Decreased hepcidin expression in dogs with induced iron deficiency has recently been validated, and this work provides incentive for further investigation of gene and protein expression of this and other iron regulating molecules in the dog.(5)
Hepatocytes: Siderosis, periportal, diffuse, severe, with periportal hepatocellular loss and bridging fibrosis.
As elaborately summarized by the contributor, there are serious consequences to a mutation of the rate-limiting step of the anaerobic glycolytic pathway. Erythrocytes, which lack mitochondria, are solely dependent on this pathway for ATP production and thus are most severely affected. However, with the compensatory extramedullary hematopoiesis that can occur following the rapid turnover of erythrocytes, it is ultimately the accumulation of iron within the liver which causes the clinical deterioration observed in this case.
Osteosclerosis with the loss of marrow observed grossly in this case is consistent with other reports in dogs and does not occur in people or cats with PK deficiencies.(8) Mutations resulting in a deficiency of the enzyme phosphofructokinase, another protein in the glycolytic pathway, has also been described in dogs. It causes a persistent hemolytic anemia with occasional episodes of intravascular hemolysis due to alkalemia from hyperventilation, such as occurs during strenuous exercise. This also leads to hepatic hemosiderosis and can affect the skeletal muscle, however, osteosclerosis and liver failure has not been observed in these cases.(9)
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